extfieldsofstudy: Biology Medicine

text:
Hair cell identity establishes labeled lines of directional mechanosensation

Directional mechanoreception by hair cells is transmitted to the brain via afferent neurons to enable postural control and rheotaxis. Neuronal tuning to individual directions of mechanical flow occurs when each peripheral axon selectively synapses with multiple hair cells of identical planar polarization. How such mechanosensory labeled lines are established and maintained remains unsolved. Here, we use the zebrafish lateral line to reveal that asymmetric activity of the transcription factor Emx2 diversifies hair cell identity to instruct polarity-selective synaptogenesis. Unexpectedly, presynaptic scaffolds and coherent hair cell orientation are dispensable for synaptic selectivity, indicating that epithelial planar polarity and synaptic partner matching are separable. Moreover, regenerating axons recapitulate synapses with hair cells according to Emx2 expression but not global orientation. Our results identify a simple cellular algorithm that solves the selectivity task even in the presence of noise generated by the frequent receptor cell turnover. They also suggest that coupling connectivity patterns to cellular identity rather than polarity relaxes developmental and evolutionary constraints to innervation of organs with differing orientation.

Author summary
Mechanosensory systems are essential for maintaining posture, gaze, and body orientation during locomotion. Such stability requires a coherent representation in the brain of the location and movement of mechanical stimuli. In fishes, mechanical stimuli at a given position activate direction-sensitive receptors called hair cells that are oriented with polarized directionality. These hair cells stimulate neurons that selectively connect with them based on polarity. We have addressed how neurons target hair cells based on polarity during development of the mechanosensory lateral line system in zebrafish. We show that neurons selectively connect based on the expression pattern of the transcription factor Emx2 in hair cells. We find that the lateral line can maintain directionality after damage and regeneration. Our data suggest a cellular mechanism that controls the formation, maintenance, and regeneration of labeled lines to enable directional mechanosensation.

Introduction
Two of the most highly conserved sensory-neural maps in vertebrates are those underlying postural control during locomotion and rheotaxis [1][2][3][4][5][6][7][8]. These sensory-motor processes originate from the action of mechanoreceptive hair cells residing, respectively, in the inner ear and in the lateral line [9][10][11][12][13][14][15][16]. Mechanosensitive ion channels in hair cells open or close according to the direction of deflection of the cells' apical hair bundle [4,17]. Consequently, the planar orientation of the hair bundles endows the sensory epithelium with directional excitability. The mammalian ear contains 3 vestibular receptive organs-the semicircular canals that detect angular shifts of head position, the sacculus that senses vertical movements, and the utricle that detects horizontal movements [18]. Utricular and saccular hair cells are divided into 2 groups of opposing hair bundle orientation, each situated across a line of polarity reversal [19,20]. Similarly, the superficial lateral line in fishes senses velocity fields in the surrounding water [3,6,7,[13][14][15]. This system is formed by a collection of discrete mechanosensory organs called neuromasts, which are distributed across the body of the animal [11]. In zebrafish, each neuromast contains 16 to 20 mechanoreceptive hair cells that occur in 2 equally numbered populations with opposite planar polarity [21]. However, unlike the utricular and saccular epithelia, hair cells of both polarities intermingle in each neuromast (Fig 1A and 1B).
In addition to this local epithelial polarity, the global orientation of the hair cells varies according to the position of the neuromast. For example, the posterior lateral line of larval zebrafish contains neuromasts whose axis of planar polarity is oriented either parallel (horizontal neuromasts) or perpendicular (vertical neuromasts) to the anteroposterior body axis (Fig 1C) [21]. Horizontal neuromasts, therefore, are formed by hair cells that are activated by water flowing in the rostrocaudal direction (rcHCs) and by hair cells of caudorostral directional tuning (crHCs) (Fig 1D). Each branch of the lateral line in larval zebrafish contains around 60 bipolar lateralis afferent neurons (LANs) that are postsynaptic to the hair cells (Fig 1C-1E). Typically, each neuromast is innervated by the peripheral axons of 4 LANs, but each axon exclusively synapses with hair cells of identical planar polarity (Fig 1D-1G) [22,23]. Because the transmission content of a sensory neuron is determined by all the inputs that it receives [24], planar-polarity-selective connectivity renders each LAN tuned to a single direction of mechanical stimulation to form a labeled-line sensory pathway from the periphery to the brain (Fig 1C and 1D).
How planar-polarity-selective synapses are established remains unknown [5,8,22,25]. Understanding this process is important because it will reveal the basic rules that direct the development of neural circuits that enable directional mechanosensation, as well as how they are maintained despite continuous turnover of receptor cells [26][27][28][29][30][31]. Previous work has led to two models to explain polarity-selective synaptic connectivity in the lateral line. The biasedselector model suggests that synaptogenesis is inherently promiscuous and that selectivity occurs by the continuous rectification of mismatched synapses through a biased interaction between hair cells and the axonal population [32]. By contrast, the scaffold model presents constitutive basal projections from the hair cells as short-range processes that provide instructive polarity-specific scaffolds to the axons [33]. This idea is attractive in its simplicity, but it is based exclusively on phenomenological descriptions that have not been experimentally tested. Although both models are consistent with most results, they do not satisfy some observations. For example, the biased-selector model is difficult to reconcile with the invariable ratio of half of the LANs innervating every identically polarized hair cell. Conversely, the scaffold model cannot explain the loss of selectivity by solitary axons. Furthermore, two model-independent basic aspects of the selectivity mechanism remain unsolved: first, whether synaptic connectivity is defined locally in each neuromast independently of its global orientation and second, whether axons synapse with hair cells of identical polarity regardless of their local orientation (synaptic fidelity) or with hair cells of a specific orientation (synaptic partner matching). This distinction is important because the rules underlying fidelity (stochastic) and partner matching (deterministic) are likely to be mechanistically different.

Hair cell basal projections are dispensable for selective connectivity
We began by experimentally testing the scaffold model using high-resolution live imaging in transgenic zebrafish expressing different fluorescent markers in LANs, hair cells, and their presynaptic active zone (Fig 2A-2C).
We first confirmed previous findings that only immature hair cells produce basal projections, up to 15 hours after they are born (Fig 2A and 2B) [33]. Synaptic connections between LANs and hair cells correlate with the stable juxtaposition of presynaptic puncta containing the active-zone constituent protein Ribeye and postsynaptic neurites ( Fig 2C) [32,[34][35][36]. Next, we assessed synapses by individualized axons marked by mosaic expression of the red fluorescent protein mCherry in single LANs, in transgenic embryos co-expressing beta1-actinenhanced green fluorescent protein (EGFP) and ctbp2a(Ribeye)-Kusabira in every hair cell (Tg[myo6b:actb1-EGFP; pou4f3:ctbp2l-mKOFP]) ( Fig 2D-2G'" and S1 Video) [22,26,37,38]. In less than one-half of the hair cells that were analyzed (N = 40), basal projections and axonal terminals physically interacted (16/40), but only a small minority of these interactions resulted in stable synapses (3/16). Moreover, although Ribeye(+) puncta were readily evident in hair cells (arrows in Fig 2D-2E'"), we could never detect them in basal projections (N = 31) (arrowheads in Fig 2D-2E'", and S1 Video). These observations suggest that apposition of presynaptic projections and postsynaptic neurites correlates poorly with synaptic stabilization but does not rule out a function of these projections in synaptic selectivity. To test this possibility, we assayed synaptogenesis by regenerating axons. To this end, we severed axons using an ultraviolet laser delivered through the imaging objective (Fig 2H-2K) [39]. We found that cut axons regenerated to re-innervate preexisting mature hair cells that did not produce basal projections (Fig 2L-2O and S2 Video). To directly assess synaptic selectivity by regenerating axons, we marked single LANs with mCherry in the transgenic lines Tg[myo6b:actb1-EGFP] or Tg [myo6b:actb1-EGFP; nkhgn39d], which reveal hair cell planar polarization in vivo (Fig 1F and  1G) [22,40]. First, we corroborated that individually marked axons selectively synapse with hair cells of identical orientation before severing (Fig 3A, 3B, 3E and 3F).
Two days after severing, every regenerated axon selectively synapsed with identical-oriented hair cells despite the conspicuous absence of basal projections (N = 35) (Fig 3C, 3D, 3G and 3H). Together, these results indicate that, under our experimental conditions, presynaptic scaffolds do not form the bases of polarity-selective connectivity in the lateral line.

Regenerating axons recreate synaptic partnership
During the course of the previous experiments, we made the surprising discovery that regenerating axons nearly always re-innervate hair cells not only of identical but also of the original planar orientation, recapitulating neuronal directional tuning ( Fig 3I, right bar). In most of these cases, when axons were cut immediately below the neuromast, they also re-innervated the original neuromast ( Fig 3J, right bar). Thus, we hypothesized that a potential memory of previous connectivity could rely on 2 possible sources of information: a cell-autonomous molecular code, or a nonautonomous cue that may include the neuronal extracellular basal lamina that remains around preexisting hair cells. Such nonautonomous blueprint of previous connectivity is reminiscent of the neuronal extracellular matrix that directs the repair of mammalian neuromuscular junctions after nerve crush and that is found in the piscine electric organs [41,42]. To test this possibility, we cut axons and, subsequently, killed hair cells pharmacologically by incubating specimens in a solution of neomycin, which eliminates local sources of information about previous synapses. Yet this manipulation did not alter the recovery of LAN directional tuning after the concurrent regeneration of axons and hair cells (N = 8) (Fig 3K-3N). In a separate set of experiments, when axons were cut furthest from the neuromasts (near the lateralis ganglion), they often did not re-innervate the original neuromast ( Fig  3J, left bar). Nevertheless, these axons formed selective synapses with identical-oriented hair cells in new neuromasts and almost always with those whose orientation was the same as the original (Fig 3I, left bar). Moreover, we found a remarkable correlation between horizontal and vertical neuromasts in that, when regenerating axons that originally innervated crHCs in horizontal neuromasts (Fig 3O-3Q) innervated a vertical neuromast, they always synapsed with ventrodorsal hair cells (vdHCs) (N = 4) ( Fig 3O, 3O", 3R and 3S). Conversely, rcHCinnervating LANs switched to re-innervate dorsoventral hair cells (dvHCs) in vertical neuromasts after regeneration (N = 3). Put together, these data reveal that LANs consistently recognize hair cells of identical polarity and of a specific orientation. These results are important because they suggest that hair cells of either orientation must have distinct marks that can be recognized by the axons during development and regeneration.

Synaptic partnership occurs independently of coherent epithelial planar polarity
It has been shown that planar-polarity-selective synapses occur independently of evoked activity [23,32]. Therefore, we hypothesized that a cell-autonomous molecular cue differentiates hair cells of either orientation in each neuromast and identifies as "same" the horizontal Ribeye-Kusabira (light blue) by a singly marked LAN expressing mCherry (red), in double transgenics Tg[pou4f3:GAP-GFP; pou4f3:ctbp2l-mKOFP]. These panels were extracted from S1 Video. Ribeye(+) puncta are readily evident in hair cells (arrows) but absent from basal projections of hair cells (arrowheads in panel D-E'"). Synapses occur when LAN neurites and Ribeye(+) puncta are persistently juxtaposed over time (panel F-G'"). Dotted line outlines the hair cells. (H) Lateral view of a transgenic Tg[nkhgn39d] larval zebrafish expressing EGFP (green) in all LANs and mosaic expression of mCherry (red) in a single LAN, whose cell body (empty arrowhead) can be seen within the posterior lateralis ganglion. A neuromast (arrow) and the site of the eventual severing of the LAN peripheral axons (solid arrowheads) are indicated. The red rhomboid below the indicated neuromast is a singly marked myofiber resulting from nonspecific expression of mCherry in muscle.  rcHCs and vertical dvHCs, as well as the crHCs and vdHCs. While considering this possibility, we noted a previous study in mice showing that a mutation in the transcription factor Emx2 eliminates the line of hair cell polarity reversal in the utricular and saccular maculae without affecting coherent hair cell orientation, and that Emx2 is exclusively expressed on one side of this line in wild-type animals [19,43]. A more recent report revealed that gain-and loss-offunction of Emx2 is sufficient to instruct hair cell orientation along a conserved planar polarity axis in zebrafish neuromasts [44]. These observations raise the possibility that Emx2 is part of a code underlying polarity-selective synaptogenesis. To test this hypothesis, we used a previously validated antibody to Emx2 to immunostain Tg[myo6b:actb1-EGFP] transgenic fish bearing individually labeled neurons. We first corroborated that Emx2-positive hair cells in wildtype fish were always rcHC and dvHC orientation, respectively, in horizontal and vertical neuromasts ( Fig 4A-4H) [44].
Next, we found that each axon synapsed with either Emx2(+) or Emx2(−) hair cells (N = 6) ( Fig 4I-4L and S5 Video). To test whether innervation determined the expression of Emx2 in hair cells, we examined zebrafish carrying a mutation in neurogenin1, which lack all innervation to the lateral line but have normal planar polarity in neuromasts [32]. We found that Emx2 was normally expressed in about half of the hair cells in mutant specimens (Fig 4M-4P).
The above results, together with the strict correlation between hair cell orientation and synaptogenesis, support the long-standing idea that synaptic selectivity and coherent epithelial planar polarity are mechanistically linked [33]. However, to examine this relationship experimentally, we took advantage of trilobite zebrafish, which harbors a loss-of-function mutation in the core planar-polarity protein van gogh-like 2 (Vangl2) [45] and displays a fully penetrant and strongly expressive randomization of hair cell orientation in the lateral line (Fig 5A and  5B) [46].
We reasoned that, if planar polarity and polarity-selective synaptogenesis are functionally linked, loss of coherent hair cell orientation will either induce neurons to innervate all, none, or random numbers of hair cells or to affect synaptic stability. First, we performed immunostaining for Emx2 in Vangl2 mutants and saw that, similarly to the wild type, approximately   half of the hair cells in neuromasts were Emx2(+), indicating that neither the core planarpolarity pathway nor coherent hair cell orientation determine the expression pattern of Emx2 (Fig 5C-5E). Second, we quantified hair cell innervation in Vangl2 mutants and found that, as in wild-type specimens, selective innervation correlated with Emx2 expression (Fig 5F-5K) and that approximately half of the hair cell population was stably innervated by each axon (Fig  5L-5O). Together, these data reveal that Emx2 expression correlates with synaptic partnership and, crucially, that selective synaptogenesis is separable from coherent planar polarity.

Synaptic partnership is determined by Emx2
To test the function of Emx2 in synaptic partnership, we used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genome editing to mutate Emx2. Specifically, to facilitate this analysis by live imaging in zebrafish carrying multiple transgenes, we used the CRISPant approach for somatic mutagenesis [47] [48]. We first corroborated that Emx2 loss-of-function renders nearly every hair cell identically oriented, always dorsally in vertical neuromasts ( Fig 5P) and rostrally in horizontal neuromasts ( Fig 5Q) [44]. Second, we found that in Emx2-deficient animals, individually marked axons synapsed with nearly every hair cell in neuromasts (Fig 5L and 5R-5T) or with a small minority to none (Fig 5L and 5U-5W). Some specimens presented partial expressivity of the Emx2 mutant phenotype, with neuromasts bearing a majority of hair cells oriented in one direction and a small minority in the opposite. Yet in these cases, individually marked axons also synapsed exclusively with hair cells of identical orientation regardless of their absolute or relative number (Fig 5L). These results reveal that loss of Emx2 homogenizes hair cell orientation without altering the ability of axons to recognize identically oriented hair cells and, importantly, that the Emx2 expression status in hair cells determines whether they will be innervated by a specific axon.

Discussion
The piscine lateral line is a prime example of a neural system that represents in the brain the entire mechanosensory scene as vectorial signals from multiple peripheral receptors [2,8,9]. Therefore, it is ideally suited to unravel how neuronal tuning to mechanical-flow direction is neuromast expressing EGFP in hair cells ( Planar polarity-selective synaptogenesis established during development and maintained in the face of synaptic instability driven by constant receptor cell turnover. Here, we uncover a simple partner-recognition mechanism that governs the strict innervation of identically plane-polarized hair cells by afferent neurons. The underlying process is robust to environmental perturbations, enabling the maintenance of labeled lines of directional mechanosensory flow in a system that undergoes frequent and lifelong renewal of receptor cells. Below, we illustrate this mechanism and discuss the supporting evidence.

Hair cell projections are dispensable for synaptic selectivity
Similarly to the mammalian utricular and saccular maculae, the hair cells in piscine neuromasts are divided into 2 populations with opposing planar orientation [11,21]. Utricular and saccular afferent neurons do not co-innervate hair cells across the line of polarity reversal [49][50][51]. Identical observations have been made in the vestibular apparatus of amphibians [51] and birds [52]. Several studies have revealed that the afferent neuronal pathway of the lateral line is organized in a similar manner [8,22,23,33,52]. This is remarkable given the absence of anatomical compartmentalization of epithelial planar polarity in neuromasts. To shed light on the synaptic selectivity mechanisms, we experimentally tested the scaffold model. We show that hair cell basal projections are dispensable for polarity-selective innervation, suggesting that they act as haptotactic cues that facilitate or accelerate the innervation of nascent hair cells in a nonselective manner, possibly by increasing the cell's surface area during the search-and-find period that precedes synaptic maturation [22,53].

Planar polarity and synaptic selectivity are separable
The above results raise 4 interwoven questions. What is the source of the selectivity cues? Do selectivity cues act locally in each neuromast or globally along the entire lateral line system? Does a synaptic selectivity code exist? If so, what is its identity? We investigated whether planar polarity per se represents a selectivity cue by using mutant zebrafish lacking the core planar-polarity component Vangl2, in which hair cells assume random orientations. We reasoned that if coherent planar polarity instructed selectivity, its loss would randomize or destabilize synapses. In Vangl2−/− animals, connectivity patterns are stable and nonrandom because, as in wild-type specimens, each LAN axon synapses with one-half of the hair cells. Furthermore, our analysis of de novo synaptogenesis by regenerating axons shows a remarkable robustness in the lateral line system in that axons invariably re-innervate hair cells of the same orientation as their original. This remains true even when regenerating axons target a neuromast different from the original. We also found a near-perfect correlation of synaptogenesis when regenerating axons re-innervate neuromasts of differing global orientation because LANs that innervate rcHCs in horizontal neuromasts synapse with dvHCs when switching to innervate vertical neuromasts. Together, these results demonstrate that synaptic selectivity is separable from the local and global orientation of the hair cells and that it is determined independently in each neuromast. It is tempting to extrapolate these results to mammals to explain the puzzling observation that, while the core planar-polarity proteins Frizzled6 and Prikle-like2 distribute identically in the entire epithelium of the sacculus and the utricle, neurons never co-innervate hair cells across the line of polarity reversal [19,51,54]. Therefore, it is likely that both in the mammalian inner ear and in the piscine lateral line, planar polarity per se does not determine polarity-selective synaptogenesis. Assessing hair cell innervation in the vestibular system of Vangl2 mutant mice may directly test the likelihood of such evolutionary conservation.

Emx2 governs the formation and homeostasis of synaptic partnership
The above results suggest the existence of a label that identifies as same caudal-oriented and ventral-oriented hair cells, as well as rostral-and dorsal-oriented cells. They also indicate that the process of planar-polarity-selective synaptogenesis can be framed as a problem of synaptic partner matching rather than of synaptic fidelity. The transcription factor Emx2 is expressed in half of the hair cells in each neuromast and correlates with their global orientation in that Emx2 is exclusively expressed in rcHCs and dvHCs. We found that Emx2 expression patterns are not affected by the loss of coherent planar polarity and that axons make strict connections according to the hair cells' Emx2 expression status, even when they are not coherently oriented. Moreover, we show that loss of Emx2 affects hair cell innervation in a predictable manner because it enables some axons to synapse with nearly every hair cell in the neuromast while remaining selective as well as simultaneously prevents other axons from making any synapse. These findings demonstrate that Emx2 establishes an identity code for synaptic partners and represents the link between planar polarity and synaptic selectivity. It transpires that there must be differences between the neurons that innervate each hair cell polarity class because, if neurons were identical, regenerating axons should not be able to regain directional tuning by recognizing previous partners or re-innervate matching populations of hair cells when switching from horizontal to vertical neuromasts. Although we currently lack the tools to directly test this hypothesis, we believe that it is likely given some evidence from the mouse, in which a common pool of Neurogenin-1(+) neuronal progenitors produces auditory afferent neurons with segregated connectivity in the inner ear [55].
We wonder how a transcription factor may mediate polarity-selective synaptic connectivity. Genetic perturbation experiments indicate that Emx2 does not control local or global planar polarity because the global axis of polarization of the hair cells is not altered in the inner ear of mice and in neuromasts of zebrafish lacking Emx2. Instead, we interpret the collective data as suggesting that Emx2 instruct hair cell identity, which in turn enables cells to implement the global polarity cue in one of the two possible directions. One possibility is that Emx2-dependent expression of high-affinity complementary transmembrane proteins determines synaptic partnership between matching subpopulations of LANs and hair cells. This will enable neurons to recognize synaptic partners locally and independently of their relative of absolute number and to regain directional tuning after recovery from injury. It also explains how individual neurons synapse with hair cells of differing global orientation when they co-innervate multiple neuromasts during the expansion of the lateral line in juvenile and adult zebrafish [56,57]. This can occur because synaptic partnership is controlled locally by Emx2, independently of the specific orientation of the hair cells. Taken together, our data strongly support the conclusion that Emx2 establishes an identity code that governs synaptic partnership between hair cells and afferent neurons.

A wiring algorithm controlling synaptic partnership
How may the rules governing synaptic partner choice be implemented? In other words, what is the underlying wiring algorithm [58]? Our data suggest a simple structure for the interaction among synaptic partners that can explain the development of planar-polarity-selective synapses, the regeneration of neuronal directional tuning, and the long-term maintenance of labeled lines of directional mechanosensation (Fig 6A).
Crucially, it also unifies all previous results leading to the biased-selector and the scaffold models [32,33]. The components of the selectivity process are 2 subpopulations of hair cells and t2wo subpopulations of LANs (Fig 6B). In hair cells, this assumption is supported by polarity-associated expression of Emx2 as well as Tmc2b [59]. Hair cells attract axons  [32]. However, the Emx2 expression status in hair cells determines the outcome of this attraction because synapses will preferentially form when the appropriate axonal subclass contacts hair cells in an Emx2-matching manner. Once formed, stable synapses repulse incorrect partnerships (Fig 6A). This explains the promiscuous synaptogenesis by solitary axons, which become doubly tuned because the absence of other axons prevents the formation of the converse correct synapses, eliminating the repulsive signals (Fig 6C). A combination of attractive and repulsive signals also explains the recovery of selectivity by the reintroduction of additional axons, which reestablish synapses with the correct partners based on Emx2 expression status, regenerating the repulsive signal that eventually pushes the promiscuous solitary axon to exclusively synapse with hair cells of identical polarity (Fig 6D) [32].
Previous work has shown that chronic loss of hair cell innervation does not affect epithelial planar polarity in neuromasts. This has led to the prediction that innervation does not determine Emx2 expression patterns, which we have now confirmed experimentally. This explains how regenerating axons are able to synapse with the original hair cells (Fig 6E). It occurs because Emx2 expression in denervated hair cells does not change, thus serving as an indelible mark of previous partnership. Our model also explains the recovery of selectivity and neuronal directional tuning after hair cell death and regeneration because the system continuously selforganizes by reenacting the developmental program that assigns different identities to sibling hair cells, in turn determining the likelihood of synaptic stabilization of specific axons (Fig 6F). Upon loss of Emx2, the entire hair cell population becomes homogeneously oriented [44]. In this case, the architecture of the wiring algorithm becomes asymmetric, directing one axonal population to synapses with every hair cell and inhibiting the other axonal population from establishing any synapse (Fig 6G). Furthermore, because loss of mechanoreception does not affect hair cell planar polarity, hair cell activity cannot affect Emx2 expression, explaining why synaptic selectivity is normal in fish with nonfunctional hair cells [23,32]. Finally, selective synaptogenesis is maintained in the absence of coherent epithelial planar polarity because Emx2 remains expressed in half of the hair cells in Vangl2 mutants (Fig 6H), and thus the architecture of the algorithm remains intact (Fig 6A).

Conclusion
Three questions in mechanosensory biology are the current focus of intense attention. First, what is the mechanism that establishes the connectivity patterns between distinct mechanoreceptors and ascending afferent neurons [1,32,60]? Second, how are the resulting labeled lines of directional mechanosensation maintained in the face of continuous turnover of receptor cells [1,32,60]? Third, how much has the anatomical blueprint for directional mechanosensation diverged during the 600 million years of evolution since hair cell-based mechanosensation appeared in the common ancestor of agnathans and mammals [50,54,61,62]? We found because the absence of the converse synapses eliminate the repulsive signals. (D) Scheme of an acutely solitary axon, which becomes transiently nonselective because the regeneration of the converse synapses recreates the repulsive signals to push the promiscuous axon to selectivity. Jagged arrow (purple) indicates axon damage. (E) Situation of axon damage and regeneration. Initially during regeneration, axons explore the presynaptic field and transiently interact with hair cells, but eventually the wiring algorithm is recreated following the same rules controlling selectivity during circuit development. (F) Situation of hair cell loss and regeneration. Initially during hair cell regeneration, axons explore the presynaptic field and transiently interact with hair cells, but once hair cells mature, the wiring algorithm is recreated following the same rules controlling selectivity during circuit development. (G) Situation of homogeneously oriented hair cell upon loss of Emx2 activity. The wiring algorithm becomes asymmetric, directing one axonal population to synapses with every hair cell and inhibiting the other axons from establishing any synapse. (H) Situation of misoriented hair cell upon loss of Vangl2. The wiring algorithm remains normal because it operates independently of coherent planar polarity. Therefore, synaptic selectivity for Emx2(+) and Emx2(-) hair cells occurs normally. crHC, caudorostral HC; crLAN, caudorostral LAN; HC, hair cell; LAN, lateralis afferent neuron; rcHC, rostrocaudal HC; rcLAN, rostrocaudal LAN; Vangl2, van gogh-like 2.
https://doi.org /10.1371/journal.pbio.2004404.g006 that selective connectivity between LANs and hair cells of identical orientation occurs independently of presynaptic projections and that it is separable from the coherent planar polarity and global orientation of the hair cells. We demonstrate a wiring algorithm that locally disambiguates hair cell identity, links identity to orientation, and establishes the synaptic partnership rules that generate neuronal directional tuning. Despite its simplicity, this algorithm readily solves the selectivity task even in the presence of constant noise generated by frequent death and regeneration of hair cells, and independently of scale differences between the receptive organ and the neuronal population. It also operates robustly upon evolutionary changes in epithelial polarity, or when neurons co-innervate organs with differing global orientation, which naturally occurs during the expansion of the lateral line that accompanies animal growth [56,57]. The striking similarities between the zebrafish lateral line and the mammalian vestibular apparatus, including their dependence on Emx2 for hair cell orientation, suggest that the wiring mechanism that we have unveiled may be ancient and conserved across vertebrates.

Ethics statement
Experiments with wild-type, mutant, and transgenic embryos of undetermined sex were conducted under a protocol approved by the Ethical Committee of Animal Experimentation of the Parc de Recerca Biomedica de Barcelona (Spain), and protocol number Gz.:55.2-1-54-2532-202-2014 by the "Regierung von Oberbayern" (Germany).

DNA constructs
The hsp70:mCherry-SILL (SILL:mCherry) construct was generated using the Tol2 kit. Entry vectors were generated as described in the Invitrogen Multisite Gateway manual. PCRs were performed using primers to add att sites onto the end of DNA fragments, using Platinum Pfx (Invitrogen). The pEntry vectors containing the UAS sequence, hsp70 minimal promoter, mCherry, and polyA are from the Tol2 kit, and the pEntry vector containing the SILL enhancer was previously generated in our laboratory [32].

Mosaic neuronal labeling
For sparse labeling of LANs, we injected 15 to 20 pg of a DNA plasmid containing the construct hsp70:mCherry-SILL (SILL:mCherry) in 1-to 4-cell-stage embryos. Resulting embryos at 3 to 4 dpf were anaesthetized and screened for red fluorescence in single neurons using a Zeiss stereomicroscope.

Immunohistochemistry
Larvae were fixed overnight at 4˚C in a solution of 4% PFA. After fixation, fish were washed with PBST and permeabilized in acetone at −20˚C for 5 minutes. Then, samples were washed with MiliQ water for 5 minutes and incubated for 1 to 2 hours at room temperature with blocking solution (1% BSA, 2% NGS, 1% DMSO). After blocking, larvae were incubated overnight with primary Ab (Ctbp2 1:100) or for 48 hours (Emx2 1:250) at 4˚C. The next day, fish were washed with PBST 5 to 6 times and incubated overnight at 4˚C with secondary Ab (GaRb 633, GaRb 555). Then, samples were washed with PBST and mounted for imaging. The Ctbp2 primary antibody was obtained from Proteintech (Manchester, UK) and the Emx2 antibody from TransGenic (Fukuoka, Japan).

Imaging and videomicroscopy
For in vivo imaging, laser-mediated axotomy, and some Emx2 immunostaining, we used a custom-built inverted spinning disc microscope (Zeiss Axioscope). Emx2 immunostainings in wild type with single neurons labeled were imaged using a Zeiss inverted confocal microscope with a 40× water immersion objective. Embryos and larvae used for in vivo imaging were anesthetized in MS-222 (tricaine) 0.16 g/L and mounted in 1% low-melting-point agarose on the cover slip of a glass-bottom dish (MatTek, Ashland, MA). Imaging dishes were bathed in Danieau's with MS-222 0.16 g/L, except for time-lapse imaging where concentration was reduced to 0.08 g/L. Acquisition was performed at 28.5˚C using a 63× water immersion objective.

Assessing synaptic connectivity
Synaptic connectivity was assessed in transgenic fish expressing different fluorescent markers in all hair cells and in individual axons. Synapses were identified as bulged postsynaptic endings adjacent to the base of hair cells. This was always done by progressing through individual focal planes of Z-stacks, from the apical end of the epithelium (to assess the planar polarization of each hair cell) to the most basal aspect of the epithelium (where apposition of neuronal endings and hair cells are found). Examples are shown in 2 supplemental videos (S3 and S4 Videos, associated with Fig 3).

Statistical analyses
The total number of hair cells is not uniform across different neuromasts. Thus, quantification of the innervation in wild-type, Vangl2, and Emx2 mutant specimens was normalized to the total number of hair cells in each neuromast. We compared the obtained fractions in the different groups using an ANOVA test. For the analysis of Emx2-positive cells in wild-type and Vangl2 mutants, we also normalized samples by dividing the number of positive hair cells by the total number of hair cells. We then compared the wild-type and Vangl2 mutant groups using an unpaired t test.

Neomycin treatment and laser-mediated microsurgery
Specimens were incubated in MS-222 (3-aminobenzoic acid ethyl ester) in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, in deionized water [pH 7]) and mounted in 1% low-melting agarose on a glass-bottom Petri dish. They were positioned using a hair loop under light from a xenon arc lamp passed through the appropriate filters to reveal green or red fluorescence. Axon severing wad done using a computer-controlled iLas-Pulse laser system (Roper Scientific SAS, Evry, France) consisting of a pulsed ultraviolet laser (355 nm; 400 ps/2.5 μJ per pulse). The axons were transected with a focused laser beam coupled to a spinning-disk inverted microscope. The laser power was set to 35 mW at the sample plane. To sever axons, a region of interest (ROI) was drawn over the nerve, and a train of laser pulses was repeatedly applied until all fluorescence disappeared within the ROI. After severing, the axons were observed repeatedly to evaluate the success of the cuts. Subsequently, specimens were removed from the agarose and left to recover in fresh E3 embryo medium in individual Petri dishes. For regeneration experiments, larvae are remounted as the above for time-lapse imaging. Z-stacks were made using ImageJ (Plugins ! Segmentation ! Simple Neurite Tracer). Hair cells were pharmacologically ablated by incubation of specimens in a 250 μM solution of neomycin for 45 to 60 minutes at room temperature and then rinsed with E3 medium [26,39,46].
Supporting information S1 Video. Developmental innervation of hair cells by a singly marked axon. This video shows an example of the innervation of hair cells expressing EGFP (green) as they develop, by a single axon marked by mosaic expression of mCherry (red). Hair cells also express the active-zone component Ribeye fused to Kusabira (orange). Basal projections from hair cells are readily evident during the first half of the video. Yet Ribeye is never detected in the projections, and synaptic stability occurs by the juxtaposition of LAN neuritis and Ribeye-containing puncta in the basal aspect of hair cells that do not produce projections. EGFP, enhanced green fluorescent protein; LAN, lateralis afferent neuron. (AVI) S2 Video. Regenerative innervation of hair cells by a singly marked axon. This video shows an example of the re-innervation of mature hair cells expressing EGFP (green) by a previously severed single axon marked by mosaic expression of mCherry (red). Basal projections are not produced by these hair cells. Yet synaptogenesis occurs normally. Thin green extensions towards the end of the video are apical kinocilia from the hair cells, which become evident due to their free movement. EGFP, enhanced green fluorescent protein.
(AVI) S3 Video. Determination of synapses in a horizontal neuromast by individualized axon before severing. This video is a Z-stack used to generate panel P-P' of Fig 3 and shows an example of the innervation of mature hair cells expressing EGFP (green) by an axon marked by mosaic expression of mCherry (red). Red dots indicate the synapsed hair cells, whose planar polarization is evident in the most apical aspect of the epithelium (beginning of the video), revealing their caudorostral orientation (as depicted in Fig 3Q). Yellow arrows indicate synaptic contacts as bulged axon endings. EGFP, enhanced green fluorescent protein.
(AVI) S4 Video. Determination of synapses by the individualized axon that initially innervated the horizontal neuromast, after severing and regeneration to innervate a vertical neuromast. This video is a Z-stack used to generate panel R-R' of Fig 3 and shows an example of the re-innervation of mature hair cells expressing EGFP (green) by an axon marked by mosaic expression of mCherry (red). Red dots indicate the synapsed hair cells, whose planar polarization is evident in the most apical aspect of the epithelium (beginning of the video), revealing their ventrodorsal orientation (as depicted in Fig 3S). EGFP, enhanced green fluorescent protein.
(AVI) S5 Video. Assessment of Emx2 immunostaining. This video shows an example of a Z-stack of a neuromast immunostained for Emx2. White circles mark 5 Emx2(+) hair cells (magenta). Numbers reflect order of appearance across the Z-stack from the epithelial apex to base. This Z-stack was used to generate the maximal-projection image shown in Fig 4K and is exemplified in drawing of Fig 4L. (AVI) S1 Table. Numerical data for the plots in Fig 5. This table contains the data point used for statistical tests plotted in Fig 5E (left) and Fig 5L (right), including all conditions that include wild-type and mutant specimens. (TIFF)


s2fieldsofstudy: Biology Medicine

text:
High RAC3 expression levels are required for induction and maintaining of cancer cell stemness

RAC3 is a transcription coactivator, usually overexpressed in several tumors and required to maintain the pluripotency in normal stem cells. In this work we studied the association between RAC3 overexpression on cancer cell stemness and the capacity of this protein to induce cancer stem properties in non tumoral cells. We performed in vitro and in vivo experiments using two strategies: by overexpressing RAC3 in the non tumoral cell line HEK293 and by silencing RAC3 in the human colorectal epithelial cell line HCT116 by transfection. Furthermore, we analysed public repository microarrays data from human colorectal tumors in different developmental stages. We found that RAC3 overexpression was mainly associated to CD133+ side-population of colon cancer cells and also to early and advanced stages of colon cancer, involving increased expression of mesenchymal and stem markers. In turn, RAC3 silencing induced diminished tumoral properties and cancer stem cells as determined by Hoechst efflux, tumorspheres and clonogenic growth, which correlated with decreased Nanog and OCT4 expression. In non tumoral cells, RAC3 overexpression induced tumoral transformation; mesenchymal phenotype and stem markers expression. Moreover, these transformed cells generated tumors in vivo. Our results demonstrate that RAC3 is required for maintaining and induction of cancer cell stemness.

INTRODUCTION
Tumorigenesis has been long known to resemble organogenesis and most tumors are heterogeneous containing many phenotypically and functionally different cells [1,2]. In fact, this heterogeneity may be a consequence of the clonal expansion and selection from cells having mutations and genomic instability, as well as the evolution trough proliferation, maturation and differentiation from an original tumor initiating cell, called cancer stem cell [3].

Research Paper
Moreover, these mechanisms don't have to be mutually exclusive and a combination of both could be contributing to the tumor development.
Whenever the case, what is really important to consider is that cancer stem cells side population is the major responsible of cancer propagation and resistance to most anti-cancer treatments. These cells have the ability to perpetuate the tumor when invade other tissues or when are transplanted, recapitulating the heterogeneity and histology of the parent tumor in the new focus.
Although there is a diversity of tumor types and treatments, most of them could be considered as targeting a systemic disease, where a definitive remission is usually not sure until several years after detection, surgery and/ or treatment. Despite some cancer types are originally sensitive to chemotherapy, giving a promising response, after some years there is a tumoral relapse usually refractory to chemotherapeutic drugs. This could be explained as a the clonal selection of some resistant cells due to acquired mutations driven for the chemotherapeutic drugs and the self-patient physiology, as well as, the clonal expansion of cancer stem cells, originally resistant to chemotherapy. Whatever the way, the consequences are the same: human death by cancer.
These cancer stem cells have specific cell markers [4][5][6], although their origin and maintenance are not clear at present. They may alternatively be derived from stem cell pools, progenitor cells or differentiated cells that undergo trans-differentiation processes [2,6].
In fact, RAC3 is an oncogene that contributes to tumor development acting as a nuclear receptor coactivator of several transcription factors [12,17] that control the expression of genes related to cell cycle progression and proliferation, inhibition of apoptosis [16,18], autophagy [19] and senescence [20].
Additional functions related to tumor progression and metastasis development [17,21], such as metalloproteinases expression, cell migration and epithelial-mesenchymal transition have also been described and attributed to the RAC3 splicing variant Delta 4-SRC3 [17,22,23].
Most of the studies that allowed to define RAC3 as an oncogene were performed in models where it is naturally overexpressed, such as tumoral cell lines, tumors and transgenic or knockout mice [8] where expression is equally affected in all the tissues. Although the transforming effect of RAC3 overexpression over non tumoral cells has not been deeply investigated up to date, we have previously demonstrated that RAC3 overexpression as a unique change, in the non tumoral human embryonic kidney cell line (HEK293) enables these cells to growth in soft agar forming colonies [24].
In addition, it was recently demonstrated that RAC3 expression is required in order to preserve the pluripotency and self-renewal of stem cells [25][26][27][28]. In normal healthy tissues RAC3 expression is downregulated in mature and differentiated cells, as well as in aged tissues [20], suggesting that changes in their levels may be playing a critical role in development. However, we have previously found that inflammatory cytokines like TNF up-regulate the expression of this oncogene [29].
Therefore, being RAC3 an oncogene and also a required factor to maintain the stem properties of normal cells, in this work, we investigated its association with cancer stem cell phenotype.

RAC3 overexpression contributes to maintain the mesenchymal phenotype
RAC3 is a known high expression molecule in several types of tumors including colorectal cancer [29]. However, it was not clearly determined at present if this overexpression could be early detected at tumor initiation and then homogeneously sustained along the cancer progression.
Therefore, we performed the analysis of microarrays from repository public data base of 19 human colorectal cancer samples. We found that RAC3 is a common elevated expression gene in this type of tumors and metastasis ( Figure 1A), which correlated with an enhanced mesenchymal phenotype, increasing Vimentin and c-MYC, but decreasing E-Cadherin expression ( Figure  1B-1D). Moreover, these changes were accompanied by an increased expression of stem cell markers OCT4 and Nanog ( Figure 1E and 1F). Concerning cancer progression, RAC3 shows to be early overexpressed and maintained at advanced degree of the sickness ( Figure 1G-1H).
In view of these results, we wanted to know if RAC3 could have a role inducing or maintaining some phenotypic properties of cancer stem cells that are shared with mesenchymal cells [30]. Therefore, we first investigated if a diminished expression of RAC3 may affect the expression pattern of Vimentin a typical marker of tumoral migratory, mesenchymal and some endothelial cells [31] and the epithelial marker E-Cadherin. In these experiments we compared the levels of both mRNA and proteins in the human cancer colon HCT116 cell line having high expression levels of RAC3 [19], stably expressing an scrambled or the shRAC3 expression vector, which significantly inhibits the RAC3 expression as shown in the Figure 2A.
We found that cells having low RAC3 expression shown an increased expression of E-Cadherin while Vimentin was downregulated respect to the control cells. Thus, these results demonstrate that RAC3 inhibition induces a mesenchymal-epithelial transition (MET) ( Figure 2B and 2C).
In addition, MET induced by RAC3 inhibition was accompanied by a decrease in the metaloprotease-2 (MMP-2) activity and expression, as shown in Figure 2D and 2E, that also correlated with a diminished migratory and invasive behavior respect to control cells ( Figure 2F). All these effects of MET, MMP-2 downregulation and migratory-invasive behavior inhibition, were also obtained in the human mammary cancer T47-D cells where its natural high RAC3 expression was stably inhibited by an shRAC3 RNA (data not shown), demonstrating that is not exclusive of colon cancer.

RAC3 overexpression contributes to maintain the cancer stem cell side population of colorectal cancer cells
Regarding the previously reported property of cancer stem cell population to quickly exclude the Hoechst staining through their high ABC-MDR expression levels [32], we performed experiments to detect differences in the stem cell amount between cells overexpressing or not RAC3 in the absence or presence of Verapamile in order to block the ABC transporters activity.
We found that in the absence of the blocking drug no differences in the amount of cells retaining the stain over 90 minutes were observed between cultures expressing high or low levels of RAC3 ( Figure 3A). However, when the ABC transporters were blocked, although the total stain retention was increased, the amount of unstained cells was significantly inhibited in shRAC3 cells respect to control, demonstrating a decrease of cancer stem cell population in cells having low expression levels of RAC3.
In order to confirm these results by additional assays, we determined the clonogenic growth of control and shRAC3 HCT116 cells after seven days of platting at low density. As shown in Figure 3B, the number of colonies was significantly diminished in cells where RAC3 was inhibited. Moreover, cells expressing low RAC3 levels were almost unable to growth as tumorspheres ( Figure 3C). This was in agreement with a diminished amount of cancer stem cells markers OCT4 and Nanog [33] (Figure 3E), while a high expression of the colon cancer stem cell marker CD133 and Nanog were detected by immunofluorescence staining of tumorspheres, as expected for their high amount of stem cells ( Figure 3D).
We then analyzed if the expression levels of stem markers could be also affected by a decreased RAC3 expression in HCT116 cells. Figure 3E shows that expression of both Nanog and OCT4 were downregulated under low RAC3 levels.
In order to determine if RAC3 overexpression could be mainly associated to one cancer stem cell marker, we first isolated the CD133+ enriched side population from wild type HCT116 cells using a specific antibody conjugated to magnetic beads. The non-retained fraction was considered as CD133− side-population. Then, the expression levels of RAC3 mRNA were measured in both fractions. As shown in Figure 3F, high expression levels of RAC3 were mainly associated to the CD133 positive side population.
Similar results were obtained when we processed the public repository microarrays data from HCT116 cells and the human epithelial colorectal adenocarcinoma CaCo cells ( Figure 3G, left and right panels, respectively).
Taken together all these results, we may conclude that RAC3 overexpression is mainly associated to colorectal cancer stem cell markers, and contributes to maintain some cancer stem properties.

RAC3 overexpression in non tumoral cells induces a mesenchymal transformation
In view of the results obtained in tumoral cells that naturally overexpress RAC3; we first investigated if RAC3 overexpression in non tumoral cells as a unique genetic introduced modification, is able to induce some of the tumoral properties that were observed in the human colorectal cancer cells.
The non tumoral HEK293 cells express limited amounts of this coactivator, similar to normal mature differentiated cells [16]. They were stably transfected with a RAC3 expression vector, thus, significantly increasing the RAC3 levels respect to the cells carrying the empty vector as shown in Figure 4A.
We found that Vimentin levels were significantly increased while E-Cadherin levels were inhibited in cells overexpressing RAC3 ( Figure 4B and 4C), supporting the results obtained in tumoral cells and the role of RAC3 overexpression favoring the mesenchymal phenotype. In addition, this transition to a mesenchymal phenotype when RAC3 was overexpressed was accompanied with an increased activity of MMP-2 as determined by zymography ( Figure 4D), which correlated with the enhanced MMP-2 gene expression ( Figure 4E) and acquired migratory and invasive behavior. Although control HEK293 cells were almost unable to migrate and invade, we found they acquired this ability under RAC3 overexpression ( Figure 4F).
We then investigated whether RAC3 overexpression could confer to these non tumoral cells the ability to proliferate in the presence of low growth factors. The diagram bars shows the mean ± SD of HCT116 colonies growth each one from one clone, when cells were plated at densities of 200, 400 or 800 per well (B). Only SC cells were capable to growth as tumorspheres in stem media. Left panel shows the mean ± SD and (a representative image at right panel) (C). High RAC3 and stem markers detected by IFI in tumorspheres from SC-HCT116 using the specific primary antibodies or unspecific control α-mouse 594 (D). Expression levels of mRNA stem markers were determined in SC or shRAC3-HCT116 cells (mean from three independent experiments ± SD) (E). The diagram bars shows the mean ± SD mRNA RAC3 expression levels in HCT116 cells from CD133 enriched (CD133+) or depleted (CD133-) side population (F). Similar to F, but from both public repository data microarrays of FACS sorted HCT116 cells and CaCo cells ( * p < 0.05 respect to CD133-). Platform GPL6244, accession number GSM932995 for HCT116 (colon cancer cells). And platform GPL96, accession number GSE24747 for CaCo (colon cancer cells), both from Affymetrix Santa Clara, CA, USA. The side population CD133+ o CD133− isolated by flow cytometry (G). Therefore, the cell number was determined daily during three days in cultures of HEK293 overexpressing RAC3 or controls growing under normal or low serum (1%) conditions. Although control cells were unable to proliferate under low serum, RAC3 overexpressing cells acquired this ability ( Figure 4G) increasing the cell number during 72 h of culture respect to control cells.

RAC3 overexpression in non tumoral cells induces tumor initiating cells
In order to investigate if this increase in cell number could be due in part to the enhanced stem cell side population in cultures overexpressing RAC3, we first investigated the ability of these cells to growth as tumorspheres. We found that although control HEK293 were almost unable to growth as tumorspheres, they acquired this ability under RAC3 overexpression ( Figure 5A), while the clonogenic growth was significantly increased ( Figure 5B). Moreover, experiments of Hoechst efflux ( Figure 5C) also demonstrate the increase of stem cells in cells overexpressing RAC3. Therefore, we analyzed the expression levels of the stem cell marker genes Nanog and OCT4. As shown in Figure 5D, mRNA levels of both were significantly increased by RAC3 overexpression.
Our findings demonstrated that RAC3 overexpression as the unique introduced genetic modification in non tumoral cells, induced a cell transformation toward a phenotype that suggests the presence of cancer stem cells.
Therefore, we then investigated whether these cells were capable to initiate tumors when they are inoculated in vivo.
Although the wild type or empty vector transfected cells were unable to induce tumors, two mice inoculated with HEK293 overexpressing RAC3 developed tumors that after 18 days of initiation acquired a size of 5,8 and 6,8 mm. The hystopathological analysis of sections from these samples, identified them as malignant tumors showing high mitosis, some necrosis, angiogenesis and invasion of the skeletal muscle ( Figure 5D).

DISCUSSION
RAC3 is an oncogene having nuclear and cytoplasmic functions usually overexpressed in several tumor types, but having very low or undetectable expression levels in normal mature differentiated cells. Moreover, a few years ago it was also demonstrated that stem cells require RAC3 expression in order to maintain the pluripotency [25][26][27][28]. Likewise, it is downregulated when stem cells are differentiated in a similar way that the other stem gene markers [33]. The fact that RAC3 is a molecule whose detection at high or moderate levels could only be found in normal stem or tumoral cells open the intriguing question regarding a possible link between them.
Although its probably association to cancer stem cells has not been previously investigated, in this work we demonstrated that RAC3 overexpression is mainly associated to a cancer stem marker enriched side population and is required to maintain some cancer stem properties of colorectal cancer cells.
The original definition of cancer stem cells comes from the functional evidences concerning the ability to regenerate the tumor. Furthermore, the identification of specific markers associated to different tumor types was used to select side populations enriched in cancer stem cells, such as CD44 and CD133 [5,34]. The assays performed with cancer stem cells enriched populations obtained by specific marker selection or limit dilution from the parental tumor allowed to deeply investigate their properties and behavior. In addition to their ability for tumor regeneration and in vitro growing as spheroids [35], these cells are the most resistant to chemotherapeutic drugs, being the high MDR expression at least one of the reasons [32,36], in agreement with our previous works demonstrating that cells overexpressing RAC3 are more resistant to apoptosis, senescence and autophagy [16,[18][19][20]. Therefore, integrating all our previous and present results it is not surprising a probably association of RAC3 overexpression with a cancer stem cells.
In this work we demonstrated that although RAC3 is a high expression molecule in human colorectal tumors, its levels could be modulated along the cancer progression together with cancer stem cell markers, being probably higher in states associated with initiation, major propagation invading secondary focus, and aggressiveness where an important amount of cancer stem cells are required, having the ability to migrate and invade new tissues in order to build the new metastatic focus. Several evidences support the oncogenic role of RAC3 [9,[13][14][15], and being RAC3 a NF-kB coactivator, and Vimentin a target of this transcription factor [24], its contribution to maintain the mesenchymal phenotype could be not surprising. However, their tumoral transforming effects when is overexpressed in non tumoral cells has not been previously investigated.
It was previously demonstrated that both RAC3 or the splicing Delta 4-SRC3 variant downregulation inhibits the oncogenic potential of cancer cells affecting several pathways related to its activity as a coactivator of nuclear receptors or the cytoplasmic signaling [23].
In general the genetic modification of a unique oncogene is not strong enough to induce a deployment of tumoral characteristics, but here we reported that RAC3 overexpression in non tumoral cells induces the cell transformation acquiring a typical tumoral phenotype with the concomitant ability to migrate, to invade, to produce metalloproteinases, to growth as spheroids even under low serum, to increase the expression of stem cell markers [3,5,33,35] and the most strong evidence of the presence of cancer stem cells, like the ability to initiate tumors in vivo showing malignant hystopathological diagnose.
Because our studies of overexpression were performed in non tumoral cell lines but not in normal primary tissues, we cannot exclude that chromosomic alterations and the immortality condition could be contributing to the RAC3 transforming effect, giving a permissive signaling context. In addition, independently of the oncogenic profile, is the association of cancer stem markers and properties with RAC3 overexpression, which was supported through our findings analyzing microarrays from tumoral tissues and cell lines. Although these RAC3 overexpressing cells induced a rapid tumor growth when were inoculated in vivo in nude mice, their ability to be differentiated toward other several tissues was not demonstrated in the present work, afterwards, their classification as "stem cells" is not completely suitable.
Concerning the origin of cancer stem cells is currently unclear. It has been suggested that cancer can be considered as a disease of unregulated self-renewal in which mutations convert normal stem cell self-renewal pathways into engines for neoplastic proliferation [37,38]. In this regard, there are evidences that support this origin, as the expression of some common stem marker genes [5,34,35,39,40]. However, there are not evidences that avoid to excluding the origin of cancer stem cells from mature differentiated cells carrying accumulated mutations or epigenetic changes.
Although the mechanism by which RAC3 is overexpressed, as a conserved signal in such amount of tumors remains unclear, it is possible to speculate this is a cancer stem cell condition, then heritable to the heterogeneous tumoral progeny. Moreover, an additional intriguing question still not solved arises from this hypothesis, concerning the origin of cancer stem cells. They can be a mutated product from normal stem cells, but the possible origin from normal differentiated mature cells acquiring mutations and epygenomic changes could not be excluded. In the first case, the increased RAC3 expression could be a heritable marker from the normal stem cell that once mutates to give a cancer stem cell, but in the second, we have to consider the possible signaling inducing this overexpression in mature differentiated cells. In this regard, inflammation is increasingly recognized as a critical component in tumor initiation and progression [41,42]. Furthermore, we have previously demonstrated that inflammatory cytokines as TNF may upregulate the RAC3 gene expression [29]. Therefore, in this context, the induction of malignant or premalignant lesions from cells acquiring this RAC3 overexpression and possible additional mutations or epygenomic rearrangements sounds as a possible source for tumor initiating cells.
Finally, as a concluding remark, our results demonstrate that non tumoral cells may acquire a cancer stem like phenotype when RAC3 is overexpressed, suggesting that this switch deserves to be investigated in vivo, being a possible early step in tumor initiation when it occurs in normal differentiated cells having or not accumulated additional mutations. Although the cancer stem cells origin is still unclear, our results demonstrate that the phenotype of cells where RAC3 is overexpressed resembles some characteristics of normal stem cells, like the expression of stem gene markers and growing behaviour. Although the requirement of RAC3 expression for normal stem cells has been demonstrated, the consequences of overexpression over them, remains as an intriguing question to be investigated in order to improve the strategies for anti-cancer treatments and also that involving stem cell therapies.

Cell culture and reagents
The non-tumoral cell line human embryonic kidney HEK293, was maintained in Dulbecco's Modified Eagle Medium (DMEM) high glucose and the tumoral cell line HCT116 (human colon cancer) was maintained in DMEM F12 (Gibco Laboratories, Grand Island, NY, USA). All cultures were supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 . The HEK293 and HCT116 cell lines were acquired from ATCC. All the cell lines are usually tested for mycoplasma contamination once a month.
HEK293 cells were transfected by the CaCl2 method and HCT116 cells using Lipofectamine 2000 (Invitrogen).
Unless stated otherwise, all reagents were obtained from Sigma Chemical co. Bs. As., Argentine or Santa Cruz Biotechnology, USA. www.impactjournals.com/oncotarget

Expression vectors and reporter plasmids
HEK293 was transfected with an expression vector for RAC3 (pCMV-Tag 2B-RAC3) or with the empty vector (EV) as control (Invitrogen) and in the case of HEK293, selected for stable expression with Neomycin. HCT116 cells were transfected with an expression vector for shRNA-RAC3 (pRV-GFP-puromycin) or the scramble control, as previously described in our laboratory and selected for stable expression with puromycin [19].
Once transfected and after selection of cells for stable expression, the cultures were amplified and preserved by frozen in liquid nitrogen. After defrost and before to each experiment the RAC3 expression levels were tested using qPCR and western blot and no more than teen passages were used in order to ensure the original heterogeneity along passages.

Western blot analysis
Western blot assays were performed as previously described [29]. Briefly, monolayers were scraped and treated with RIPA buffer containing pepstatin A, phenymethylsulfoyl fluoride and dithiothreitol, solubilized in SDS-PAGE sample buffer, separated on 8% SDS-PAGE, and electro-transferred to a nitrocellulose membrane.
Membranes were blocked for nonspecific binding with TBS 5% milk and 0.05% Tween-20 (T-TBS) and incubated overnight in T-TBS/0.5% BSA with 0.05 µg/ml of anti-RAC3, anti-Vimentin or anti-E-Cadherin, primary antibodies. Subsequently, membranes were washed and incubated for 1 h with HRP-conjugated secondary antibody, developed by chemiluminescence (Santa Cruz Biotechnology).

Clonogenic assay
Clonal cell growth was performed as previously described [43]. Briefly, single-cell suspensions containing 200-800 cells were seeded onto 6 well plates in MEM with 10% of FCS. Medium was changed every 72 h. After 7 days of culture the number of colonies was determined using an inverted microscope.

Tumorspheres
The tumorspheres assay was performed as previously reported [44]. Briefly, 5 × 10 4 HEK293 RAC3 or EV and HCT116 SC or shRAC3 cells were maintained in DMEM supplemented with B27 (Stem Cell Supplemented Invitrogen), EGF 20 ng/ml (Invitrogen) and penicillin/ streptomycin in six well plates pre-coated with 1% agarose. Cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 . The presence of tumorspheres was evaluated daily using light microscope and its number determined after 14 days.

Invasion assay
The invasion assay was performed as previously described [45]. Briefly, 5 × 10 4 HEK293 or HCT116 cells were seeded in the upper transwell chamber (24 inserts, 8 μm, Corning) coating with matrigel (Corning) in DMEM high glucose or DMEM-F12 and 0.01% BSA without FBS. DMEM supplemented with 10% FBS was used as chemoattractant in the lower chamber. After being incubated for 16 h, the invasive cells through the matrigel layer were fixed with formaldehyde, stained with gelred (Biotium) and counted. Images were captured by fluorescence microscope (Olympus BX51).

Proliferation assay
The proliferation assay was performed as previously described [20]. Briefly 1 × 10 3 cells were seeded onto 96 well plates. After 24 h, the medium was substituted by fresh medium in two different conditions: normal (DEMEM + 10% SFB) or low (DEMEM + 0.1% SFB) serum condition. The cell number was determined at 24, 48 and 72 h by spectrophotometry at 570 nm after staining with crystal violet.

Hoechst efflux
Hoeschst efflux assay was performed as previously described [32], with some modifications. Briefly, cells were stained with 5 µg/ml Hoechst 33342 in presence or absence of 50 µM Verapamil (Sigma), for 90 minutes at 37°C. Then, the cells were washed and suspended in PBS. The Hoechst efflux in the presence or the absence of Verapamil was determined by spectrophotometry at 350 nm and cell number determined by counting under fluorescence microscopy.

MMP activity assay
MMP2 enzymatic activity was determined by zymography, as previously described by [43]. Briefly, semiconfluent cell monolayers growing in 35-mm plates were extensively washed with PBS. Serum-free medium (1 ml) was added and incubation was continued for 24 h. Conditioned media containing MMP activity were harvested and frozen, while the remaining monolayers were lysed with 1% Triton X-100 in PBS, and cell protein content was determined (Bio-Rad Protein Assay). Samples were run on 9% SDS polyacrylamide slab gels containing 1 mg/ml of gelatin, under non-reducing conditions. After electrophoresis, gels were washed for 30 min using in 2.5% Triton X-100 and subsequently incubated for 48 h at 37°C in a buffer containing 0.25 M Tris-HCl pH 7.4, 1 M NaCl, and 25 mM CaCl 2 . Non-specific activity was detected using gels incubated in the same buffer solution but supplemented with 40 mM EDTA. After incubation, gels were fixed and stained with 2% Coomassie Brilliant Blue G-250 in methanol/acetic acid/H2O (30:10:60). The white bands corresponding to MMP2 activity were determined and analyzed using Image J program after Coomassie Blue (Sigma) staining, where the densitometry values for each band was relativized to the protein contents of conditioned medium as determined by Bradford assay.

CD133-positive or depleted side population preparation
Side-populations were prepared following the instructions of the magnetic separation kit from Milteni company. Briefly, 2 × 10 8 HCT116 cells in buffer (PBS + 0,5% BSA+2 mM EDTA) were mixed with 2 mg of anti CD133 antibody (AC133, Milteni) for 15 minutes, then, cells were centrifuged and resuspended in the same buffer and 15 ml of magnetic A/G beads were added and slowly mixed in orbital shaker for 30 minutes. The complexes were washed two times and centrifuged at 300 × g for 10 minutes and resuspended in buffer. CD133+ and CD133-depleted side-populations were prepared by using of µMACS columns. The fraction obtained after two washes of cells bound to magnetic beads that were retained in the column; correspond to CD133+ side-population, while the CD133− sidepopulation was collected from the excluded fraction after a second passage into the columns.

Analysis of microarrays
The RAC3 expression was analyzed in pools of GEO (Gene Expression Omnibus) public repository microarrays data bank: I) Platform GPL570 (Affymetrix Santa Clara, CA, USA), accession number GDS4718, Analysis of homogenized colorectal cancer (CRC) tumors representing various stages and metastases. II) Platform GPL96 (Affymetrix Santa Clara, CA, USA), accession number GSE24747 for CaCo (colon cancer cells). The side population CD133+ o CD133− were isolated by flow cytometry.

In vivo experiments
Animal experiments were performed in accordance with Guide for Care and Use of Laboratory Animals (National Institute of Health, US), Britannic Committee research animals (United Kigdom Coordinating Committee on Cancer Research, 1998; Woekman et al., 2010) and approved buy our Animal Ethics Committee.
Male Nude (nu/nu) mice, 7-10 weeks old, were housed in the animal facility of the School of Sciences, University of Buenos Aires with food and water ad libitum. Mice were randomly divided into two groups and inoculated subcutaneously at the right dorsal flank with 2 × 10 6 of RAC3-HEK293 or EV-HEK293 transfected cells. No changes in weight were detected. Tumor size was calculated as W 2 × L/2, where W = width and L = length. Tumor volume was normalized to tumor volume at the start day of exponential tumor growth [46].
Histopathology was performed in sections from two tumors by staining with hematoxylin and eosin after 20 days of inoculation. Furthermore, under our experimental conditions, the inoculated cells did not produce any toxic effect as animals gained weight as control animals, and the autopsy of animals did not reveal any signs of toxicity as confirmed by the pathologist. www.impactjournals.com/oncotarget

Statistics analysis
Each experiment was performed in triplicate and repeated at least three times. The differences in mean values among groups were evaluated and expressed as the mean ± SD. T-test were used for data analysis.

Author contributions
Laura C. Panelo, she performed almost all the experiments that are included in this paper, Mileni Soares Machado, she performed all the experiments in order to determine the levels of stem gene markers that are shown in Figure 4 (Immunofluorescence and PCR assays), she also cooperated with the experiments of fluorochrome exclusion and growth of tumorspheres.
María F. Rubio, she generated the HCT116 cell line stably expressing shRAC3, cooperated with the in vivo experiments, qPCR assays and also performed the clonogenic assays.
Felipe Jaworski, he performed the in vivo assays. Cecilia V. Alvarado, she cooperated with the analysis of microarrays.
Leonardo A. Paz, as a pathologist, he performed histophatology analysis of tumor sections.
Alejandro J. Urtreger, he supervised the invasion assays and cooperated with paper writing and figures making.
Elba Vazquez, she supervised the in vivo assays cooperated with the interpretation of all the results and suggested experiments in order to solve problems arising from the in vivo assay and also cooperated with the paper writing.
Mónica A. Costas, the director of this project, mentor of the work and who wrote the paper. All authors read and approved the final manuscript.


s2fieldsofstudy:
Biology
Environmental Science

text:
Molecular Evidences of a Hidden Complex Scenario in Leporinus cf. friderici

The megadiversity of the neotropical ichthyofauna has been associated to recent diversification processes, reflecting in subtle or lacking morphological differentiation between species, challenging the classical taxonomic identification. Leporinus friderici occurs in several river basins of South America, and its nominal taxonomic validity has been questioned. Its wide distribution within the Brazilian Shield suggests that this species could be genetically structured among the hydrographic basins, despite a sharp morphological similarity. In this study, we performed phylogenetic analyses, based on three nuclear (recombination activating gene 1, RAG1, recombination activating gene 2, RAG2, and myosin heavy chain 6 cardiac muscle alpha gene, Myh6) and two mitochondrial (COI and Cytochrome b, Cytb) markers, in specimens morphologically similar to L. friderici and related species from different hydrographic basins in South America. Our phylogenetic tree identified four well-supported clades, which point out to the existence of taxonomic inconsistencies within this fish group. A clade named L. cf. friderici sensu stricto included eight Molecular Operational Taxonomic Units recently diversified in the Brazilian Shield basins. These results were also confirmed by a single-gene species delimitation analysis. It is suggested that this clade includes a species complex, characterizing taxonomic uncertainties. Another clade recovered only L. friderici from the Suriname rivers, validating this nominal species in its type locality. A third no-named clade, characterized by deeper species divergence, recovered five different nominal species interleaved with other undescribed forms previously also recognized as L. cf. friderici, indicating taxonomic errors. The fourth clade only included L. taeniatus. Our results showed a complex scenario involving the morphotype L. cf. friderici and allowed us to address aspects related to evolutionary diversification of this fish group and historical processes involved with, highlighting the importance of revealing hidden biodiversity for the taxonomy and conservationist action plans of these fish.

INTRODUCTION
South America freshwater fish represent one-third of the world continental ichthyofauna (Reis et al., 2016). However, this huge biodiversity is relatively recent, mostly due to extensive speciation events during the last 10 Ma (Hubert et al., 2007;Albert and Reis, 2011). Several taxa diverged <1 Ma during the late Pleistocene because of the Quaternary activity that led to topographic changes such as recent drainage rearrangements (Ribeiro, 2006). These events probably reflected in subtle or lacking morphological differentiation between the emerged species, challenging the classical taxonomic identification.
Molecular analyses have been largely used to aid species identification and delimitation within neotropical fish (e.g., Pereira et al., 2011Pereira et al., , 2013Ramirez and Galetti, 2015;Machado et al., 2016), contributing in revealing hidden biodiversity (Pires et al., 2017;Ramirez et al., 2017a). Both DNA barcoding and phylogeny studies have provided important contributions for better understand the phylogenetic relationships within taxa, and historical and evolutionary processes involved in the species diversification (e.g., Hebert et al., 2003;Carvalho-Costa et al., 2011;Ramirez et al., 2017b). In this way, mitochondrial and nuclear DNA data can be used to define discrete genetic lineages, detecting reciprocal monophyly, and characterizing a Molecular Operational Taxonomic Unit (MOTU, cluster of orthologous sequences generated by an explicit algorithm), representing a monophyletic lineage that could or not correspond to a taxa (Blaxter et al., 2005;Jones et al., 2011).
Leporinus, from the Anostomidae family, is considered one of the richest genus within Characiformes, a predominant freshwater fish order in South America (Garavello and Britski, 2003). Recent morphological and molecular studies have shown Leporinus as a non-monophyletic genus (Sidlauskas and Vari, 2008;Ramirez et al., 2016), highlighting the need of a deep taxonomic revision on this group. Freshly, an integrated morphological, chromosomal, and molecular approach described the new genus Megaleporinus, gathering the largest body sized Leporinus species in a monophyletic clade (Ramirez et al., 2017b).
Among the remaining Leporinus species, Leporinus friderici Block (1794) has a wide geographic distribution occurring in most rivers of South America (Garavello et al., 1992). This large distribution has been investigated by several authors, who have found morphological (Géry et al., 1987;Renno et al., 1990Renno et al., , 1991Garavello et al., 1992;Sidlauskas and Vari, 2012) and genetic (Renno et al., 1990(Renno et al., , 1991 variations among populations, suggesting that this L. friderici morphotype may contain a species complex (Sidlauskas and Vari, 2012). While L. friderici from Suriname and French Guiana rivers has been recognized as the type species (Sidlauskas and Vari, 2012), the provisional nomenclature Leporinus cf. friderici has been used to refer to the remaining individuals of this morphotype. However, because of its wide distribution within the Brazilian Shield, one could expect some levels of genetic differentiation within the L. cf. friderici among hydrographic basins. Morphological variations in the eyes, body length, and color patterns have been already reported between L. cf. friderici from the Amazon and Paraná-Paraguay basins (Géry et al., 1987;Garavello et al., 1992).
In this study we tested the hypothesis that L. cf. friderici consists of a monophyletic group formed by different MOTUs currently separated in distinct basins within the Brazilian Shield. We performed a phylogenetic analysis using mitochondrial and nuclear markers to confirm L. cf. friderici as a monophyletic group, and a single-gene species delimitation analysis to characterize MOTUs. Finally, we infer on the evolutionary historical processes possibly responsible for the diversification within this group.

DNA Extraction, Gene Amplification, and Sequencing
Total DNA was extracted using the conventional phenolchloroform/proteinase K protocol (Sambrook et al., 1989). For the phylogenetic analysis at least one individual per each major sampled hydrographic basin was used. Cytochrome b (Cytb) and cytochrome oxidase subunit 1 (COI) mitochondrial regions were amplified according to Ramirez and Galetti (2015). The nuclear recombination activating gene 1 (RAG1), recombination activating gene 2 (RAG2), and myosin heavy chain 6 cardiac muscle alpha gene (Myh6) were amplified following Oliveira et al. (2011). PCR products obtained for both DNA strands were sequenced on an ABI 373xl sequencer (Applied Biosystems, Little Chalfont, United Kingdom).

Data Analysis
The obtained sequences were manually edited and aligned using Bioedit (Hall, 1999) and Clustal W (Thompson et al., 1994), respectively. All sequences were checked for indels and stop codons. The haplotypes of the nuclear genes were combined into a consensus sequence by coding polymorphic sites with the IUPAC ambiguity codes (IUPAC, 1974).
A multilocus Bayesian species tree (BST) was estimated by * BEAST (Star-BEAST) (Heled and Drummond, 2010) using 150 million generations, sampled every 5000, and a burn-in of 300. A nucleotide substitution model selected was based on the Bayesian criterion, using JModeltest 2 (Darriba et al., 2012). The models chosen were HKY+I+G, HKY+G, K80+I, K80+I, and K80+I for COI, Cytb, Myh6, RAG1, and RAG2, respectively. The two mitochondrial gene tree topologies were linked and set to have an effective population size of onequarter from that of nuclear genes. A lognormal relaxed clock was used for all partitions. Bayesian trees using all sequences were established for each gene, separately. These trees were estimated with 10 million of generations, sampling every 5000, and a burn-in of 10%. Yule speciation model was used and nucleotide substitution models followed the same criteria above cited (Supplementary Figures S1-S5). The convergence (sample size >200) and stationarity of the values were checked in TRACER v1.6 (Rambaut et al., 2014).
Two different analyses, using a single-gene species delimitation approach based on COI sequences, were performed to determine the number of MOTUs within the clade widely distributed in the Brazilian Shield (L. cf. friderici sensu stricto, see below). First, the General Mixed Yule-Coalescent (GMYC) model (Pons et al., 2006) was used to determine the clustering of the COI haplotypes. This analysis was performed with a single threshold that was implemented in the SPLITS package using R 3.3.3 statistical software (R Core Team, 2017). For this analysis an ultrametric tree was generated using BEAST 2.3.2 (Bouckaert et al., 2014), with a lognormal relaxed clock, a birth and death model, and a HKY substitution model chosen by jModeltest 2 (Darriba et al., 2012). A total of 50 million MCMC generations and a burn-in of 10% were used. In second, a Bayesian analysis of genetic structure was implemented using BAPS software (Corander et al., 2008). The maximum number of genetically diverged groups (K) was firstly set up for 10 replicates, 10 times. The obtained groups containing samples from different basins were submitted to a second layer of analysis in BAPS using K = 1-3, replicated also 10 times. This hierarchical approach to DNA sequence clustering provides a useful way to increase statistical power and detect separated haplogroups that are assigned to conservative clusters (Cheng et al., 2013). The most likely K was chosen based on log (likelihood) and posterior probability values. Next, we consider the concordant groups between the two different analyses, and the groups presenting allopatry and reciprocal monophyly as the final number of MOTUs.
The genetic distances between MOTUS were calculated based on K2P model using MEGA 6.06 (Tamura et al., 2013). Finally, a haplotype network was generated using Median Joining (Bandelt et al., 1999) in the POPART software (Leigh and Bryant, 2015).

RESULTS
We successful obtained a total of 127 sequences for the specimens studied herein [63 for the COI gene (557 bp) and 16 for each remaining amplified marker -Cytb (1005 bp Table S2).
The phylogenetic trees, generated by MP, ML, and BST analyses (Figure 2), strongly supported four clades with maximum support values. A clade recovered L. friderici from the Suriname basin alone. A monophyletic clade characterized by a recent diversification within the Brazilian Shield, named herein as L. cf. friderici sensu stricto, included specimens of L. cf. friderici from Amazonas (main channel), Madeira, Upper Tapajós, Tocantins, Paraguay, and Paraná basins, L. agassizii, and specimens of L. piau from São Francisco basin. A no-named clade, showing older diversification, joined specimens of L. cf. friderici, from Mearim, Tocantins, Turiaçu, Xingu, and Madeira basins, interleaved with L. boehlkei, L. lacustris, L. cf. parae, L. piau from Jaguaribe basin, and L. venerei. Lastly, a fourth clade recovered only L. taeniatus.
The GMYC analysis, considering 23 parsimony-informative sites and no insertions or deletions within the COI sequences, identified seven MOTUs (CI: 6-7), with a significant likelihood ratio of 10.97 (P < 0.005) within L. cf. friderici sensu stricto. From these, six MOTUs corresponded to L. agassizii, L. cf. friderici Amazon 1, L. cf. friderici Madeira 1, L. cf. friderici Paraná, L. cf. friderici Paraguay, and L. cf. friderici Upper Tapajós. The seventh MOTU joined L. piau São Francisco and L. cf. friderici Tocantins 1 (Figure 3). The results of the first BAPS layer presented four as the most likely K with log (ml) = −554.0008 and 0.93 posterior probability values. In addition, the results of the second BAPS layer were similar to the GMYC MOTUs, but recovered L. piau São Francisco and L. cf. friderici Tocantins as two different MOTUs. The hierarchical analysis of BAPS could separate these populations, since information related to sample locations were given. Differently, GMYC method considers no prior information. Despite this little divergence in detecting MOTUs, both these lineages presenting recent divergence are reciprocally monophyletic and geographically isolated in nature.
The mean genetic distance COI K2P values among the MOTUs ranged from 0.4 to 2.4%. The maximum intra-MOTU distance (0.5%) was observed in L. agassizii, while the minimum inter-MOTU distance (0.4%) was between L. cf. friderici Tocantins and L. piau São Francisco (Supplementary Table S3).
A total of 27 haplotypes was obtained within L. cf. friderici sensu stricto, in which each MOTU was represented by a haplogroup, except for the MOTUs from Paraná and Paraguay that shared one haplotype. L. piau São Francisco and L. cf. friderici Upper Tapajós were separated by only one mutational step, while the other haplogroups were connected by at least two mutational steps (Figure 4).

DISCUSSION
Our phylogenetic analyses showed that specimens morphologically identified as L. friderici constitute a polyphyletic group, widely distributed along the South America (Figure 2). The individuals collected as L. friderici across the Brazilian Shield basins are not conspecific with L. friderici from the type locality, representing a different species. L. cf. friderici sensu stricto constitutes a monophyletic species complex distributed in Amazon, Madeira, Upper Tapajós, Tocantins, São Francisco, Paraná, and Paraguay river basins. This finding can represent a typical situation of recent diversification forming a strictly related group composed of potential cryptic species, revealing typical taxonomic uncertainties (Ramirez et al., 2017a). On the other hand, in the no-named clade the five nominal species L. boehlkei, L. cf. parae, L. lacustris, L. piau Jaguaribe, and L. venerei were interleaved with individuals that morphologically fit with the description of L. cf. friderici. In this clade, characterized by an older diversification, the use of the term L. cf. friderici hides undescribed cryptic species. All species from this clade share discriminative morphological general pattern with L. friderici (i.e., one-three spots on the body along the lateral-line, and dental formulae 4/4, except L. venerei that has 4/3, unique in Anostomidae), hindering the identification of these cryptic species. Moreover, three (L. lacustris, L. parae -L. cf. parae -, and L. venerei) of the five nominal species of this clade have been already considered as very similar in morphology due to their deep body, terminal mouth, anal fin long and dark, and three blotches on the lateral line (Britski and Birindelli, 2008).
Overall, the results obtained for L. cf. friderici sensu stricto confirm our hypothesis that there are different MOTUs within L. cf. friderici, currently separated in distinct river basins, but not all provisionally recognized as L. cf. friderici, that can be joined in a single monophyletic group.
Within the clade L. cf. friderici sensu stricto, L. agassizii is clearly recognized as a valid species different from L. friderici, mainly due to the presence of a longitudinal stripe, extending from dorsal fin to just before the caudal fin (Birindelli et al.,  2013). L. agassizii was firstly described for the Iça river, Upper Amazon basin (Steindachner, 1876), and posteriorly it was also found in the Tefe lake, Nanay, Negro, and Branco rivers (Birindelli et al., 2013), from the same basin. This species has been described as restricted to the Upper Amazon basin. A parsimony analysis of endemism in the South America reported the Upper Amazon as a separate clade from the Amazon drainages (Hubert and Renno, 2006), suggesting that uplift of the paleoarches has promoted allopatric divergence in the ichthyofauna of this region, which was enhanced by marine incursions.
In our study, all individuals collected in rivers above Madeira river falls were joined in the single MOTU L. cf. friderici Madeira 1, while individuals downstream the Amazon basin were recovered in the MOTU L. cf. friderici Amazon 1 (Figure 3), with the exception of individuals from Upper Tapajós (see below), indicating these falls as possible barrier that limits the fish species distribution in the region. Previous studies had already reported evidences of structuring along the Amazon basin (Goulding, 1979;de Queiroz et al., 2013). This subdivision was attributed to geomorphological agents that allowed allopatric fragmentation and diversification events (Albert and Reis, 2011). In the Madeira river, the Teotônio fall seems to play a relevant role on the ichthyofauna diversification of the Amazon basin. This fall, besides other rapids, has been keeping apart the rivers from Upper and Lower Madeira, and has been considered a geographic barrier by limiting the fish species distribution in the region (Zanata and Toledo-Piza, 2004;Hubert et al., 2007;Torrente-Vilara et al., 2011).
Barrier effects can also explain the presence of L. cf. friderici Upper Tapajós joining individuals caught in the Juruena -Teles Pires sub-basin, in the Upper Tapajós (Supplementary Tables S1, S2). Waterfalls and rapids along the Tapajós river, and in its tributaries seem acting as barriers to fish dispersal (Britski and Garavello, 2005;Britski and Lima, 2008;Dagosta and de Pinna, 2017). The region above the Juruena -Teles Pires confluence river has been characterized by an endemic ichthyofauna different from other Amazon rivers (Carvalho and Bertaco, 2006;Britski and Lima, 2008), which could account to the separation of L. cf. friderici Upper Tapajós.
In turn, the Tocantins basin is considered an independent system from the Amazon basin, since its waters flow directly into the Atlantic Ocean (Albert and Reis, 2011). This fact was reflected in our analyses, in which the Tocantins individuals corresponded to a different genetic group (L. cf. friderici Tocantins 1). The final establishment of modern course of Tocantins (1.8 Ma) separated definitively this basin from Amazon (Rossetti and Valeriano, 2007), and the differentiation of the Tocantins ichthyofauna has been often associated to the rise of Gurupá arch, the Tucurui rapids, or the limited connectivity (Hubert et al., 2007;Hrbek et al., 2014).
The relationship between L. piau São Francisco and L. cf. friderici Tocantins 1 observed here can be accounted for a biogeographic history between the Tocantins and São Francisco basins, and the low genetic divergence (0.4%) between them likely represents a recent diversification. These two hydrographic basins share an extensive watershed, where the Sapão river (São Francisco basin) shares headwaters with the Galheiros river, Tocantins basin (Lima and Caires, 2011). The existence of these common headwaters can allow a fauna exchange between these basins. Geological evidence shows that the western border of Serra Geral from the Goiás plateau has been gradually eroded and could have potentially promoted headwater capture events between the São Francisco and Tocantins rivers (Lima and Caires, 2011). Geodispersal events (i.e., headwater capture) from Amazon river to eastern basins of the Brazilian Shield (as São Francisco river) have been already claimed in studies using molecular approaches (Hubert et al., 2007;Ramirez et al., 2017b).
While the L. piau specimens from São Francisco was linked to the L. cf. friderici sensu strito clade, L. piau from Jaguaribe was grouped in the no-named clade (Figure 2), revealing a clear taxonomic inconsistency. Fowler (1941) claimed the Salgado river (Jaguaribe basin) from Ceará state as type locality of L. piau, and included one paratype from Jatobá river (São Francisco basin). Consequently, specimens from São Francisco river have been usually cited as L. piau (Garavello and Britski, 2003;. Our results pointed that specimens from São Francisco basin indeed constitute a different species from the L. piau from the Jaguaribe river, the type basin. Still within L. cf. friderici sensu stricto, a well-supported differentiation between specimens from the Upper Paraná and Paraguay basins was also observed, although some individuals from the Upper Paraná showed haplotypes from the Paraguay basin (Figures 3, 4). It is possible that both L. cf. friderici Paraná and L. cf. friderici Paraguay reached their current distribution through ancient geodispersal events, as headwater captures between Amazon rivers and the Paraná and Paraguay basins. In the modern river basin landscape, the Paraguay basin has a watershed with the Guaporé, Tapajós, and Xingu rivers, while the Upper Paraná shared a watershed at the headwaters of the Tocantins basin (Albert and Reis, 2011). These hydrographic systems have experienced a long history of major capture events and formation of semipermeable barriers (Lundberg et al., 1998) that can support this hypothesis. The Upper Paraná ichthyofauna was separated from the Lower Paraná by the Sete Quedas Falls, a natural geographic barrier which no longer occurs. In the past this barrier isolated the Upper Paraná, where the ichthyofauna has been diverging, as already reported in Megaleporinus (Ramirez et al., 2017b) and Salminus (Machado et al., 2016). The shared haplotypes between L. cf. friderici Paraná and L. cf. friderici Paraguay are probably resulting of the removal of the natural barrier when the Itaipu hydroelectric was built. The resulted dam flooded an extensive area, including the no longer existent Sete Quedas Falls, allowing the connection between both ichthyofauna from Lower and Upper Paraná facilitating contact between mitochondrial lineages since the formation of Sete Quedas Falls (Júlio et al., 2009;Prioli et al., 2012).

CONCLUSION
Our study showed that L. cf. friderici as provisionally used hides at least two major situations. First, L. cf. friderici sensu stricto, a monophyletic clade joining eight MOTUs, potentially includes a true species complex, characterized by recent diversification across the Brazilian Shield basins. According to our initial expectations, L. cf. friderici sensu stricto is genetically structured along the Brazilian shield basins, and this structure appears to be related to geomorphological agents, determining the current hydrographic structure. Its taxonomic significance is an open question, requesting complementary studies for resolving this typical situation of taxonomic uncertainties. Second, a no-named clade, characterized by relatively older diversification, in our opinion, hides undescribed cryptic species under L. cf. friderici denomination, likely due to the morphology similarities that characterize the clades here studied (except L. taeniatus clade). However, this new MOTUs show deep phylogenetic divergence and they are interleaved with other nominal valid species (L. venerei, L. boehlkei, L. lacustris, L. piau, and L. cf. parae), supporting them as potential new species.
Overall, our results have important significance for the taxonomy and evolutionary knowledge of this fish group as well as for its conservation. Moreover, this scenario indicates that L. cf. friderici sensu stricto can constitute an excellent phylogeographic model in studying evolutionary and speciation processes acting in the South America basins. Despite its migratory behavior, L. cf. friderici cannot be considered as a single genetic stock even within the same basin (i.e., Amazon basin) and needs to be well known for having its whole diversity considered in any conservation effort. For a more complete understanding, the taxonomic status of each MOTU that was revealed herein needs to be evaluated using preferentially morphological and molecular data in an integrative approach.

AUTHOR CONTRIBUTIONS
RS-S and JR collected the data, reviewed the literature, and achieved the bioinformatic analyses. All authors contributed to design the research, article writing and discussion, and approved the final version of the manuscript.