BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

Distal outgrowth/expansion and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT AV valvuloseptal morphogenesis. At the onset of EMT, endocardial endothelial cells transform into cushion mesenchymal cells. Transformed cushion mesenchymal cells (CMCs) invade into the acellular cardiac jelly. After EMT, during distal elongation of the AV cushions CMCs continue to migrate and spread within the expanding mesenchymalized cushions. Distal outgrowth and expansion, which begin at HH stage-25/26 in the chick, largely involve i) CMC migratory behavior and ii) expression of valvulogenic molecules by CMCs, which include extracellular matrix (ECM) components [15,16]. We and others have proposed regulatory roles for BMP in CMC differentiation after EMT [14,17]; however, the role of BMPs in regulating the valvulogenic processes, i. e. CMC migration and expression of ECM components, has not been fully elucidated in post-EMT AV cushion distal outgrowth and expansion.
Two of the prominent ECM components in AV cushions, hyaluronan (HA) and versican interact and make macromolecular complexes in developing cartilage [18,19]. Regarding cardiac development, mouse models with deficiencies in versican (hdf mouse) [20,21] and in HA synthase 2 (Has2 KO mouse) [22] are characterized by severe cardiac defects resulting from abnormal formation of the cardiac cushion mesenchyme at the EMT stage, indicating that these two ECM components are essential for valvuloseptal morphogenesis. However, because of the early lethality of mice with Has2 and versican deficiencies at the cushion-formation stage, studying the role of these ECM molecules during later valvuloseptal morphogenesis is hampered.
Valvuloseptal morphogenesis is thought to be regulated by a coordination of growth factor signaling and ECM interactions [23,24]; however, despite the relevance, little is known about the functional relationship of BMP and ECM and their roles in CMC migration during distal outgrowth and maturation of AV cushions. In our previous work, we demonstrated that BMP-2 and the BMP signaling pathway induced AV CMC migration [14]. In the present work, we explored the expression patterns of two major ECM components, versican and HA, during AV cushion expansion and distal outgrowth and investigated the role of BMP-2 in versican and HA production. Using a welldefined 3D CMC aggregate culture on hydrated collagen gels, we provide evidence that BMP-2 induces production of HA and versican and that these ECM components contribute to BMP-2stimulated CMC migration during post-EMT AV cushion expansion and distal outgrowth.

Versican and HA are intensely expressed in AV cushion mesenchyme during AV cushion expansion and maturation
HA, versican and aggrecan were localized in HH stage-17, -24, and -29 chick embryo hearts. HA localization was histochemically detected with hyaluronan binding protein (HABP) following the protocol described previously [25]. Production and specificity of the anti-chick versican and antichick aggrecan antibodies were described previously [26]. At the early stage of cardiac cushion expansion (HH stage-17), intense immunostaining of versican was observed in the basement membrane of the endocardial side of myocardium, most prominently at the distal end of the cardiac inlet and outlet (arrows in Figure 1A). HA and versican were localized in the AV cardiac jelly (double arrows in Figure 1A), whereas aggrecan expression was restricted to cartilage in the developing vertebra (an arrow in Figure 1B) but not in the myocardium. HA staining images we present in this paper do not differentiate intracellular from extracellular staining. At the middle stage of AV cushion expansion (HH stage-24), HA, versican and aggrecan were localized in the AV and OT endocardial cushions (ED in Figure 1C, 1D). In addition, intense versican expression was detected in the myocardial basement membrane most prominently in the subendocardial space of the atrial and ventricular myocardium (arrows in Figure 1C) and myocardial-cushion interface in the OT and AV cushions (arrows in Figure 1C), whereas aggrecan expression was confined to the mesenchymalized AV and OT cushions (ED in Figure 1D). We observed robust deposition of HA and versican in the AV cushion up to the cushion maturation stage at H-H stage-29 ( Figure 1E, 1F). Versican expression was also prominent in the myocardial basement membrane (endocardial-myocardial interface) in the atrial myocardium and ventricular trabeculae (arrows in Figure 1E), whereas aggrecan expression was not detectible in the atrial and ventricular walls (Figure 1 F). Robust HA staining persisted in the AV cushions from the early cushion-forming stage (HH stage-17) to the cushion maturation stage (HH stage-29). Consistent with a previous report indicating declining mRNA expression of aggrecan in the heart after HH stage-20 [26], we found that aggrecan protein was most intensely expressed at HH stage-24 but was less abundantly expressed at HH stage-29 in the heart.

BMP-2 Treatment Increases Expression of Versican Protein and mRNA by AV Endocardial Cushion Mesenchymal Cells (CMCs)
Versican expression was assessed (both mRNA and protein) in AV CMC aggregates cultured on hydrated collagen gels. Versican protein expression was detected by immunostaining (Figure 2A-2F) and quantitatively evaluated by immunointensity analysis ( Figure 2G) following the procedure described previously [14]. The expression of versican was significantly increased when AV CMC aggregates were cultured with concentrations as low as 20 ng/ml BMP-2 ( Figure 2A-2G). Incubation with noggin, an antagonist of BMP, abolished BMP-2-induced versican expression in cultured AV CMC aggregates ( Figure 2E, 2G). Incubation with noggin alone also significantly repressed endogenous versican expression in CMC aggregates when compared to the untreated control, i.e. incubation with Medium 199 alone (Figure 2A, 2F, 2G).
We also assessed versican mRNA expression in AV CMC aggregates cultured on collagen gels ( Figure 2H). Quantitative (Q) RT-PCR data revealed an approximately 7-fold increase of versican mRNA expression by AV CMC aggregates treated with BMP-2 over the untreated control ( Figure 2H). As low as 20 ng/ml of exogenously added BMP-2 induced versican mRNA expression ( Figure 2H). Incubation with noggin in the presence of exogenously added BMP-2 significantly inhibited BMP-2-stimulated expression of versican mRNA in AV CMC aggregates in culture. Incubation with noggin alone also significantly repressed endogenous versican mRNA expression in AV CMC aggregates cultured on the collagen gels ( Figure  2H).

BMP-2 treatment induces HA deposition and Has2 mRNA expression by AV CMCs
stain intensity analysis ( Figure 3G). Significant promotion of HA deposition was detected when AV CMC aggregates were treated with as low as 20 ng/ml BMP-2 ( Figure 3A-3G). Noggin addition to the medium inhibited the potential of BMP-2 to stimulate HA deposition ( Figure 3E, 3G). Incubation with noggin alone also significantly repressed endogenous HA HA deposition was detected using HA binding protein (HABP). A, C and E show HA (green) and versican (red) staining. MF20 immunostaining is shown in blue. B, D and F show HA (green) and aggrecan (red). HA is abundantly localized in outflow tract (OT) and atrioventricular (AV) cushion mesenchyme throughout early endocardial cushion forming stage (HH stage-17) to endocardial cushion maturation stage (HH stage-29). Versican expression is evident in myocardial basement membrane in the distal end of outflow (OT) and inflow tracts (arrows in A) as well as in the cushion mesenchyme (double arrows in A) at HH stage-17. Intense versican expression is seen at the myocardial-cushion mesenchyme interface (arrows in C), subendocardial space in the ventricular trabeculae (arrows in C, E) and in the OT and AV cushions (ED in C) at HH stage-24. Aggrecan expression is restricted to forming vertebra at HH stage-17 (an arrow in B). Aggrecan expression is confined to AV and OT cushions (ED in D) but not seen in the atrial and ventricular myocardium. Aggrecan expression is most prominently seen at HH stage-24 and less abundantly expressed at HH stage-29 (F). * blood in the cardiac lumen. Scale bars=50 µm. The Has's are enzymes localized in the cell membrane that are responsible for HA production. Has2 is the only known Has expressed in the embryonic heart [23]. Therefore, Has2 mRNA expression was used as an index of HA production by CMCs in this study. Has2 mRNA expression was assessed with QRT-PCR in AV CMC aggregates cultured on collagen gels ( Figure  3H). QRT-PCR data revealed an approximately 5-fold increase of Has2 expression by CMC aggregates treated with 50-200 ng/ml BMP-2 over the untreated controls ( Figure 3H). As low as 20 ng/ml of BMP-2 induced Has2 expression ( Figure 3H). BMP-2-stimulated Has2 mRNA expression was abolished when noggin was added to the culture. Incubation with noggin

BMP-2-promoted AV cushion mesenchymal cell migration is impaired by versican siRNA treatment
Our previous study indicates that BMP-2 induces AV CMC migration in a dose dependent manner. As low as 50 ng/ml BMP-2 promoted CMC migration into collagen gels [14]. To examine whether versican expression plays an important role in BMP-2-induced AV CMC migration, we treated CMCs with versican siRNA. Target sequences of the versican siRNA are located in the chick versican G1 domain, thus targeting all chick versican isoforms. First, the efficacy of versican siRNA treatment for versican gene silencing was verified by versican immunointensity analyses and QPCR analysis for versican mRNA expression. Versican siRNA significantly reduced versican expression, whereas control scrambled RNA did not alter versican expression by AV CMCs ( Figure S1). Secondly, with versican siRNA we examined the significance of versican expression in BMP-2-promoted AV CMC migration. AV CMCs cultured with BMP-2 migrated deeper into the collagen gels ( Figure 4b, 4f) than the control untreated CMCs (Figure 4a, 4e). Quantitative measurements, as shown in Figure 5B, confirmed that BMP-2-treated CMCs indeed migrated deeper (360 µm from the surface) into the gels than the control untreated CMCs (280 µm from the surface). Effects of BMP-2 were reduced when CMCs were treated with versican siRNA (100 nM) and cultured with the BMP-2-containing medium (Figure 4c  CMCs treated with BMP-2 (200 ng/ml) migrated deeper into the collagen gels (b and arrows in f) than the control cultures (M199, a and arrows in e). Versican siRNA treatment (100 nM) significantly reduced cell migration (j, l), whereas, treatments with control RNA (scrambled RNA, 100 nM) did not alter CMC migration (i and arrows in k). BMP-2-stimulated cell migration was significantly reduced when versican siRNA was added to the medium (c and g), whereas control RNA did not alter BMP-2-stimulated cell migration (d and arrows in h). doi: 10.1371/journal.pone.0077593.g004

BMP-2-promoted AV cushion mesenchymal cell migration is impaired by HA oligomer treatment
To examine whether blockade of HA signaling alters BMP-2induced AV CMC migration, HA oligomers were added to the AV CMC aggregates cultured on the collagen gels. Many studies have shown that the HA oligomers used in this study (A) CMC aggregate cultures were treated with versican siRNA (100 nM) or control (scrambled) RNA (100 nM). Versican siRNA treatment significantly reduced cell migration, whereas control (scrambled) RNA treatment did not significantly alter cell migration. *p<0.01; **p<0.001 as compared to the untreated control (M199). (B) BMP-2 (200 ng/ ml)-stimulated CMC migration. BMP-2-stimulated cell migration was significantly reduced by adding versican siRNA (100 nM) to the CMC aggregates cultures, whereas control (scrambled) RNA (100 nM) did not reduce BMP-2-stimulated mesenchymal cell migration. The asterisks on the green curve indicate significant differences between BMP-2 treatment and the untreated control (M199) (blue curve). The asterisks on the purple curve indicate significant differences between treatment with BMP with BMP-2+ versican siRNA and treatment with BMP-2 (green curve). Vertical bars indicate ± SD of the mean. *p<0.01; **p<0.001. A and B represent quantitation of results for each culture condition from a minimum of three experiments, in each of which three separate aggregates were examined. doi: 10.1371/journal.pone.0077593.g005 effectively reduced HA signaling [27] and consequently cell invasion [28,29]. In the present study, we observed that BMP-2-promoted cell migration (Figure 6a, 6b, 6e, 6f) was largely reduced when CMCs were treated with HA oligomers (100 µg/ml) in the presence of BMP-2 (Figure 6c, 6g). HA oligomers alone also repressed migration of CMCs (Figure 6j, 6l) when compared to control chitin oligomer-treated CMCs (Figure 6i, 6k). Chitin oligomers did not alter the effect of BMP-2 stimulation upon cell migration (Figure 6d, 6h). Quantitative analysis confirmed that HA oligomer treatment significantly reduced endogenous and BMP-2-promoted AV cushion cell migration into the collagen gels ( Figure 7A, 7B).
Consistent with our previous finding [14], BMP-2 did not induce AV CMC proliferation. Also, treatments with HA oligomers, chitin oligomers, versican siRNA or control (scrambled) RNA did not significantly alter CMC proliferation ( Figure S2, S3). Therefore, it is unlikely that the cell migration data we present in this paper reflect altered cell proliferation of CMCs. Moreover, only a few cells (less than 0.1%) underwent apoptosis in the CMC aggregate cultures (Table S1, S2). Therefore, it is reasonable to conclude that the reduced cell numbers found in the collagen gels after treatment with versican siRNA or HA oligomers were due to fewer cells migrating into the gels from the CMC aggregates cultured on the gel surface.

Discussion
Studies with Nkx2.5-cre/BMP-2 cKO mice showed that myocardially-derived BMP-2 was essential for AV cushion formation [7,8]. Moreover, data from Tie1-cre/Alk3 and Flk1cre/Alk3 cKO mice indicated a critical role for the Type I BMP receptor, Alk3 in endothelial cells with respect to AV cushion formation [9,10]. Over all, these data revealed a crucial paracrine signaling role for myocardially-derived BMP-2 in AV cushion formation. However, subsequent lethality of the cKO embryos at the initial cushion formation stage has hampered analyzing the role of BMP signaling in later stages of valvulogenesis. Although myocardial expression of BMP-2 is down regulated in the AV myocardium in the later stages, BMP-2 is expressed in AV cushion mesenchyme in mice [5] and chicks [12]. Also, Type I BMP receptors, Alk3, Alk2 and Alk6 are all expressed in the AV cushion mesenchyme [14], suggesting potential autocrine regulation by BMP-2 in CMCs during post-EMT expansion and maturation of AV cushions. However, the roles of BMP signaling have not been fully understood in CMCs during AV cushion expansion and maturation. Our present work has revealed critical roles for BMP-2 in regulating cell biological processes necessary for distal outgrowth/expansion of AV cushions, valvulogenic ECM production and mesenchymal cell migration by using a welldefined 3D AV CMC aggregate culture system.
It is critical to use a well-defined 3D culture system that recapitulates the in vivo microenvironment through which AV cushion cells migrate to access their cellular biological function. An advantage of the 3D AV CMC aggregate culture that we developed is that, because CMCs are dissociated during preparation, only minimal amounts of ECM protein remain upon initiation of the cultures; hence, assessment of BMP-dependent changes in ECM protein expression can be monitored unambiguously. Another advantage is that the original cell number is easily quantified for subsequent monitoring in response to BMP signaling. Thus our culture system satisfies the criteria necessary to assess the role of BMP signaling in AV cushion expansion and maturation. Using this culture system we successfully performed immuno-intensity analysis of ECM component expression, QRT-PCR of ECM components and cell migration assays and found that BMP-2 played significant roles in CMC migration and the production of valvulogenic ECM components by CMCs.
Versican, one of the major ECM components in AV cushions, is a large chondroitin sulfate proteoglycan (CSPG) consisting of a core protein with 12-15 chondroitin sulfate (CS) side chains covalently attached. Versican belongs to a family of extracellular proteoglycans (hyalectins) that bind to HA of which aggrecan, the major proteoglycan of cartilage, is the prototype [30]. The linear glycosaminoglycan, HA, a major constituent of cardiac jelly [31], is synthesized at the plasma membrane by HA synthase (HAS) enzymes HAS1, -2, and -3.
Has2 is the only known HA synthase in the embryonic heart and is essential for AV cushion morphogenesis [22]. Has2 and versican mRNAs were shown to be intensely expressed in the myocardium of the AV canal at the onset of AV cushion BMP-2-stimulated CMC migration was significantly reduced when HA oligomers (100 µg/ml) were added to the medium (c and g). HA oligomer treatments alone reduced mesenchymal cell migration relative to the control cultures (j and l), whereas chitin oligomers (control oligomers) (100 µg/ml) did not alter CMC migration (i, arrows in k). Chitin oligomers did not reduce BMP-2-stimulated CMC migration (d, arrows in h). doi: 10.1371/journal.pone.0077593.g006 BMP2 and ECM in Post-EMT AV Cushion Cell Migration PLOS ONE | www.plosone.org formation at ED 9.5 and in AV cushion mesenchyme following EMT at ED10.5 in mice [22]. Versican protein and HA were also localized in mouse AV and OT cushions at ED 9.5 to ED 12.5 [32,33]. In chick, Has2 mRNA is expressed in the AV canal and OT at the onset of AV cushion formation and (100 µg/ml). HA oligomer treatment significantly reduced cell migration, whereas control chitin oligomer treatment did not significantly alter cell migration. *p<0.01; **p<0.001 as compared to the untreated control (M199). (B) BMP-2 (200 ng/ml) promoted cell migration. BMP-2-promoted cell migration was significantly reduced by adding HA oligomers (100 µg/ml) to the CMC aggregate cultures, whereas chitin oligomer treatment did not significantly reduce BMP-2-promoted cell migration. The asterisks on the green curve show significant differences between BMP-2 treatment and the untreated control (M199) (blue curve). The asterisks on the purple curve indicate significant differences between treatment with BMP-2+ HA oligomers and treatment with BMP-2 (green curve). Vertical bars indicate ± SD of the mean. *p<0.01; **p<0.001. A and B represent quantitation of results for each culture condition from a minimum of three experiments, in each of which three separate aggregates were examined. doi: 10.1371/journal.pone.0077593.g007 restricted to cushion mesenchyme at HH stage-20 [24]. Here we present for the first time that HA and versican protein are intensely expressed in the AV cushion mesenchyme up to the cushion maturation stage (HH stage-29) in the chick. Consistent with semi-quantitative RT-PCR data [26], we found that aggrecan protein expression was less intense in the AV cushion mesenchyme at HH stage-29 than at stage-24. Others found that BMP-2 induced aggrecan expression from AV cushion mesenchymal cells in culture [17], suggesting that BMP-2 can regulate cartilage-associated genes and phenotypes in AV cushions. Our localization data provide the basis for studies to test the hypothesis that BMP signaling in cushion mesenchyme regulates HA and versican during post-EMT AV cushion expansion and maturation.
A few studies have focused on the regulation of versican and HA/Has2 expression in cardiac development. TGFbeta2 was found to promote Has2 expression and HA production during epicardial cell differentiation and invasion [34]. Aberrant accumulation of versican protein was also reported in developing pulmonary valves in ADAMTS5 deficient mice in later stage valvulogenesis [35]. Regarding AV cushion formation, myocardial misexpression of Tbx2 induced Has2 expression in the myocardium and accelerated abnormal endocardial cushion formation from the ventricular and atrial myocardium with excessive deposition of HA [36]. Intriguingly, BMP2 was shown to selectively induce cTbx2 in noncardiogenic mesoderm in chick embryos [37], suggesting the presence of a BMP--Tbx2--Has2 regulatory axis in cardiac development. Moreover, recent studies revealed that myocardial specific conditional deletion of Tbx2 and Tbx3 led to deficiencies in AV cushion formation in mice [38]. Taken together, these findings suggest that Tbx2 in the AV myocardium regulates Has2 expression and HA deposition into the cardiac jelly prior to the onset of endocardial EMT and early formation of cushion mesenchyme. However, Has2 expression was shown to be independent of BMP-2 expression in the precardiac mesoderm [39], suggesting that regulation of HA/ Has2 expression is largely dependent on cell type and developmental context. In this regard, our present work is the first to indicate that BMP-2 induces mRNA expression of Has2 and versican and production of HA and versican protein by CMCs during AV cushion expansion and maturation. It is known that mRNA of versican isoforms, V0, V1 and V2 are expressed in the mouse embryo hearts from ED 10.5-ED 16.5 during development [32]. Because the QRT-PCR primers we used for the present study are not isoform specific but designed to detect all isoforms of chick versican it is of future interest to examine whether BMP-2 induces chick versican mRNA in an isoform-specific manner in post-EMT AV CMCs. In addition, because our mouse developmental data indicate that versican protein expression and HA deposition remain intense in all four cardiac valves at the newborn stage [40], subsequent investigations are needed to determine whether HA and versican play significant roles in later valve leaflet formation and stratification of mature valves.
Cell migration through ECM after formation of AV cushion mesenchyme is an essential and critical biological process during AV cushion expansion and maturation. HA and versican are known to regulate migration and invasion of many cell types including tumor cells [41,42], hypaxial muscle cells and neural crest cells [43][44][45]. The role of a primary HA receptor, CD 44 in cell migration and motility has also been extensively studied in tumor cells [46][47][48] and other cell types [47,49]. HA and versican are prominent ECM components of AV cushions; however, the role of HA and versican in AV CMC migration has received little attention. A mouse model with versican deficiencies (hdf mouse) dies at ED 10.5 and exhibits complete failure in endocardial cushion formation; however, excised AV regions of the hdf mouse have the ability to produce mesenchyme in a 3D collagen gel environment [20]. Intriguingly, Vcan ∆3/∆3 mice whose versican lacks a subdomain of the G1 domain exhibit impaired CMC migration in ED 12.5 AV explant cultures [50]. These data suggest that versican may not be directly involved in CMC formation through an EMT but may regulate CMC migration after EMT in AV cushions. Using a well-defined 3D cell aggregate culture, we demonstrate in the present study that HA production and versican expression contribute to BMP-2-induced CMC migration. Our data also suggest that endogenous HA and versican may play significant roles in CMC migration because cell migration was reduced when AV CMC aggregates were cultured with versican siRNA or HA oligomers in untreated control medium. However, it is still not clear how BMP and/or HA/versican signaling regulate changes in cytoskeletal organization associated with AV endocardial cushion cell migration. HA binding to astrocytes is reported to stimulate Rac1-dependent PKNgammakinase activity, which, in turn, activates the cytoskeletal protein, cortactin and regulates cell migration [51]. Myosin-X was shown to be responsible for BMP-6-dependent filopodial extension and migration of endothelial cells [52]. More recently, actin cytoskeleton reorganization via the p38/MK2/Hsp25 pathway was shown to be critical for BMP-2-induced migration of C2C12 myoblasts [53]. It is of future interest to determine how BMP-2 and /or BMP2-induced HA/versican regulates cytoskeletal organization and filopodial extension of CMCs during post-EMT AV cushion expansion and maturation.
In conclusion, our current work provides evidence that BMP-2 induces expression and production of versican and HA by AV CMCs and that versican and HA contribute to BMP2induced CMC migration by using a well-defined 3D CMC aggregate culture. BMP-2 also induces other valvulogenic molecules including an ECM protein, periostin [14] which is reported to regulate cellular migration, collagen fibrillogenesis and compaction in post-EMT AV cushion maturation [54]. Because many other molecules are expressed in the post-EMT cushion mesenchyme and involved in valvulogenesis [2][3][4]24], further studies are warranted to better characterize the intersection of BMP signaling with other valvulogenic regulatory molecules and their regulatory cascades in post-EMT AV valvulogenesis.

Chick embryos
Fertilized eggs of White Leghorn (Gallus gallus domesticus) chicken were purchased from Pilgrim's Pride (Sumter, SC) and incubated in a humid atmosphere at 37°C. Stages of embryonic development were determined by using the criteria of Hamburger and Hamilton (HH) [55].

Histochemical Localization of Versican, Aggrecan and Hyaluronan (HA)
Preparation and specification of antibodies to anti-chick versican and aggrecan were previously reported [26]. The antichick versican antibody used in the present study was prepared against a synthetic peptide whose sequence was found in the alternatively spliced GAG-alpha region of versican and should recognize the V0 and V2 forms of versican but should not recognize the V1 form [26]. HA deposition was histochemically detected by using a specific hyaluronan binding protein (HABP) following the procedure previously described [25]. Biotinylated HABP (bHABP) was purchased from Seikagaku America (East Falmouth, MA).

3D AV endocardial cushion mesenchymal cell (CMC) aggregate culture
3D AV CMC aggregate culture was performed as described previously [14] with minor modifications. An advantage of our 3D aggregate culture is that mesenchymal cells are dissociated with trypsinization before the hanging-drop procedure, therefore cells are carrying minimal amount of endogenous extracellular matrix (ECM) proteins that can hamper the assessment of ECM production. Cushion mesenchyme was dissected out from HH stage-24 chick AV cushions by carefully removing the associated myocardium. AV CMCs were dissociated with trypsinization and 40,000 CMCs were cultured as a hanging drop overnight with serum-free Medium199 (Invitrogen) supplemented with ITS (5 µg/ml insulin, 5 µg/ml transferring, 5 ng/ml selenium, BD Sciences) and antibiotics (100 units/ml penicillin and 100µg/ml streptomycin, Invitrogen). Resultant cell aggregates were placed on the hydrated collagen gels (1mg/ml, rat tail tendon, BD Biosciences, Bedford, MA) and cultured with Medium 199 supplemented with 1% chick serum (Sigma, St. Louis, MO), ITS and antibiotics. Recombinant human BMP-2 and Noggin were purchased from R&D Systems (Minneapolis, MN). BMP-2 was used at final concentration ranging from 20 to 200 ng/ml and Noggin was used at final concentration of 500 ng/ml. Cultures were observed daily under an inverted microscope with Hoffman optics (Olympus, IMT-2). Cell migration, expression of versican and HA deposition, and mRNA expression of versican and Has2 were evaluated at 72 hrs. For each AV CMC culture experiment, three CMC aggregates were examined for each culture condition and the experiments were repeated a minimum of three times.

Cell migration assay
Seventy-two hours after placing the AV CMC aggregates on collagen gels, cell migration was assessed under an inverted microscope with Hoffman optics (Olympus, IMT-2). Cell numbers were counted in a series of focal planes at 40µm intervals within the collagen gels from 40µm below the surface of the gels to the bottom. Quantitative analysis data for cell migration assay represent a minimum of three independent experiments with cultures performed in triplicate.

BrdU incorporation assay
BrdU incorporation was used as an index of cell proliferation. 5'-bromodeoxyuridine (BrdU, Sigma) was added to a final concentration of 50 µM for 2 hrs before the termination of the CMC culture. CMC aggregates cultured on the collagen gels were fixed with 4% paraformaldehyde in PBS, pH 7.4 and rinsed with PBS. CMC aggregates were permeabilized with 0.1% Triton X-100/PBS and treated with DNase I (0.1 U/µl; Roche Diagnostics). Then, CMC aggregates were washed, blocked with 3% BSA/0.1% TritonX-100/PBS and processed for immunohistochemical detection of BrdU in the nuclei with anti-BrdU (BD Sciences) as described previously [14]. All nuclei were stained with propidium iodide. Stained samples were observed under a Leica TCS SP5 confocal microscope. Pictures for the BrdU incorporation assay were taken from the middle of the entire thickness of the CMC aggregates. A total of 500 nuclei was evaluated on photographs taken from 5 random fields per each CMC aggregate culture. Quantitative analysis data for BrdU incorporation assay represent a minimum of three independent experiments with cultures performed in triplicate.

TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect and quantify apoptosis at single cell level using the In Situ Cell Death Detection Kit (Roche). Following manufacturer's recommendation, CMC aggregates cultured on collagen coated wells were fixed with 4% paraformaldehyde in PBS and after being rinsed with PBS and permeabilization, CMC aggregates were processed for TUNEL reaction. All nuclei were stained with propidium iodide. TUNEL positive cells in CMC aggregates were observed and counted under a Leica TCS SP5 confocal microscope. Quantitative analysis data for TUNEL assay represent a minimum of three independent experiments with cultures performed in triplicate.

Immunostaining, histochemical detection and staining intensity analysis of AV CMC aggregate culture
Immunostaining and staining intensity analysis for CMC aggregates cultured on collagen gels were performed as described before [14] with minor modifications. Briefly, cultured CMC aggregates were fixed in 100% methanol at -20 °C and processed for immunostaining with anti-chick versican and histochemical detection of HA using bHABP. Stained samples were observed under a confocal microscope (Leica TCS SP5). The staining intensity of versican and HABP was evaluated by measuring the intensity of the fluorescence of the cell aggregates on photographs using computer software, Adobe Photoshop CS2. Results of staining intensity analysis represent at least three independent experiments with cultures performed in triplicate.

Quantitative RT-PCR for versican and Has2
Total RNA was extracted and purified using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) from CMC aggregates that had been cultured for 72 hrs on the collagen gels. After DNase digestion to remove genomic DNA, mRNA was further purified by Arcturus PicoPure RNA Isolation kit (AB applied biosystems, Foster City, CA). Complementary DNA was prepared using the iScript TM cDNA synthesis kit (BIO RAD, Hercules, CA) following the manufacturer's instruction. The following PCR primer pairs were designed to specifically amplify chick versican (NM_204787) (Forward 5'-aacatatggacgccgttttc-3'; Reverse 5'cattcctccaggccacatac-3') and chick Has2 (AF106940) (Forward, 5'-ccctgaaaaagtgcgatttg-3'; Reverse 3'gcagtcctttggtcatagagg-5'). Versican primer sequences used in this study are located in the versican G1 domain and detecting all isoforms of chick versican. Real-time PCR reactions were performed using 40 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 2 min. Chick versican and Has2 amplification was normalized to chick ß-actin amplification for each experimental treatment. The PCR products were verified via the thermal cycle sequencing using TagDNA polymerase and fluorescent dye-labeled termination. (MUSC, Biotechnology Resources Laboratory). QRT-PCR results represent at least three independent experiments with cultures performed in triplicate.

HA oligomer treatment
HA oligomers were fractionated from testicular hyaluronidase digests of purified hyaluronan polymer by differential filtration as described previously [57]. The oligomers used in this study were a mixed fraction of average molecular weight ~2.5 X 10 3 and were composed of 3-10 disaccharide units. The specificity of HA oligomers was analyzed as described before [27]. Chitin oligomers, used as control to HA oligomers, were obtained from Seikagaku America (Falmouth, MA). CMC aggregates were treated with 100 µg/ml HA or chitin oligomers in serumfree Medium 199 for 4 hrs and cultured in Medium 199 supplemented with 1% chick serum and ITS as indicated above.

Versican siRNA treatment
Nucleotide RNA duplexes corresponding to the chick versican mRNA (NM_204787) were designed using BLOCK-iT TM RNAi Designer (Invitrogen, Carlsbad, CA) and generated by Invitrogen (Carsbad, CA). The chick versican siRNA oligonucleotides used for this study were stealth103, targeting chick versican mRNA 333-357 and stealth 208, targeting chick versican mRNA 438-462. A scrambled (control) oligonucleotide (sense 5'-CAAGGCUAAGAUGGUAAUCGUAGAU-3' antisense 5'-AUCUACGAUUACCAUCUUAGCCUUG-3') was also generated by Invitrogen. Target sequences are located in the chick versican G1 domain which contains HA-binding sites and targeting all chick versican isoforms. Two siRNAs were combined and a final siRNA concentration of 100 nM was used with Lipofectamine 2000 (Invitrogen) with serum-free Medium 199 for 4 hrs to transfect CMC aggregates following the manufacturer's recommendation. A scrambled (control) oligonucleotide was also used at a final concentration of 100 nM with Lipofectamine 2000. To measure the transfection efficiency, the BLOCK-iT TM Fluorescent (FL) Oligo (Invitrogen) was co-transfected. More than 75% of CMCs on average showed FL oligo transfection (not shown). After a 72 hrs culture with 1% chick serum and ITS as described above, the reduction of versican protein and mRNA expression by versican siRNA transfection with CMCs was confirmed by real time RT-PCR and versican immunointensity analysis ( Figure  S1).

Statistical analyses
Analysis of variance (ANOVA) was used to determine significant differences between groups. When differences were found among groups, the Turkey-Kramer's t-test was used to determine if the difference was statistically significant. Differences were considered significant if p<0.05. Versican immunointensity was significantly reduced in versican siRNA-treated culture, whereas scrambled RNA-treated culture did not show significant reduction of versican immunointensity. (E) QRT-PCR data revealed an approximately 80% reduction of versican mRNA in CMC aggregate culture treated with versican siRNA, whereas scrambled RNA treatments did not significantly reduce versican mRNA expression. Vertical bars indicate ± SD of the mean. *p<0.05, as compared to the untreated control (M199). (PSD) Figure S2. BrdU incorporation assay for HA oligomer treatment. The CMC aggregates were cultured in untreated control (M199) (A, D), or treated with HA oligomers (100 µg/ml) (B, E) or chitin oligomers (control oligomers) (100 µg/ml) (C, F) in the presence (D, E, F) or absence (A, B, C) of BMP-2 (200 ng/ml). All nuclei were stained with propidium iodide (red). Note that incidence of BrdU-positive (green) nuclei appears to be the same in all CMC cultures. (G) Quantitative analysis of BrdU incorporation assay with AV CMCs. BrdU-positive andnegative nuclei in the cultured CMCs were counted to determine the percentage of cells in cell-cycle transit. A total of 500 nuclei in each culture was evaluated in 5 random fields.

Supporting Information
Vertical bars indicate ± SD of the mean. There were no significant differences between treated (BMP-2, HA oligomer, and chitin oligomer) and untreated control (M199) cultures. (TIF) All nuclei were stained with propidium iodide (red). Note that incidence of BrdU-positive (green) nuclei appears to be the same in all CMC cultures. (G) Quantitative analysis of BrdU incorporation assay with AV CMCs. BrdU-positive andnegative nuclei in the cultured CMCs were counted to determine the percentage of cells in cell-cycle transit. A total of 500 nuclei in each culture were evaluated in 5 random fields.
Vertical bars indicate ± SD of the mean. There were no significant differences between treated (BMP-2, versican siRNA, and scrambled RNA) and untreated control (M199) cultures. (TIF)


extfieldsofstudy: Biology Medicine

text:
The LIM-Homeodomain transcription factor Islet-1 is required for the development of sympathetic neurons and adrenal chromaffin cells

Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2β were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.

Introduction
The neural crest is a transient embryonic structure that gives rise to many different cells types, including neuronal and glial cells of the peripheral nervous system, endocrine cells of the adrenal medulla, thyroid C cells, and melanocytes (for review see Le Douarin and Kalcheim (1999)). During the past decade considerable progress has been made in deciphering the molecular networks underlying the segregation, differentiation and maturation of neural crest derived cells.
Sympathetic neurons of the para-and pre-vertebral ganglia and the endocrine adrenal chromaffin cells originate from the trunk neural crest. Though distinct in function, they share many characteristics as they both possess the machinery to synthesize, store and release the catecholamines noradrenaline (and adrenaline in a sub-population of chromaffin cells). Originally both cell types were thought to be derived from a common bi-potential catecholaminergic sympathoadrenal (SA) precursor, that develops from neural crest cells in the primary sympathetic ganglia close to the dorsal aorta (Anderson et al., 1991;Anderson and Axel, 1986). Though recent studies have suggested that the neuronal and endocrine SA subline-age may segregate before the onset of catecholaminergic differentiation, both nevertheless employ similar early developmental programs to establish the traits of catecholamine releasing cells (for review see Huber (2006) and Huber et al. (2009)). This is reflected by their expression of a common set of transcription factors, including the homeodomain transcription factors Phox2A and B, the basic helix-loop-helix transcription factors Mash-1 and Hand2 and the zinc-finger transcription factor Gata3 and Insm1. These transcription factors are induced by BMP −2/4/7 and can be first detected shortly after the primary sympathetic ganglia have formed from a subset of neural crest cells that have migrated to the vicinity of the dorsal aorta (for a recent review see Rohrer (2011)). Phox2B has been identified as master regulator of SA cell differentiation (Pattyn et al., 1999). Its loss leads to the lack of expression of any genes characteristic for neuronal and catecholaminergic function and it abrogates the expression of the above mentioned transcription factors apart from Mash-1, which is prematurely downregulated (Hendershot et al., 2008;Huber et al., 2005;Pattyn et al., 1999;Tsarovina et al., 2008;Wildner et al., 2008). Mash-1 Hirsch et al., 1998;Pattyn et al., 2006), Hand2 (Morikawa et al., 2007;Hendershot et al., 2008;Schmidt et al., 2009), Gata3 (Moriguchi et al., 2006;Tsarovina et al., 2008) and Insm1 (Wildner et al., 2008) have more discrete functions and together with Phox2B they act as a network rather than in linear cascade to promote the differentiation of SA cells. Loss of function studies have revealed that during sympathetic neuron development they control catecholaminergic and generic neuronal differentiation, proliferation of progenitors and immature neurons, survival of postmitotic neurons (Gata3) and maintenance of differentiated sympathetic neuron properties (Hand2, Phox2B, for review see Rohrer (2011)). Similar, but not identical functions of this gene regulatory network have been proposed for the development of adrenal chromaffin cells (for review see Huber et al. (2009)).
The LIM-Homeodomain transcription factor Islet-1 is expressed in distinct neuronal and non-neuronal populations and it is known as a key regulator of the development of spinal motoneurons (Pfaff et al., 1996), the pancreas (Ahlgren etal., 1997)and theheart (Cai etal., 2003). Multiple aspects of motoneuron development depend on Islet-1, including specification (Pfaff et al., 1996;Song et al., 2009), axonal growth (Liang et al., 2011) and patterning (Kania and Jessell, 2003). In the peripheral nervous system Islet-1 is widely expressed, including sensory neurons of the dorsal root ganglia (DRG) and sympathetic ganglia (Avivi and Goldstein, 1999;Sun et al., 2008). Loss of function studies have revealed an essential role of Islet-1 for the developmentof the sensory neurons of the dorsal root ganglia, with the TrkA-positive nociceptive neurons being most prominently affected by Islet-1 deficiency. While the early development of dorsal root ganglia appeared grossly normal following ablation of Islet-1 in the neural crest, later in development a selective loss of nociceptive markers and enhanced apoptosis was observed in dorsal root ganglia. Gene expression analysis further revealed a prolonged expression of transcription factors that are normally restricted to early sensory neurogenesis and ectopic expression of genes that are normally present in the CNS, but not in sensory ganglia (Sun et al., 2008). Islet-1 has been suggested to regulate sensory neuron gene expression epistatically with Brn3A through binding to an enhancer in the neurod4 gene (Dykes et al., 2011). Direct transcriptional downstream targets of Islet-1 also include Mef2C (Dodou et al., 2004) and Hb9 (Lee and Pfaff, 2003), which are important for heart and motoneuron differentiation, respectively.
Though the expression of Islet-1 in developing sympathetic neurons is well documented, its function has remained unclear up to date. In this study we have investigated the role of Islet-1 for the development of sympathetic neurons and chromaffin cells. Our data indicate that Islet-1 acts genetically downstream of Phox2B and does not essentially affect the initial differentiation of SA cells, but is required for subsequent survival and correct maturation of sympathetic neurons and chromaffin cells.

Experimental animals
The generation and genotyping of wnt1cre conditional Islet-1 deficient mice containing a Rosa26 LacZ reporter gene (Sun et al., 2008) and Phox2B Lacz mice (Pattyn et al., 1999) were described previously. Embryos were staged considering midday of the day of the vaginal plug as embryonic day 0.5. Embryos with the genotype Islet-1 flox/flox /wnt1cre are referred to as Islet-1 wnt1cre mouse embryos and embryos with the genotype Islet-1 flox/+ / wnt1cre are referred to as control mouse embryos.

Histology
Pregnant mice were killed by cervical dislocation. Embryos were removed from the uterus, briefly rinsed with phosphate-buffer (PB, pH 7.4) and fixed in PB containing 4% paraformaldehyde (PFA) overnight. Tissues were then rinsed 3 times with PB and transferred into 30% sucrose in PB for cryoprotection. After immersion in sucrose overnight the tissue was coated with OCT ™ compound (Tissue Tek), frozen on a metal bridge that was placed in liquid nitrogen, and stored at −70 °C until further processing. Tissues were then cut into 10 μm serial sections, mounted on Superfrost ™ slides, and air dried for 30 min, before performing in situ hybridization, immunfluorescence staining or TdT dUTP nick end labeling analysis, respectively.
For immunfluorescence-staining sections were pretreated with 10% serum corresponding to the secondary antibody in PBS containing 0.1% Triton X-100. Primary antibodies were incubated overnight at 4 °C. After rinsing in PBS sections were incubated with Cy2 ™conjugated or biotinylated secondary antibody for 2 h at room temperature. Sections were then rinsed in PBS, incubated with Cy3 ™ -conjugated streptavidin (in case a biotinylated secondary antibody was used) and after final rinsing in PBS mounted with Fluoro-Mount containing DAPI (Dianova, Hamburg, Germany).

TdT dUTP nick end labeling (TUNEL) analysis
For detection of apoptotic cells we performed TUNEL on 10 μm cryosections using an ApoTag ™ In Situ APOPTOSIS Detection Kit (Millipore) following the manufacturer's instructions. Subsequently, TH-immunofluorescence staining was carried out as described above. TUNEL positive cells were counted in every 10th section through the thoracic sympathetic chain within the TH-immunoreactive area. The size of the TH immunoreactive area was subsequently measured by using ImageJ and the number of TUNEL positive cells per TH immunoreactive area was determined.

Electronmicroscopy
For electronmicroscopy, adrenals from E16.5 embryos were fixed by immersion in a mixture of glutaraldehyde (1.5%) and paraformaldehyde (1.5%) in 0.1 M phosphate buffer (PB) at pH 7.4 for 48 h and rinsed several times with PB. Organs were then postfixed in 1% OsO 4 in PB, rinsed in PB and block-stained with 1% uranyl acetate. After dehydration through increasing concentrations of ethanol, the tissue was embedded in Durcupan (Fluka). Ultrathin sections (50 nm) were examined with a Zeiss EM10.

Microarray analysis
Whole adrenal glands of E14.5 control and Islet-1 wnt1cre mouse embryos were dissected and stored in RNAlater® (Qiagen, Düsseldorf, Germany) at −80 °C until further processing. After genotyping adrenal glands were pooled (four adrenal glands per sample, four samples per group) and homogenized in Trizol. Total RNA was extracted from the homogenized tissue according to manufacturer's protocol and eluted in RNAse free water. Microrarray analysis was performed using mouse Gene ST 1.0 Arrays (Affymetrix UK, High Wycombe, UK) as previously described (Spittau et al., 2013).

Statistical analysis
To assess the statistical significance a Student's t-test for a two-tailed distribution and a twosample unequal variance was performed.

Expression of Islet-1 in SA cells
We first investigated the expression of Islet-1 in developing mouse sympathetic ganglia and adrenal glands. Islet-1 expression is initiated in the primary sympathetic ganglia around E9.5-E10 (not shown) and can be detected throughout all axial levels by E10.5 (Fig. 1A). The expression of Islet-1 is maintained in sympathetic ganglia at least until E16.5 (Fig. 1D).
Adrenal Chromaffin cells share a common origin from the neural crest with sympathetic neurons and express a similar set of genes during their early development. We could detect Islet-1 expression in the developing adrenal gland as soon as it is colonized by chromaffin progenitors at E12.5 (Fig. 1B). Islet-1 expression is maintained in the adrenal medulla at least until E16.5, albeit at lower levels than in sympathetic ganglia (Fig. 1F).

Islet-1 expression in SA cells depends on Phox2B
The homeodomain transcription factor Phox2B controls the development of distinct neuronal populations and it is associated with catecholaminergic differentiation as well as the establishment of autonomic reflex pathways (Dauger et al., 2003;Pattyn et al., 1999Pattyn et al., , 2000. The differentiation of SA cells crucially depends on Phox2B Pattyn et al., 1999) and most other components of the transcription factor network that governs the development of SA cells, including Hand2, Phox2A and Insm1, act downstream of Phox2B (for review see Huber (2006) and Rohrer (2011)). To explore, whether Phox2B is acting upstream of Islet-1 in the SA lineage we analyzed Islet-1 expression in Phox2B Lacz/Lacz mouse embryos. As anticipated, Islet-1 expression was not detectable in LacZ positive cells accumulating at the dorsal aorta of E11.5 Phox2B LacZ/LacZ embryos ( Fig. 2), indicating that Islet-1 acts genetically downstream of Phox2B in the SA lineage.

The initial differentiation of SA cells does not essentially depend on Islet-1
In order to define a putative role of Islet-1 for the development of SA cells we employed Islet-1 wnt1cre mice. This mouse line has been shown to efficiently eliminate the expression of exon 3/4 of the Islet-1 transcript in neural crest derivatives, as previously described (Sun et al., 2008). Due to the early onset of wnt1 expression in the neural plate before the delamination of neural crest cells (Echelard et al., 1994) wnt1cre mediated excision of Islet-1 should occur before the expression of Islet-1 is initiated in cells of the SA lineage.
The size of primary sympathetic ganglia of Islet-1 wnt1cre mouse embryos appeared grossly normal at E10.5, using Phox2B ( Fig. 3A and B) or LacZ (not shown) as a marker. In order to identify a putative impact of Islet-1 on the initial differentiation of SA cells we analyzed the expression of the transcription factors Mash-1, Hand2, Phox2A, Gata3, and Insm1 as well as genes indicative of neuronal and catecholaminergic differentiation, i.e. neurofilament (NF) 68, SCG10 (not shown), tyrosine-hydroxylase (TH, the rate limiting enzyme for catecholamine synthesis), dopamine-β-hydroxylase (DBH, the enzyme that converts dopamine into noradrenaline) and the proto-oncogene c-ret in primary sympathetic ganglia of E10.5 Islet-1 wnt1cre mouse embryos and control littermates ( Fig. 3A-T). This analysis did not reveal any striking differences between control and Islet-1 wnt1cre mouse, suggesting that Islet-1 is not essentially required for the initial differentiation of SA cells. Furthermore, in contrast to the sensory neurons of the dorsal root ganglia (Sun et al., 2008), sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes that are normally present in the CNS like LHX1, LHX2 and LBXCOR1 at E10.5, E11.5 (not shown) or E12.5 (see Supplementary Fig. S1).

Sympathetic ganglia of Islet-1 wnt1cre mice become progressively atrophic
At E11.5 and later stages the size of sympathetic ganglia of Islet-1 wnt1cre mouse embryos appeared considerably smaller as compared to control littermates. This could be observed throughout all axial levels and regardless whether Phox2B (Fig. 4A-J), LacZ, NF68 or TH ( Fig. 4K-N) was used as a marker. Like in control mouse embryos, the vast majority of Phox2B positive cells in sympathetic ganglia of Islet-1 wnt1cre mouse embryos were also THimmunoreactive at E11.5 and E13.5 (Fig. 4K-N), indicating that the transition of Phox2B +/TH− progenitors to Phox2B+/TH+ SA cells was not significantly impaired in Islet-1 deficient mice. In order to quantify the effect of Islet-1 deficiency on sympathetic neuron numbers, we determined the number of TH-positive cells throughout the thoracic sympathetic chain in every 10th section (Fig. 4O). This analysis revealed a progressive reduction of the relative number of TH positive cells in sympathetic ganglia of Islet-1 wnt1cre mouse embryos as compared to control mouse embryos from E11.5 (amounting to 44% of control values) to E16.5 (amounting to 4% of control values).
To determine the cause of the cell number reduction in sympathetic ganglia of Islet-1 wnt1cre mouse embryos we performed TUNEL analysis and assessed the proliferative activity in sympathetic ganglia by using the M-phase mitotic marker pH3. As demonstrated in (Fig.  5A-C we observed a significant increase of the number of TUNEL positive cells in the area of developing sympathetic ganglia of E11.5 Islet-1 wnt1cre mouse embryos as compared to control mouse embryos. In addition the percentage of TH positive cells that were also positive for pH3 was diminished in Islet-1 wnt1cre mouse embryos (Fig. 5D-F). Thus enhanced cell death and reduced proliferation account for the reduced size of sympathetic ganglia.

The expression of TrkA and c-Ret is altered in sympathetic ganglia of Islet-1 wnt1cre mice
At E16.5 we could still detect small clusters of sympathetic neurons in a few sections throughout the whole sympathetic chain of Islet-1 wnt1cre mouse embryos. These cells were LacZ positive (not shown) and still displayed apparently normal expression of Phox2B ( Fig.  6A and B), DBH ( Fig. 6G and H), NF 68 ( Fig. 6I and J), TH, SCG10, Phox2A, Hand2, and Gata3 (not shown).
Accordingly the expression of Mash-1, which is transiently expressed in early developing sympathetic ganglia, had virtually disappeared in both control and Islet-1 wnt1cre mouse embryos by E16.5 ( Supplementary Fig. S2) and it was unaltered at other developmental stages investigated (E10.5-E16.5). Furthermore, in contrast to spinal sensory neurons, sympathetic neurons of Islet-1 wnt1cre mouse embryos did not show an apparent upregulation of Insm1 expression at any age investigated (E10.5-E16.5, not shown).
The proto-oncogene c-Ret is a receptor typosine-kinase, which mediates signal transduction of members of the glial-cell line derived neurotrophic factor (GDNF) superfamily (for review see Airaksinen and Saarma (2002)). It is expressed homogenously in SA cells that aggregate at the dorsal aorta, but later in development its expression becomes restricted to the cholinergic sub-population of sympathetic neurons (Burau et al., 2004;Pachnis et al., 1993). As shown in Fig. 6E in E16.5 control mouse embryos high expression of c-Ret is limited to a subpopulation of sympathetic neurons, while the residual sympathetic neurons in Islet-1 wnt1cre mouse embryos display homogenously high expression of c-Ret (Fig. 6F). These results suggest that the few remaining cells in sympathetic ganglia of Islet-1 wnt1cre mice might not have matured correctly or that a TrkA negative, c-Ret positive subpopulation of sympathetic neurons is selectively spared in Islet-1 wnt1cre mouse embryos. Since the cholinergic subpopulation of sympathetic neurons expresses c-Ret, we performed in situ hybridization using probes for the cholinergic markers vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) (not shown). Our findings did not support the notion that the cholinergic subpopulation of sympathetic neurons specifically survives in Islet-1 wnt1cre mouse embryos.

The correct maturation of chromaffin cells is impaired in Islet-1 wnt1cre mouse embryos
In contrast to the massive atrophy of sympathetic ganglia, the numbers of adrenal chromaffin cells in Islet-1 wnt1cre mouse embryos as identified by TH-immunoreactivity were unaltered at E13.5 and relatively mildly affected at E16.5, amounting to 60% of control values (Fig.  7E). In situ hybridizations for TH ( Fig. 8A and E) and DBH (C and G) did not reveal an apparent alteration of the expression of these enzymes in the adrenal medullae of Islet-1 deficient embryos at E16.5.
As chromaffin cell precursors mature, the adrenergic subpopulation initiates the expression of the enzyme phenyl-N-methyl-transferase (PNMT) that converts noradrenaline into adrenaline. As demonstrated in Fig. 8B, substantial numbers of chromaffin cells expressed PNMT in control embryos at E16.5. Strikingly, PNMT expression was virtually absent in the adrenal medullae of Islet-1 wnt1cre mouse embryos (Fig. 8F). In addition the expression of chromogranin (Chg) A, a secretory protein located in chromaffin vesicles, appeared diminished ( Fig. 8D and H). These data suggest that some aspects of chromaffin cell maturation were impaired in Islet-1 wnt1cre mouse embryos. To assess the overall differentiation of chromaffin cells in Islet-1 wnt1cre mouse embryos we analyzed their ultrastructure. As shown in Fig. 8J adrenal medullary cells of Islet-1 deficient mice like control littermates displayed the typical secretory chromaffin granules in their cytoplasm (Coupland, 1972;Coupland and Tomlinson, 1989) indicating that the acquisition of an endocrine cell phenotype was not substantially impaired.

Microarray analysis
To better understand the genetic program that is controlled by Islet-1 in cells of the SA lineage we performed gene expression profiling by microarray analysis. As stated above, sympathetic ganglia were substantially reduced in size from E11.5 onwards. Since dissection of sympathetic ganglia prior to E11.5 was not feasible, we focused on the adrenal gland. Microarray analysis was performed on RNA extracted from whole adrenal glands of Islet-1 wnt1cre and control mouse embryos obtained at E14.5, the age when PNMT expression is initiated in mouse chromaffin cells (Lohr et al., 2006). As summarized in Table 1 the overall alterations of gene expression levels were relatively mild and the only mRNA transcript showing a more than 3 fold change was that of the PNMT gene (13 fold down regulation). In contrast the expression levels of TH, DBH, Gata3, Mash-1, Phox2A, Phox2B and Insm1 were not significantly altered, consistent with the observation that chromaffin cell numbers were normal at early developmental ages and corroborating the finding that Islet-1 does not significantly influence the initial differentiation of SA cells.
However, the expression levels of some other transcription factors that are associated with SA cell development were reduced in the adrenal glands of Islet-1 wnt1cre mouse embryos, i.e. Gata2 (2.9 fold), Hand1 (2 fold) and AP-2β (2.5 fold). In cells of the SA lineage Gata2 and Hand1 act downstream of Gata3 and Hand2, respectively (Tsarovina et al., 2004;Vincentz et al., 2012). AP-2β, in contrast, is already expressed in migrating neural crest cells and it is required for sympathetic neuron survival and the maturation of chromaffin cells.
We assessed the expression of these transcription factors in primary sympathetic ganglia and the adrenal glands of Islet-1 wnt1cre mouse embryos and control littermates by in situ hybridization. While we did not obtain unequivocal results regarding the expression of Hand1 and AP-2β, we could show that Gata2 expression is markedly diminished in sympathetic ganglia of E10.5 Islet-1 wnt1cre mouse embryos ( Fig. 9C and D). Likewise at E13.5 Gata2 expression was apparently reduced in the adrenal gland and the remaining sympathetic neurons of Islet-1 wnt1cre mouse embryos as compared to control littermates ( Fig. 9E and F). These findings show that Islet-1 deficiency affects the expression levels of distinct molecular markers and transcription factors in chromaffin cells, including PNMT and Gata2.

Islet-1 is required for the development of sympathetic neurons and chromaffin cells
The development of the nervous system is controlled by a complex network of intrinsic and extrinsic signals that govern cell fate specification, choice of neurotransmitter identity and axonal patterning. Islet-1 is a LIM-Homeodomain (LIM-HD) transcription factor, initially identified as a protein binding to the insulin gene enhancer (Karlsson et al., 1990). It regulates gene expression by binding via its homeodomain to the consensus sequence YTAATGR, while it can recruit co-factors mainly via the Limdomain (for review see Hobert and Westphal (2000)).
Islet-1 is required for the development of distinct neuronal populations in the peripheral and central nervous system, but it has also important functions outside the nervous system, e.g. during the development of the heart and the pancreas (Ahlgren et al., 1997;Cai et al., 2003). Neuronal populations that require Islet-1 for their development include spinal motoneurons (Pfaff et al., 1996), sensory neurons of the DRG (Sun et al., 2008), cholinergic forebrain neurons (Elshatory and Gan, 2008), and retinal ganglion cells (Elshatory et al., 2007). In the spinal cord specific combinations of LIM-HD proteins are considered to constitute a so called "Lim-code" that determines cell fate and axonal patterning (for review see Lumsden, 1995;Sockanathan, 2003). Gene ablation studies have revealed that Islet-1 is required for the initial generation of motoneurons as well as for multiple aspects of their subsequent development, including axonal pathfinding of specific subsets of motoneurons (Kania and Jessell, 2003;Liang et al., 2011;Pfaff et al., 1996).
In this study we show that Islet-1 also has important functions for the correct development of the derivatives of the SA cell lineage, i.e. sympathetic neurons and the endocrine adrenal chromaffin cells. Our data indicate that the initial differentiation of SA cells and the onset of catecholaminergic and neuronal differentiation does not essentially depend on Islet-1. Yet, in Islet-1 wnt1cre mouse embryos the survival and proliferation of sympathetic neuroblasts were impaired, resulting in massive atrophy of the sympathetic ganglia from E11.5 onwards. The few sympathetic neurons surviving up to E16.5 had apparently not matured correctly, as they lacked the high affinity NGF-receptor TrkA. In contrast to the severe loss of sympathetic neurons, chromaffin cell numbers in the adrenal gland of Islet-1 wnt1cre mouse embryos were relatively mildly reduced and the expression levels of the noradrenaline synthesizing enzymes TH and DBH were normal. However, the adrenaline synthesizing enzyme PNMT was virtually absent and chromogranin A expression levels were reduced, indicating a role of Islet-1 in establishing in more mature chromaffin cell traits. Thus, this study establishes Islet-1 as a transcription factor that acts relatively late during the differentiation of cells derived from the SA cell lineage.

The role of Islet-1 for the development of sympathetic neurons versus DRG neurons
Like sympathetic neurons the sensory neurons of the dorsal root ganglia are neuronal derivatives of the neural crest. Loss of Islet-1 has been shown to predominantly affect the nociceptive TrkA-positive subpopulation of sensory neurons and the dorsal root ganglia of Islet-1 deficient mice exhibited enhanced apoptosis, similar to our observation in sympathetic ganglia (Sun et al., 2008). Apart from this, our study revealed profound differences between the developmental programs regulated by Islet-1 in sympathetic and sensory neurons. While sensory neurons of Islet-1 deficient mouse embryos displayed ectopic expression of genes specific for subpopulations of CNS neurons, this could not be observed in sympathetic ganglia. Furthermore, in DRG neurons the lack of Islet-1 leads to a prolonged expression of genes normally restricted to early sensory neurogenesis, as e.g. the basic helix-loop-helix transcription factors NeuroD4 and neurogenin1 (Sun et al., 2008). In contrast, as shown in this study, sympathetic neurons of Islet-1 wnt1cre mouse embryos normally downregulated Mash-1, a proneural transcription factor transiently expressed during their early development. The zinc-finger transcription factor Insm1 is expressed in both sensory and sympathetic neurons as well as in the endocrine pancreatic Islet cells, which also require Islet-1 for their development (Ahlgren et al., 1997). Interestingly, Insm1 showed enhanced expression in sensory but not in sympathetic neurons. Together these data imply that distinct gene regulatory programs are addressed by Islet-1 in sensory neuronal and sympathoadrenal neural crest derivatives and they underscore the context specificity of Islet-1 action as well as intrinsic differences between these neural crest derivatives.

Islet-1 and the sympathoadrenal transcriptional network
The differentiation of SA cells is controlled by a cross-regulatory network of transcription factors including Phox2A/B, Mash1, Gata3, Hand2 and Insm1. The components of this network jointly control the expression of generic and subtype-specific neuronal genes, but they also reciprocally regulate their own expression. Phox2B is a master regulator of the development of SA cells and it is essentially required for the expression of all members of this network, apart from MASH-1 (for review see Huber (2006) and Rohrer (2011)). We show here that Islet-1 expression is absent in primary sympathetic ganglia of Phox2B deficient mice. This finding establishes Islet-1 as an additional member of the group of transcription factors acting downstream of Phox2B in the SA lineage. Islet-1 apparently does not influence the expression levels of Phox2A/B, Hand2, Mash-1 and Gata3 in sympathetic neurons and chromaffin cells.
However, according to our microarray data, we found three transcription factors among the gene products that were significantly down-regulated in adrenal glands of Islet-1 wnt1cre mouse embryos known to be associated with the development of SA cells, i.e. Gata2, AP2β, and Hand1. Out of these genes, Gata2 displayed the most pronounced alteration of its expression level (2.9 fold down) and we could confirm its down-regulation in sympathetic neurons and chromaffin cells of Islet-1 wnt1cre mouse embryos by in situ hybridization. In murine SA cells Gata2 is a direct or indirect downstream target of Gata3 with functions most likely identical to or at least overlapping with that of Gata3. In the chick, however, only Gata2 is present (Lim et al., 2000;Moriguchi et al., 2006;Tsarovina et al., 2004). Interestingly, Islet-1 wnt1cre mouse embryos and Gata3 knockout mice display some similarities with regard to the developmental deficits observed in sympathetic ganglia. This includes the timing and extent of sympathetic neuron death and the fact that the initial differentiation of SA cells is grossly normal, though, unlike in Islet-1 wnt1cre mice, the expression levels of TH were markedly reduced in sympathetic neurons and chromaffin cells of Gata3 mice (Moriguchi et al., 2006;Tsarovina et al., 2004). It should be noted that Gata transcription factors and Islet-1 have been shown to synergistically regulate some aspects of heart development (Dodou et al., 2004;Golzio et al., 2012). Together these observations point to the possibility that Islet-1 may co-regulate some functions of Gata3 during the development of SA cells.

Functions of Islet-1 for the development of neuronal versus endocrine SA derivatives
Though sympathetic neurons and chromaffin cells share a common set of transcription factors during their early development, gene ablation studies have revealed distinct deficits in sympathetic neurons and chromaffin cells following the null mutation of various individual transcription factors (for review see Huber (2006) and Huber et al. (2009)). Therefore it was not surprising that the loss of Islet-1 affects neuronal and endocrine SA derivatives in a different way. Yet it should be noted that Islet-1 in general, like other LIM-HD proteins, appears to execute a very diverse array of functions depending on cellular context specific co-regulators (for review see Hobert and Westphal (2000)).
Sympathetic neurons, as we show here, undergo apoptosis in the absence of Islet-1 after having accomplished a grossly normal initial differentiation. How does Islet-1 deficiency affect sympathetic neurons survival? Islet-1 may simply interfere with the basic apoptotic machinery as suggested as a mode of action of Gata3 in maintaining the survival of mature sympathetic neurons (Tsarovina et al., 2010). Alternatively, cell death in Islet-1 wnt1cre mouse embryos may be the result of a lack of correct further differentiation of sympathetic neurons beyond the stage of a catecholaminergic neuroblast. The fact that the few sympathetic neurons that survive up to E16.5 in Islet-1 wnt1cre mouse embryos fail to correctly express the receptor tyrosine kinase TrkA and instead maintain the expression of c-Ret, a feature of early sympathetic neuroblasts, corroborates the latter notion. Nevertheless, we cannot exclude the possibility that the TrkA-negative and c-Ret-positive subpopulation of sympathetic neurons is specifically spared in Islet-1 wnt1cre mouse embryos. However, it should be noted that expression of TrkA in sympathetic ganglia is not initiated before E13.5 (Fagan et al., 1996), i.e. after the onset of substantial cell loss in sympathetic ganglia of Islet-1 wnt1cre mouse embryos.
The numerical deficits of chromaffin cells in Islet-1 wnt1cre mice were much less pronounced than that of sympathetic neurons at least up to E16.5. The most striking alteration that we found in the adrenal gland of Islet-1 wnt1cre mice was the virtual lack of PNMT expression. The distinct survival rates of sympathetic neurons and chromaffin cells in the absence of Islet-1 and the fact that in the adrenal gland primarily the expression of PNMT was affected may indicate that Islet-1 exerts fundamentally distinct functions in the two cell types. However, it is also conceivable that the molecular mechanisms underlying Islet-1 action is similar in sympathetic neurons and chromaffin cells, but leads to a distinct outcome depending on the cellular context. Different survival spans of intra-adrenal and extra-adrenal SA cells upon developmental impairment have been described before (for review see Huber (2006) and Huber et al. (2009)) and may be caused either by intrinsic differences between endocrine and neuronal SA cells or by a survival supporting environment provided by the adrenal gland.
The virtual lack of PNMT expression in the adrenal gland of Islet-1 wnt1cre mice could be due to an impairment of overall chromaffin cell differentiation to a point where PNMT expression can be enabled. However, based on their ultrastructure and gene expression profile chromaffin cells of Islet-1 wnt1cre mouse embryos appeared to have developed relatively normal in general, with some minor alterations. Furthermore, our microarray analysis did not reveal massive changes of the expression level of genes known to regulate PNMT expression, as e.g. the glucocorticoid receptor, Egr1 or AP-2 (for review see Wong (2003)). Thus it is also conceivable that Islet-1 may directly co-regulate PNMT expression or possibly interact with the glucocorticoid receptor, which is essentially required for PNMT expression in chromaffin cells (Finotto et al., 1999). It should be pointed out here, that physical interactions of Islet-1 with other nuclear steroid receptor have been described before (Gay et al., 2000).
In conclusion, our data show that Islet-1 is required for distinct aspects of the development of neuronal and endocrine SA lineage derivatives beyond the stage of a catecholaminergic neuroblast. In the future it will be intriguing to identify the mechanisms and co-regulators underlying Islet-1 function in cells of the SA lineage. Possible candidates include Gata transcription factors and the glucocorticoid receptor. Islet-1 is expressed in primary sympathetic ganglia (arrowheads) of E10.5 mouse embryos (A). In situ hybridizations for NF68 (C) and TH (E) were performed on near adjacent sections. In the developing adrenal gland Islet-1 can be detected at E12.5 (B). To identify the area of the developing adrenal gland the adreno-cortical marker Sf1 was used as a marker on an adjacent section (not shown). Islet-1 expression can be detected at least until E16.5 in sympathetic ganglia (D, thoracic sympathetic ganglion) and in adrenal chromaffin cells (F) albeit at lower levels. Insert: higher magnification. The dashed line indicates the border of the adrenal gland anlage in (B) and the adrenal medulla in (F). (ac) Adrenal cortex, (ag) adrenal gland anlage, (am) adrenal medulla (da), dorsal aorta, (sg) sympathetic ganglion, (srg) suprarenal ganglion; bars: (A, B, C, E, and F) 100 μm, (D) 50 μm. Islet-1 expression is lacking in the area of sympathetic ganglia of Phox2B LacZ/LacZ mice at E11.5. Photomicrographs show cross sections through thoracic sympathetic ganglia (arrowheads) of E11.5 wildtype (A and B) and Phox2B LacZ/LacZ mouse embryos (C and D). In-situ-hybridizations for Phox2B, LacZ and Islet-1 were performed in near adjacent sections. (da) Dorsal aorta. Bar: 100 μm.  Sympathetic ganglia of Islet-1 wnt1cre mouse embryos become progressively atrophic. The size of sympathetic ganglia, as visualized by Phox2B ISH, is reduced in Islet-1 wnt1cre mouse embryos (B, D, F, H, and J) as compared to control embryos (A, C, E, G, and I) at E11.5 (A and B) and E13.5 (C-J). (K-N) Double immunofluorescence-staining using antibodies against TH (red cytoplasmic stain) and Phox2B (green nuclear stain) of sympathetic ganglia of control and Islet-1 wnt1cre mouse embryos at E11.5 (K and L) and E13.5 (M and N). Note that in both control and Islet-1 wnt1cre mouse embryos virtually all Phox2B-immunoreactive cells are also positive for TH. (O) Counts of TH-immunoreactive cells in the thoracic sympathetic chain of control and Islet-1 wnt1cre mouse embryos. Data represent the number of TH positive cells per section and are presented as means±s.e.m. Every 10th section throughout the thoracic sympathetic chain of at least 3 embryos per group was analyzed. Bars: (A-J) 100 μm, (K-N) 50 μm. ** p<0.01; *** p<0.01. Apoptosis is enhanced and proliferation is reduced in sympathetic ganglia of Islet-1 wnt1cre mouse embryos at E11.5. (A and B) Photomicrographs showing TUNEL (green) and THimmunofluorescence-staining (red) of sympathetic ganglia of a control (A) and an Islet-1 wnt1cre mouse embryo (B) at E11.5. (C) Relative number of TUNEL-positive cells per TH-positive area in thoracic sympathetic ganglia of control and Islet-1 wnt1cre mouse embryos. (D and E) Double immunofluorescence-staining using antibodies against TH (red) and phosphohistone 3 (pH3, green) of sympathetic ganglia of control (D) and Islet-1 wnt1cre mouse embryos (E). (F) Number of pH3-positive cells (% of TH positive cells) in thoracic sympathetic ganglia of control and Islet-1 wnt1cre mouse embryos. Data are presented as means±s.e.m. Every 10th section throughout the thoracic sympathetic chain of at least 3 embryos per group was analyzed. Bar: 50 μm. ** p<0.01. In situ hybridization for Phox2B (A and B) and TrkA (C and D), c-Ret (E and F), DBH (G and H) and neurofilament 68 (I and J) on sections through the thoracic sympathetic chain of E16.5 control and Islet-1 wnt1cre mouse embryos. Note that the majority of cells in control sympathetic ganglia express TrkA and a small subpopulation of cells expresses high levels of c-Ret. In contrast the few remaining sympathetic neurons in Islet-1 wnt1cre mouse embryos lack TrkA and uniformly express high levels of c-Ret, while the expression levels of DBH and neurofilament 68 appear unaltered. The area of the sympathetic ganglion is demarcated by a dashed line in (D). Bar: 100 μm. Photomicrographs showing cross sections through the adrenal glands of control (A and C) and Islet-1 wnt1cre mouse embryos (B and D) at E13.5 (A and B), and E16.5 (C and D) stained with an antibody against TH (red cytoplasmatic stain) and DAPI (blue nuclear stain). (E) Counts of TH-immunoreactive cells in sections of the adrenal glands of Islet-1 wnt1cre mouse embryos and control littermates. Data are presented as means±s.e.m. Every 5th section of at least 6 adrenal glands of 3 different animals for each group has been analyzed. Bars: 100 μm. ** p<0.01. In situ hybridization for TH (A and E), PNMT (B and F), DBH (C and G) and chromogranin A (D and H) on sections of the adrenal glands of E16.5 control (A-D) and Islet-1 wnt1cre mouse embryos (E-H). Note that in the adrenal gland of Islet-1 wnt1cre mouse embryos the expression of PNMT is virtually lacking and expression levels of chromogranin A appear reduced. The approximate border of the adrenal medulla is demarcated by a dashed line. (I and J) Electronmicrographs of adrenal medullae of E16.5 control (I) and Islet-1 wnt1cre mouse embryos (J). SA cells in the adrenal gland of Islet-1 wnt1cre mouse embryos have undergone grossly normal chromaffin cell differentiation as indicated by the presence of chromaffin granules (arrowheads); insert: higher magnification. Bars: (A-H) 100 μm and (I and J) 2 μm. (A-D) In situ hybridization for Gata2 on sections through thoracic primary sympathetic ganglia (arrowheads) of E10.5 control (C) and Islet-1 wnt1cre mouse embryos (D). Phox2B in situ hybridizations (A and B) have been carried out on adjacent sections. (E and F) In situ hybridization for Gata2 on sections of the adrenal gland of E13.5 control (E) and Islet-1 wnt1cre embryos (F). The border of the adrenal gland is demarcated by a dashed line. Bars: 100 μm.