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Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

INTRODUCTION
RNAi is a process in which small noncoding RNAs silence gene expression in a post-transcriptional manner. 1,2 MicroRNAs (miRNAs) are endogenous effector molecules in mammals, and they exert their repressing function in the RNA-induced silencing complex (RISC) by binding to target mRNA. 3,4 Conventional miRNA biogenesis involves consecutive cleavage of a longer folded primary microRNA (pri-miRNA) precursor transcript, by first Drosha and then Dicer, that results in the formation of a duplex composed of two individual $22-nt RNA strands with 3 0 overhangs. [5][6][7][8] While both miRNA strands may direct silencing, only one strand is preferably loaded into RISC. 9 The miRNA pathway can be exploited using several entry points for the targeted downregulation of gene expression, and RNAi remains an attractive method for research or therapeutic purposes. 10 One popular entry point is the expression of artificial short hairpin RNAs (shRNAs) that mimic the hairpin structure of the pre-miRNAs produced in the nucleus by the Microprocessor complex Drosha/ DGCR8. [11][12][13] In the cytoplasm, Dicer cleaves of the loop and terminal duplex stability favors preferential loading of the guide strand, which is designed to be fully complementary to the target mRNA. 9,14 However, RISC loading of the passenger strand can occur, and this contributes to the cytotoxicity associated with RNAi, as off-target genes can be repressed by partial complementarity and binding within the seed region. [15][16][17][18] Recent discoveries have shown that shortening the stem of expressed shRNAs below 20 bp results in alternative processing that bypasses Dicer. 19,20 These Dicer-independent shRNAs rely on slicing of the 3 0 arm by Argonaute-2 (Ago2) between nucleotides 10 and 11 from the 5 0 end, which yields a single RNAi-active strand and eliminates unwanted activity of the 3 0 arm. 19,20 Further trimming by the poly(A)-specific ribonuclease (PARN) results in shorter RNA species that effectively guide target knockdown. 21 Dicer-independent shRNAs mimic the recently described non-canonical biogenesis of miR-451, which also depends on Ago2 and trimming-tailing processes rather than Dicer. [22][23][24] The Dicer-independent shRNA expression systems reported so far have been limited to RNA polymerase III (Pol III) promoters, 25 and tunable systems based on RNA polymerase II (Pol II) promoters are lacking.
In this study, we investigated if Dicer-independent shRNA structures embedded within pri-miRNA transcripts could elicit target knockdown when expressed from Pol II promoters. We demonstrate that both U1 and cytomegalovirus (CMV) promoter-derived miR-324 and miR-451 can be equipped with hairpins that potently target a gene of choice, without any passenger strand activity.

RESULTS
Identification of the Potent Dicer-Independent shRNA agsh12. 3 To optimize existing shRNA constructs, 26 we set out by re-configuring a potent shRNA from the T.J.C. lab for Dicer-independent processing. This shRNA, named sh9, is targeted toward site 9 in human (h) and murine (m) vascular endothelial growth factor (VEGF), and, expressed from the H1 promoter, it suppresses a transiently expressed reporter by $90%. 26 As illustrated in Figure 1A, the sh9 design is conventional, with a sense-loop-antisense structure, a 21-bp stem, and a frequently used UUCAAGAGA loop, 11 which likely extends the stem further.
Inspired by the work from others, 20,27 we designed two different Dicerindependent shRNAs, entitled agosh9 and agsh9 (see Figure 1A), based on the guide RNA strand from sh9 as predicted by the 5 0 counting rule for Dicer processing. 28 While agosh9 utilizes the 19/5 design as proposed by Liu and colleagues, 20 agsh9 mimics pre-miR-451 with an unpaired basal A-C fork 27 (Figure 1A). Dedicated luciferase reporters, sensitive to either guide or passenger strand release (T9-sense and T9-antisense, respectively; Figure S1A), demonstrated high activity of both sh9 strands when expressed from a modified U6 promoter 29 (see Figure S1B). As expected, both Dicer-independent shRNA designs eliminated passenger strand activity; however, in the context of VEGF target 9, neither of them retained effective target silencing ( Figure S1B). We also tested the activity of agosh9 and agosh9/3, a 19/3 variant with reduced loop size as described by Liu et al.,20 when expressed from the H1 promoter, and we compared it directly to the published pSUPERretro-based sh9 construct (see Figure S1C). While passenger strand activity was abolished from agosh9 and agosh9/3 (data not shown), we again observed only limited activity of the guide strand in our luciferase reporter assay ( Figure S1D).
We decided to proceed only with the agsh design, 27 due to the simplicity of design and the invariant 5 0 A nucleotide, which is ideal in terms of precision and efficacy of transcription when using the Pol III promoter U6. 30 Using a Renilla luciferase (Rluc) reporter fused to full-length cDNA sequence of mVEGF for screening knockdown efficiency, we cloned five additional VEGF targeting hairpins based on the agsh design in our pFRT-U6 vector 29 ( Figure 1B). Two of these agshRNA, entitled agsh12.3 (illustrated in Figure 1C) and agsh13, suppressed the Rluc reporter by approximately 90% ( Figure 1D). Given the potent knockdown efficacy, we next verified lack of passenger strand activity of the agsh12.3 construct by using luciferase reporters encompassing $100 nt of mVEGF sequence around the 12.3 target site in either sense or antisense orientation (T12-sense and T12-antisense reporter, respectively, as illustrated in Figure 1B). For comparison, we engineered a conventionally designed sh12.3 targeting the same VEGF sequence (illustrated in Figure 1C). As shown in Figure 1E, both designs effectively suppressed the T12-sense luciferase reporter, while the T12-antisense reporter, as expected, demonstrated an absence of passenger strand activity from agsh12.3. Northern blot analysis supported the absence of RNA in the size range of mature miRNA or small interfering RNA (siRNA) for the agsh12.3 construct ( Figure 1F).
Using probes complementary to the guide (antisense probe) or the passenger strand (sense probe), as illustrated in Figure 1G, we detected a prominent $30-nt band for agsh12.3, which is consistent with Ago2 processing ( Figure 1F, bands b and f). The antisense probe also detected a slightly shorter band (labeled c), which is consistent with 3 0 end tailing and PARN-directed trimming. 21,25,31 We note that our 17-nt locked nucleic acid (LNA)-doped sense probe, complementary to the entire agshRNA 3 0 stem region, may likely detect the 30-nt Ago2 cleavage product, which encompasses part of the 3 0 stem. Shortening the sense probe 4 nt in the 3 0 end reduced detection of the $30-nt band, although not fully (data not shown). The conventional sh12.3 gave rise to a precursor band of $35 nt ( Figure 1F, bands a and e), and it showed release of both guide and passenger strand species in the size range consistent with Dicer processing ( Figure 1F, bands d and g). Surprisingly, we could not detect the predicted fulllength sh12.3 precursor transcript ($49 nt), and we speculate that this may reflect aberrant processing due to overexpression.

Efficient Target Knockdown by agsh12.3 Embedded in Long Pol II-Expressed pri-miRNA Transcripts
Using the potent agsh12.3-mediated silencing of VEGF as a model for shRNA-encoded Dicer-independent RNAi, we next wanted to explore whether we could transfer the benefits of passenger strandfree knockdown from small Pol III transcripts to larger Pol II-expressed systems. Since the agsh design mimics miR-451 in terms of the stem length and the basal A-C fork, exploiting the flanking sequences of pri-miR-451 was an obvious choice (see Figure 2A). We decided to include five additional miRNAs as potential scaffolds for heterologous expression of agshRNAs (see Figure S2). All stem loop structures and prominent Drosha/Dicer cleavage sites shown in Figure S2 are based on data from miRBase 32 and the RNA secondary structure prediction algorithms MFOLD. 33 Human miR-215, miR-324, and miR-409 were chosen as they are predicted to have a bulge structure at the native Drosha cleavage site, which makes it convenient to design the pri-miRNA or agshRNA break point. Human miR-30a has often been used to derive potent shRNA-miRNA mimics, 34,35 and we have previously utilized the miR-106b cluster for the expression of shRNAs. 36 However, due to the sequence composition of miR-30a, we embedded the agsh12.3 part either to maintain the predicted secondary structure of miR-30a (miR30a-v1) or to mimic the bulge present in miR-451, which allows keeping the unpaired basal A-C fork (miR30a-v2). Similarly, we made a structural miR-106b mimic after using the natural Drosha 5 0 cut site as the break point (miR106b-v1) or keeping the A-C fork from agsh12.3 or miR-451 in an asymmetric or symmetric bulge (miR106b-v2 and -v3, respectively). We created two Pol II expression vectors based on the pcDNA3.1 with an identical multiple cloning site (MCS) flanked by either the human U1 small nuclear RNA (snRNA) promoter and a U1 termination signal or the CMV promoter and the bovine growth www.moleculartherapy.org hormone polyadenylation signal. Synthetic DNA fragments encoding the pri-miRNA-agshRNA chimeras, including $90to 130-nt flanking sequences, were cloned into the U1 or CMV expression vector.
Using the target 12.3 sensitive reporter T12-sense ( Figure 1B) in a cotransfection assay, we found potent knockdown from the U1-driven miR324-12.3 and miR451-12.3 vectors and intermediate activity from miR215-12.3 ( Figure 2B). Of note, overexpression of the wildtype miRNA transcript in some cases affected the activity of the internal Firefly luciferase (Fluc) control, as, for instance, reflected in the low Rluc:Fluc ratio for miR-451 in Figure 2B. Due to unspecific (and reporter-dependent) effects of miRNA overexpression in our dual luciferase reporter assay, we in all cases in this study estimated and report knockdown efficacy relative to the empty control.

3'
agosh9 agsh9 Predicted seed site Predicted seed site  26 based on a conventional sense-loop-antisense design (sh9, left side); Ago2-dependent designs, based on the 19/5 agoshRNA structure, as reported by the B. Berkhout lab 20 (agosh9, middle part); or the agshRNA design, as described by the Yen group 27 (agsh9, right side). Arrows indicate predicted Dicer and Ago2 cleavage sites, and position 2-7 of the guide RNA (seed site) is shown with braces. The guide and passenger strands are depicted in green and red, respectively. The frequently used Brummelkamp loop 11 is shown in blue. The invariant unpaired A-C fork, as also present in miR-451, is highlighted in purple. (B) Schematic diagram of the psiCHECK2-based reporter encoding a Renilla luciferase-mVEGF cDNA fusion transcript (used for screening purposes) and the dedicated T12-sense or T12-antisense reporters (used for testing guide and passenger strand activity, respectively), which are used in combination with the various U6-driven shRNA expression plasmids. Name (T9, target site 9) and location (484, 3 0 end of target site in the cDNA) of six chosen targets are indicated at the top. The illustration at the bottom shows the 105-nt-long cDNA region of mVEGF (dubbed T12) encompassing site 12, which was cloned into the psiCHECK2-based reporter shown above in sense or antisense orientation (numbers indicate cDNA position). (C) Predicted secondary structure of the conventional sense-loop-antisense sh12.3 (left side) and the Ago2-dependent agsh12.3 (right side) targeting site 12.3 in VEGF. Arrows and colors as indicated in (A). (D) Knockdown activity of agshRNAs designed to target the mVEGF sites, estimated by co-transfection with the mVEGF-fused reporter, as illustrated in the top part of (B). Renilla luciferase (Rluc) and Firefly luciferase (Fluc) activities were measured in relative units of light (RLU). The Rluc:Fluc ratio was normalized to the empty control (black bar) and plotted as the mean of three replicates plus SDs. An irrelevant agshIRR was included as a non-targeting control (gray bar). (E) Guide and passenger strand activity for agsh12.3 and a conventionally designed shRNA predicted to target site 12.3 using the dedicated T12-sense and T12-antisense reporters, respectively (see B). The Rluc:Fluc ratio was normalized to the empty control and plotted as the mean of three replicates plus SDs. (F) Northern blot analysis of small RNA from transfected HEK293 cells using probes detecting guide strand RNA (antisense probe, autoradiogram on the left side) or passenger strand RNA (sense probe, autoradiogram on the right side). Size of 20-, 30-, and 40-nt bands of the RNA decade marker is indicated, and agsh12.3-and sh12.3-specific bands are marked by arrows and labeled a-g. A cropped image with detection of the native U6 snRNA band (loading control) is shown below. (G) Diagram indicating probe specificity. Antisense probe, light green; sense probe, light red; Fluc, Firefly luciferase; HSV-tk, Herpes simplex virus thymidine kinase promoter; mVEGF, murine vascular endothelial growth factor; pA, polyadenylation signal; Rluc, Renilla luciferase; shRNA, short hairpin RNA; SV40, simian virus 40 promoter; T6, T-rich Pol III termination signal; U6, human U6 snRNA promoter.
Embedding the agsh12.3 hairpin crude (v1) in pri-miR-30a, -106b, or -409 showed limited knockdown of the reporter, although mimicking the symmetric loop at the Drosha cleavage site improved silencing substantially (mir30a-v2 and miR106b-v3). Screening of our panel of 12.3 targeting miRNA-agshRNA chimeras expressed from the CMV promoter showed the same tendency and rank as the U1-driven transcripts ( Figure S3A; data not shown).
Based on these results we decided to proceed with the miR-324 and miR-451 scaffolds only. First, we investigated if the miRNA-agshRNA vectors retained a lack of passenger strand activity, as demonstrated for the U6-expressed version. Using the T12-antisense reporter (Figure 1B), both the U1-and CMV-expressed vectors showed no sign of active passenger strand release ( Figure 2C; Figure S3B). Northern blot analysis using the sense probe (as used to detect passenger strand release for the U6-driven hairpins) detected a prominent $30-nt band for both miRNA scaffolds ( Figure 2D, band c). This is consistent with Drosha processing of the primary miRNA transcript and cleavage of an agshRNA-like molecule by Ago2, which in theory would leave eight bases in the 3 0 end for (partial) hybridization with our sense probe. Importantly, no miRNA or siRNA-sized bands were detectable with the sense probe. The antisense probe ( Figure 2D, left side) detected two specific bands of processed (guide) RNAs, which we estimated to be approximately 29-30 nt and $25 nt long (bands a and b, respectively). This is consistent with detection of the Ago2-cleavage product (band a), the 3 0 -trimming-tailing processes, 21,25,31 and the generation of shorter RNA species (band b) not detected by the sense probe. However, the resolution does not allow unequivocal distinction between the sizes of band a and band c, and we cannot rule out that these bands have undergone partial 3 0 trimming or tailing. Finally, we verified the targeting ability of the U1-miR451-12.3 construct in an independent assay using western blot analysis of cellular and secreted murine VEGF in co-transfected HEK293 cells, which resulted in substantial knockdown ( Figure 2E).
Functional knockdown and detection of siRNA-sized species strongly suggested that the miRNA-agshRNA transcripts mature in the nucleus by the Drosha/DGCR8 complex. If the native Drosha cleavage site is faithfully used, a short pre-miR-451-or U6-agshRNA-like hairpin-structured intermediate is released, which may subsequently be exported and processed further in the cytoplasm in accordance with the non-canonical Ago2-dependent pathway (as illustrated in Figure 2A). Hence, we would predict functionality of the miRNA-agshRNA system to be completely independent of Dicer.

Northern blot analysis (293T cells)
U6 control  To test this, we took advantage of 293T-derived Dicer-knockout cells 37 (NoDice 2-20 and 4-25) for our co-transfection assays. As expected, the U6-expressed agsh12.3 construct silenced the T12-sense reporter just as efficiently in Dicer-deficient cells as the parental 293T cells ( Figure 3A; data not shown for NoDice 4-25 cells). In contrast, the conventional sh12.3 lost activity in Dicer-deficient cells, albeit only partially using our standard experimental conditions. The ability of sh12.3 to silence the reporter target in the absence of Dicer could be a result of miR-451-like Ago2-dependent processing, or it may also be a result of sheer overloading and aberrant shRNA processing into antisense molecules acting in a RISC-independent manner. Lowering the shRNA plasmid DNA dose from 34 to 0.5 ng resulted in a more prominent loss of knockdown activity in Dicer-deficient cells ( Figure 3A, hatched bars), which tentatively argues that aberrant processing may be in play. As predicted, the Pol II-driven miRNA-agshRNA vectors were equally potent in the absence of cellular Dicer activity ( Figure 3B; Figure S3C). Consistent with Dicer-independent processing, we obtained a similar pattern of bands in northern blots of RNA from transfected Dicerknockout cells as for parental 293T cells (compare Figure 2D   Based on the potent silencing achieved with our VEGF-targeting miRNA-agshRNA vectors, we next wanted to examine whether the miRNA-based platform could be generalized and used as scaffolds for insertion of heterologous (Dicer-independent) shRNAs designed to target other genes. We took advantage of two published and potent hairpins based on the agshRNA design scheme (agsh887 and agsh1354), both targeting the Ribonucleotide reductase regulatory subunit M2 (RRM2) gene. 27 As for our VEGF-targeting agsh12.3 hairpin, we simply replaced the endogenous pre-miRNA sequence of miR-324 or miR-451 with the agsh887 and agsh1354 structure, modifying only the 3 0 UU tail to either GG or UG, respectively, to maintain base pairing of the distal part of the natural stem. We also included the miR-215 and miR-409 scaffolds, which in the context of VEGF silencing showed moderate or no activity, respectively.
To assess the potency of these constructs, we designed luciferase reporters encompassing a minimal-sized target region for RRM2 target 887 and 1354, and we co-transfected these with the corresponding U1-miRNA-agshRNA constructs. Indeed, miR-324 and miR-451 again showed efficient target silencing. In the context of the RRM2 targets, miR-215 and miR-409 showed improved knockdown performance as compared to silencing of VEGF ( Figure 4A). Of note, the RRM2 target 1354, is highly compatible with our Pol II expression system, and it displays a hyper-potent knockdown reminiscent of Pol III-based expression.
To further study the targeting flexibility of the Pol II-expressed miRNA-agshRNA system, we decided to test if a conventionally designed (and Pol III-expressed) shRNA could be directly converted for our U1-or CMV-driven miRNA scaffolds ( Figure 4B). We took advantage of a previously published shRNA construct (shS1) that potently targets the HIV-1 tat-rev transcript (site 1). 36 As illustrated in Figure 4C, the shS1 has a conventional sense-loop-antisense design and an elongated stem of 27 bp. Based on the predicted guide strand of shS1, we designed miR324-and miR451-agshS1 vectors (Figure 4C). Both miRNA-agshS1 vectors potently suppressed the HIV-1 site 1-fused luciferase reporter, and they showed comparable efficacy when using either the U1 or the CMV promoter ( Figure 4D). A knockdown efficacy of approximately 90% is comparable to the potency of the published shRNA construct (shS1) using our experimental conditions ( Figure S5).

DISCUSSION
Vector-encoded shRNAs are often used for gene therapy or long-term gene suppression directed by the encoded guide strand. However, unwanted silencing of off-targets remains an important side effect that needs careful attention for any RNAi application. Conventional shRNA design inherently co-delivers a passenger strand with potential off-target-silencing capacity, 38 and several strategies to limit this unwanted effect have been reported. These include the following: (1) co-expressing tough decoys to sequester or sponge for the passenger strand, 39 (2) adapting structural features such as bulges to favor accurate Dicer processing and asymmetric loading, 17,40 and (3)

5'
Inferred Ago2 cut In accordance with previous studies, 20,27 we have designed and tested two designs for Pol III-based expression of Dicer-independent shRNAs. Using strand-specific reporters and northern blot analysis, we confirmed the lack of passenger strand activity. In terms of optimal target specificity, it is preferable to have exact processing and, if possible, release of a single guide strand species with high silencing potency. For Ago2-matured shRNA vectors, the 5 0 end of the guide strand, which dictates target recognition and seed site match, is dependent on precise transcriptional initiation. The U6 promoter initiates on A or G as compared to the more sloppy H1 promoter. 30 Conventional shRNAs, encoding the guide strand on the 3 0 arm, in contrast, rely on Dicer cleavage for 5 0 end determination, a process that may be imprecise. 40,42 We favor the agshRNA design 27 in combination with the U6 promoter, since the invariant 5 0 A nucleotide (in the A-C fork) (1) likely favors accurate transcriptional initiation, 30,43 (2) reflects the abundant presence of 5 0 As in mature miRNAs 44 and may enhance Ago2 binding, 45,46 and (3) imitates the pre-miR-451 structure.
Elimination of the passenger strand may not only alleviate off-target activity but also potentially improve safety by limiting overloading of RISC-Argonaute proteins and Dicer, thereby reducing cell toxicity www.moleculartherapy.org and competition with endogenous miRNAs. [47][48][49][50] Hence, it is crucial to identify hyper-potent target sites and not least control the shRNA dose. Balanced expression using Pol II promoters, combined with tight processing and nuclear export, has been suggested to improve safety as compared to strong Pol III-based systems. 51 By incorporating flanking pri-miRNA sequences, we are the first to demonstrate that Dicer-independent shRNAs can be adapted for expression using Pol II promoters. Among six different miRNA scaffolds tested, we demonstrated that miR-324 and -451 can readily be equipped with shRNAs based on the agshRNA design 27 and direct potent target suppression. Notably, the miRNA-agshRNA system showed no passenger strand activity, as suggested by northern blot analysis and strand-specific reporters.
The Pol II miRNA-agshRNA transcripts are presumably cleaved by Drosha and Ago2 and trimmed by PARN, in accordance with miR-451 biogenesis and Dicer-independent shRNAs. 21,25,31 In support of this notion, we found that the miRNA-agshRNA-mediated knockdown and the pattern of RNA processing were unaffected in 293Tknockout cells lacking Dicer. Imprecise Drosha cleavage could potentially result in the release of hairpin structures with a stem length %20 bp and, hence, canonical shRNA properties. 52,53 The efficient on-target knockdown, the complete lack of passenger strand activity in reporter assays, and the absence of siRNA-sized species on northern blots when probing for the passenger strand collectively suggest that the Drosha/DGCR8 complex releases an agshRNA-like structure from both the miR-324 and the miR-451 scaffolds. Exact mapping of Drosha cleavage sites is currently ongoing and requires experimental validation, such as deep sequencing. Northern blot analysis using a guide strand-specific probe indicates that both the miR-324-and miR-451-derived agshRNA transcripts give rise to two abundant RNA classes with the approximate sizes of 25 nt and 29-30 nt. This is consistent with the trimming-tailing processes of miR-451 and supported by processing studies of artificial substrates, which led to mainly 23-to 26-mer guide RNA species. 21,27,31,54,55 While structural features and sequence composition in the pre-miRNA region may influence precision and efficiency of Drosha, [56][57][58][59] we believe that the miR-324 and -451-based vectors are robust for pre-miRNA replacement, as all the artificial agshRNA structures we tested resulted in efficient target suppression.
We have used the viral CMV promoter and the U1 snRNA promoter to drive miRNA-agshRNA expression. Both promoters display a comparable efficacy in terms of target knockdown. The ability to control Dicer-independent shRNA expression by Pol II promoters evidently allows researchers to use a wide range of application-specific promoters. We have achieved doxycycline-inducible miRNA-agshRNA expression using the CMV-TetO promoter in T-REx-293 cells, and we restricted miRNA-agshRNA-mediated knockdown to retinal pigment epithelium (RPE) cells by utilizing the VMD2 promoter (L.A. and T.J.C., unpublished data).
We believe that our novel Pol II-based miRNA-agshRNA vectors are of general use, as we successfully silenced four independent targets, and may support efficient knockdown of previously identified shRNA targets by simply converting the design. Parameters underlying efficient target suppression by Dicer-independent shRNA are not fully understood, and identifying potent targets sites may be a trial-anderror process. As also noted in the recent review by Herrera-Carrillo and Berkhout, 25 current algorithms for predicting efficient siRNAs are not suited for the design of Dicer-independent shRNAs. A very recent paper suggests that synthetic sli-siRNAs (dependent on Ago2 slicing) are more potent than conventional siRNAs but may also be more mismatch tolerant and, hence, display increased off-targeting. 54 It remains to be established if this is also the case for vectorencoded Dicer-independent shRNAs and for our Pol II-based miRNA-agshRNA vectors in particular. We speculate that our miRNA-agshRNA vectors may favor RISC incorporation compared to previous Dicer-independent shRNAs, as Drosha cleavage results in a pre-miR-451-like agshRNA structure with a 5 0 phosphate group in contrast to the triphosphate group present on Pol III-expressed transcripts. [60][61][62][63] Moreover, if Drosha processes our miRNA-agshRNA accurately, well-defined 3 0 overhangs may also facilitate handoff to Ago2, as compared to the 3 0 end of Pol III transcripts with varying numbers of uracils, 61,64 although the details of RISC loading for this non-canonical pathway remain to be clarified.
In summary, we demonstrate that U1-and CMV-driven miR-324 and miR-451 can be equipped with Dicer-independent shRNAs and elicit efficient target knockdown, without concomitant passenger strand activity. Combining our miRNA-agshRNA system with the plethora of alternative Pol II promoters would facilitate vectorization and allow restricted or controlled expression, which may advance the safe and efficient use of Dicer-independent shRNAs for fundamental research, biotechnology applications, or therapeutic purposes.

Plasmid Construction
RNAi targets were cloned into the psiCHECK2.2 vector (Promega, Nacka, Sweden) using the multiple cloning site in the 3 0 UTR of the SV40-RLuc gene cassette, using standard cloning procedures. Two $100-nt regions of the mouse VEGF gene (cDNA positions 300-405 and 463-563) were PCR amplified using NotI-tagged primers and psiCHECK-mVEGF as template, 26 and they were ligated into NotI-linearized psiCHECK.2 plasmid in both orientations to generate sense and antisense reporters with target sites surrounding VEGF site 9 and site 12. The resulting reporter plasmids are abbreviated psiCHECK-mVEGF-T9-sense and -antisense and psiCHECK-mVEGF-T12-sense and antisense, respectively (see Figures 1B and  S1B). Reporters for the RRM2 gene were constructed on the basis of the reported sequence of agsh887 and agsh1354 27 and cloning complementary target sequences into XhoI/NotI-linearized psiCHECK2.2 plasmid. Oligonucleotide cassettes bearing XhoIand NotI-compatible 4-nt overhangs at each end were generated by annealing two in vitro-phosphorylated partially overlapping oligonucleotides, as described previously. 65 psiCHECK.2-HIV-S1 and psiCHECK-mVEGF have been described previously. 26,36 The shRNA expression vector pFRT-U6 harbors the human RNA Pol III promoter U6 modified at positions -2 and -4 to create a BglII site that allows direct oligonucleotide-mediated cloning of shRNAs. 29 The shRNA constructs were cloned directionally into BglII/XhoI sites of pFRT-U6 or pSUPER.retro.puro 26 as double-stranded oligonucleotide cassettes with 4-nt compatible overhangs, as described previously. 65 The pri-miRNA expression vectors pcDNA3-CMV-MCS and pcDNA3-U1-MCS harbor the viral Pol II CMV promoter and the human snRNA promoter U1, respectively. pcDNA3-CMV-MCS was constructed from pcDNA3.1+1 (Invitrogen, Life Technologies, Carlsbad, CA) by cloning an oligonucleotide cassette into the NheI/ ApaI sites, creating a new MCS with the following unique restriction sites (5 0 -3 0 order): NheI, XhoI, BamHI, EcoRI, BspEI, AgeI, and ApaI. pcDNA3-U1-MCS was made from pcDNA3.1+1 by substituting the MluI/ApaI fragment holding the CMV promoter with a PCR amplicon harboring the U1 promoter, an MCS, and the U1 terminator, prepared using the pSK-U1-MCS-U1stop plasmid as template (generous gift from the J.J. Rossi lab, City of Hope).
For expression of the endogenous human pri-miR-30a, pri-miR-106b, and pri-miR-451 genes, we amplified and cloned 255-, 281-, and 243-nt regions, respectively, from human genomic DNA using XhoI-tagged forward primer and AgeI-tagged reverse primer for directional cloning into the MCS of pcDNA3-CMV-MCS and pcDNA3-U1-MCS. Chimeric miRNA-agshRNA sequences were purchased as synthetic DNA (GenScript, Piscataway, NJ) in the form of XhoIand AgeI-flanked inserts in the pUC57 plasmid, and they were subcloned into the MCS of pcDNA3-CMV-MCS and pcDNA3-U1-MCS. We included 80-120-nt 5 0 and 3 0 pri-miRNA-flanking sequences similar to the endogenous control genes. All primers were purchased from MWG/Eurofins (Ebersberg, Germany) or TAG Copenhagen (Frederiksberg, Denmark), and oligonucleotide and synthetic DNA sequences are available in Table S1. All vector plasmids have been verified by sequencing of inserts and restriction enzyme digest.
Dual Luciferase Assay HEK293, 293T, or 293T-derived Dcr KO 37 (NoDice 2-20 or 4-25) cells were seeded at a density of 3,000 cells/well in white 96-well plates (Nunclon Delta surface, Thermo Scientific, Denmark). DNA co-transfections were carried out the following day using 34 ng (or 0.5 ng when indicated) shRNA-expressing plasmid and 6 ng psiCHECK.2 reporter plasmid, using X-tremeGENE 9 (Roche, Basel, Switzerland), according to the manufacturer's protocol. At 2 days after transfection, Rluc and Fluc expression levels were analyzed using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA), according to the manufacturer's protocol. A multisample platereading luminometer (MicroLuminat plus LB 96V, Berthold Technologies, Bad Wild-bad, Germany) was used for measurements of the luminescence. Rluc expression levels were normalized to the Fluc expression levels and presented relative to a negative control. At least three biological replicates were made for all experiments.

VEGF Detection by Western Blot Analysis
HEK293 cells were seeded in 6-well plates at approximately 15% confluency. The next day cells were transfected with 2.1 mg DNA/well (700 ng pT2/CMV-mVEGF164-SV40-neo plasmid 26 encoding mVEGF and 1,400 ng plasmid encoding the RNA species) using X-tremeGENE 9 transfection reagent at a DNA-to-transfection reagent ratio of 1:3, according to the manufacturer's protocol. At 24 hr post-transfection, media were changed to serum-free media. Approximately 48 hr post-transfection, cells and media were harvested. Media were filtered through a 0.20-mM filter (Sarstedt, Nürnbrecht, Germany). Cells were lysed using 150 mL RIPA Lysis and Extraction Buffer/well (Thermo Scientific) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Sigma-Aldrich), according to the manufacturer's protocol, except that incubation time on ice was 15 min. Total protein concentration of the lysates was determined using Bio-Rad protein assay. 40 mg cell lysates and 32 mL media were electrophoresed on a Mini-PROTEAN TGX 4%-15% stain-free gel (Bio-Rad) in 1Â Tris-Glycin-SDS (TGS) buffer (Bio-Rad) and activated for 45 s or two times 45 s using the ChemiDoc MP imaging system (Bio-Rad), before transfer to a polyvinylidene difluoride membrane (Bio-Rad) using the Trans-Blot Turbo Transfer system (Bio-Rad). Total protein was quantified after transfer using the ChemiDoc MP imaging system, and background was subtracted using Image Lab Software (Bio-Rad). The membrane was blocked in 5% w/v skim milk powder (WVR, Radnor, PA, USA) in 1Â Tris-buffered saline (TBS) buffer (Thermo Fisher Scientific) with 0.1% v/v Tween20 (Sigma-Aldrich) (TBS-T) for 1 hr at room temperature (RT), and it was incubated with rabbit anti-VEGF antibody (ab46154, Abcam, Cambridge, UK) at a concentration of 1:2,000 overnight at 4 C with constant rocking. The membrane was washed 3 times 5 min in TBS-T before incubation with the secondary horseradish peroxidase (HRP)-conjugated goat-anti-rabbit antibody (Dako, Glostrup, Denmark) for 1 hr at RT. The membrane was washed again, and detection was done using the Clarity Western ECL substrate (Bio-Rad). Imaging was done using the ChemiDoc MP imaging system.

Small RNA Purification and Northern Blot Analysis
For detection of the U6-expressed agsh12.3 and sh12.3, HEK293 cells were transfected with 0.094 pmol inhibitor DNA plasmid and 30 ng GFP DNA plasmid in 6-well plates at approximately 15% confluency. For detection of the U1-expressed transcripts 293T or 293T-derived Dcr KO, 37 (2-20 and 4-25) cells were transfected with 26.25 mg DNA in culture flasks (surface area, 75 cm 2 ) at approximately 15% or 20% confluency, respectively, in all cases using X-tremeGENE 9 (Roche Applied Science). Media were replaced after 24 hr.
RNA enriched for small RNAs was isolated 48 hr after transfection using the mirVana miRNA Isolation Kit (Ambion, Austin, TX). For detection of the U1-expressed transcripts, RNA samples were precipitated and resolubilized in 22 mL Gel Loading Buffer II (Ambion), and 10 mL of each sample was electrophoresed in two separate lanes. For the detection of U6-driven siRNA expression, 250 ng small enriched RNA was electrophoresed. Eletrophoresis was done in 10% denaturing polyacrylamide gels (Criterion Precast gel 10% Tris-borate-EDTA [TBE]-urea; Bio-Rad, Copenhagen, Denmark) in 1Â TBE buffer for 55-60 min at 150 V, together with a RNA size marker (decade marker RNA; Ambion). The RNA was transferred to a positively charged nitrocellulose membrane, Amersham Hybond-N (GE Healthcare, Little Chalfont, UK) by electroblotting (Trans-blot SD semi-dry transfer cell; Bio-Rad). Ultraviolet-fixed membranes were hybridized overnight in PerfectHyb solution (Sigma-Aldrich, St. Louis, MO) at 42 C with purified 5 0 32P-labeled oligos complementary to the guide strand, the passenger strand, or U6 snRNA. The oligonucleotide probes (10 pmol) were end-labeled with polynucleotide kinase (PNK) (10 units; New England Biolabs) and y32P-ATP (10 mCi) in 10-mL reactions for 1 hr, and they were purified with G-25 Sephadex spincolumns (GE Healthcare). Hybridized membranes with LNA-doped oligonucleotide probes were washed three times with 6Â salinesodium citrate (SSC)/0.2% SDS and then twice with 2Â SSC/0.1% SDS, both at 38 C, before exposure to film (CL-XPosure Film, Rockford, IL). Hybridized membranes with DNA oligonucleotide probes were washed twice with 6Â SSC/0.2% SDS at 38 C, before exposure to film. DNA probes were purchased from MWG/Eurofins (Ebersberg, Germany), and LNA-doped oligonucleotide probes were purchased from QIAGEN (Copenhagen, Denmark). Probe sequences are available in Table S1.

Statistical Analysis
Data are presented as the mean ± SD. Statistical differences between two groups were evaluated using a two-tailed Student's t test assuming equal variance. p < 0.05 was considered statistically significant.

SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures and one table and can be found with this article online at https://doi.org/10.1016/j.omtn. 2018.11.013.

AUTHOR CONTRIBUTIONS
E.K. and L.A. designed the RNAi vectors, and E.K., C.R.C., and S.A. carried out luciferase assays. S.A. performed northern blot and western blot analyses. The Dcr KO cells were a kind gift from Bryan Cullen. The study was conceived by L.A. and supervised by C.K.D., T.J.C., and L.A. The manuscript was written by L.A., and it was reviewed and edited by E.K., S.A., C.K.D., and T.J.C. All authors read and approved the manuscript.

CONFLICTS OF INTEREST
The authors declare no competing interests.


extfieldsofstudy: Biology Medicine

text:
A giant dapediid from the Late Triassic of Switzerland and insights into neopterygian phylogeny

A new Triassic neopterygian is described on the basis of a large three-dimensional neurocranium from the Rhaetian (Late Triassic) of the Kössen Formation (Schesaplana, Grisons, Switzerland). CT scanning reveals neurocranial features similar to Dapedium, suggesting that this new genus, Scopulipiscis saxciput gen. et sp. nov., was deep-bodied and potentially durophagous, although no associated dental material is known. An expanded phylogenetic analysis of actinopterygians resolves Dapediidae as a clade (inclusive of Tetragonolepis), although fails to recover any characters supporting the monophyly of the genus Dapedium. Dapediids are resolved as stem holosteans, filling a conspicuous gap in early neopterygian relationships. Pycnodonts, previously suggested as either stem teleosts or the sister group to dapediids, are resolved as a clade on the neopterygian stem. Similarities between the new taxon described here and Dapedium provide insights into morphological disparity within early members of the group—suggesting that the ecological expansion of dapediids originated prior to the End-Triassic extinction—as well as contributing to a growing understanding of endocranial anatomy in Palaeozoic and Early Mesozoic actinopterygians.

Introduction
Ray-finned fishes (Actinopterygii) constitute the majority of living vertebrate diversity, displaying a huge disparity of body forms filling a wide variety of ecological niches [1,2]. Almost all extant actinopterygians (more than 30 000 species) belong to a single group, the Teleostei, with non-teleostean actinopterygians comprising just 56 living species [3]. The distribution of extant species belies past diversity: teleosts  the Cretaceous [4,5]. Indeed, their sister group, the holosteans, outstripped teleosts in terms of diversity and disparity in the earliest part of their history [5], although only eight species survive in the modern. Despite this, the understanding of relationships among early holosteans remains poor, in particular with regard to taxa branching outside of the living radiation. A bare stem is typically recovered in most analyses (e.g. [6][7][8][9]), although a stem holostean identity has recently been suggested for Dapedium [10].
Representing an early experiment in body form and ecological diversity, Dapedium is a modestly diverse (approx. 20 species) radiation of neopterygians known from the Late Triassic (Rhaetian) to mid Jurassic (earliest Aalenian), most notable for its adaptations for durophagy. Members of this genus possess thick, interlocking ganoid scales; dense, pustular ornament; and a small mouth with styliform teeth borne on the stout lower jaw. Other genera closely associated with Dapedium, and typically united into Dapediidae (or Dapediiformes), include Aetholepis [11], Dandya [12], Hemicalypterus [13,14], Heterostrophus [15], Paradapedium [16], Sargodon [12] and Tetragonolepis [16,17], although the dapediid affinity of at least Heterostrophus and Dandya has been called into question [13,16]. Most Dapedium species have a standard length of around 15 -35 cm [18], although dapediids range in size from approximately 8 cm (Dapedium noricum [12]) to approximately 1 m (Sargodon tomicus [12]). Only around five of the 30 or so known species of dapediid are present in the Triassic, and are exclusively represented by isolated teeth [19] or flattened [12,14,20] remains. Consequently, their adaptation for durophagy is thought to have facilitated the group's success in the aftermath of the End-Triassic extinction, a time at which many other durophagous taxa went extinct [21,22].
The equivocal phylogenetic signal of dapediids is in part due to the conflicting datasets used to resolve relationships. The phylogenetically informative endoskeleton [32,52,53], in particular the braincase, is described for only a small number of dapediids (Dapedium punctatum, D. caelatum [18]), and exhaustively in just a single taxon (Dapedium sp. [27,38,40,54,55]). While three-dimensional crania are known in a number of taxa (e.g. Tetragonolepis semicincta [17]; Dapedium granulatum [22], the majority of dapediids are preserved as laterally compressed fossils, with heavy dermal ornament obscuring most of the internal skeleton. In particular, no neurocrania are described from the Triassic, when the group first appeared. Here we present a new, large (approx. 17 cm length) braincase from the Rhaetian (Late Triassic) of the Kö ssen Formation (Schesaplana, Grisons, Switzerland; figure 1). Marine reptiles and pterosaurs have previously been described from this locality [56][57][58]. Actinopterygian remains attributed to Birgeria have been described from a lateral extension of the formation into Austria [59], but no dapediids have previously been identified. This new specimen expands our knowledge of the taxonomic richness of the locality and increases the size range known for dapediids, with implications for the diversification of the group and the timing of their radiation. Furthermore, CT scanning of this well-preserved neurocranium presents an opportunity to revisit the phylogenetic affinities of the group.

Phylogenetic analysis
Our dataset is based on Giles et al. [8], with 25 additional characters both novel and taken from the literature [10,14,33,36,45,46,49]. Eighteen taxa were added to the matrix in order to increase the sampling of dapediids and investigate the relationship of pycnodonts to other neopterygians. Brachydegma caelatum was removed pending a redescription of this taxon. This gives a total of 291 characters and 110 taxa. Full details are given in the electronic supplementary material. An equally weighted parsimony analysis was conducted using a heuristic search in PAUP* v. 4.0a158 [60] with the following settings: 1000 random addition sequences, five trees held at each step, maxtrees set to automatically increase, nchuck ¼ 10 000, chuckscore ¼ 1, tree bisection and reconstruction strategy enabled. Seven characters (c.89, c.91, c.151, c.171, c.241, c.247, c.251) were ordered following Giles et al. [8]. Taxonomic equivalence [61] was assessed using Claddis [62]

Etymology
Generic name describes the marine deposits on the alpine cliff sides of Schesaplana Scopuli-from scopulus (Latin)-meaning a lookout place, cliff or crag, in or under the sea-, piscis (Latin) for fish. In the specific epithet, sax-(Latin) is for 'rock' and -ciput from caput (Latin) for head because the holotype specimen PIMUZ A/I 3026 is a well-ossified neurocranium.

Holotype
PIMUZ A/I 3026 (figure 2), a neurocranium and associated dermal elements infilled by a fine carbonate matrix including small (approx. 2-8 mm) bivalves. Collected in 1976 by Dr Heinz Furrer, PIMUZ, the specimen was subsequently prepared mechanically.

Locality and horizon
The specimen was collected as float from the Kö ssen Formation of Schesaplana Mountain, in Canton Grisons/Graubü nden, Switzerland (figure 1), in the lowest part of the Alplihorn Member, Late Triassic [56], where the boundary between the Late Norian and Early Rhaetian is currently undefined (Heinz Furrer 2016, personal communication). Actinopterygians from the same locality include fragmentary elements and teeth from Birgeria [64] and isolated elements in the collections at PIMUZ assigned to Sargodon and Lepidotes. The Austrian portion of the Kö ssen Formation has produced other fish remains [59]. Other taxa from Schesaplana include marine reptiles [56,58] and pterosaurs [57].

Diagnosis
Large neopterygian with a well-ossified neurocranium and the following combination of characters: spiracular canal extending through the postorbital process to open on the ventral surface of the braincase; elongate hyomandibular facet approximately one-fourth of neurocranial length; pronounced median occipital crest, large sinus between lateral cranial canals, parasphenoid keel extending dorsally between orbits.

General
PIMUZ A/I 3026 preserves a partial skull roof and a near-complete neurocranium (17 cm preserved length), missing the nasal capsules and anterior portion of the orbit. There is some distortion, with the right orbit partially crushed and the basiocciput sheared slightly to the right. The skull roofing bones have also collapsed in the region of the anterior dorsal fontanelle. The complete ossification of the braincase and partial obliteration of dermal sutures suggests the cranium belonged to an adult specimen. Sutures between braincase ossifications are not apparent; the neurocranium appears to be ossified as a single unit. Some areas of perichondral bone were weathered away prior to collection, exposing cancellous bone, particularly in the occipital region and along the parasphenoid.

Skull roof
The dermal bones of the skull roof are heavily ornamented with pustular tubules, from 0.5 to 1 mm in diameter, radiating from ossification centres and increasing in size towards element margins (figures 2 and 3). The tubules are particularly large around the midline where the frontals and parietals meet. Such dense ornament means that sutures between bones are typically difficult to trace, although they  can sometimes be followed in CT scans. The frontal is relatively short, accounting for a little under half the length of the skull roof (fr, figure 3d). It is widest anterior to the parietal contact, and tapers anteriorly. There is no pineal foramen, and the frontals appear to be fused at the midline. Only a small fragment of the left nasal is preserved, but an unornamented area at the anteriormost point of the left frontal represents the overlap area for this bone (n.ov, figure 3d). A series of pores marks the passage of the supraorbital sensory canal through the frontal and into the parietal (soc, figure 3d ), and the path of the canal can be followed in CT sections. Very little of the midline suture is preserved between the elongate parietals ( pa, figure 3d ), which have partially collapsed into the anterior dorsal fontanelle (adf, figure 3c). The suture between the rectangular dermopterotic and parietal (dpt, figure 3d) is obscured by ornament externally, but can be traced in CT sections. However, it is unclear whether the dermopterotic contacts the frontal anteriorly. As with the supraorbital canal, the path of the otic sensory canal through the dermopterotic and into the dermosphenotic is indicated by a line of pores (ioc, figure 3d ), with the canal visible in CT slices.

Occipital region
The occipital region is the deepest part of the braincase, with the basiocciput accounting for half the vertical height of the specimen (figures 4c,d and 5). It is narrower than either the otic or orbitotemporal   regions (figure 6a,b). A 'bulge' on the midline projects dorsally above the skull roof (bul, figures 4d and 5b,d ) and houses a large cavity within the perichondrium of the braincase (cav, figure 7a). The posterior face of the occiput is developed into a broad median occipital crest, punctuated by a large ligamentous pit (lig, figures 4d and 7a). A process lateral to this pit is pierced by small anastomosing branches for the occipital nerve (focn, figures 4d and 5d). Medial to the posterodorsal angle of the braincase is a deep posttemporal fossa ( ptf, figures 4d and 5b,d), which is continuous anteriorly with the fossa bridgei (fb, figure 4d). The foramen magnum is small and triangular, and both its floor and roof are well-ossified (fm, figures 4d and 7a). Two foramina for the occipital arteries open onto the lateral face of the braincase just above the parasphenoid (foca, figures 4d and 5d), with the smaller opening for the occipital nerve dorsal to these (focn, figures 4d and 5d). The notochordal facet is large and rounded (not, figure 4d), and leads into a narrow notochordal canal that pinches out approximately halfway along the length of the basiocciput (not, figures 7a,b and 8b). Ventral to the opening for the notochord is the circular aortic canal, which is enclosed ventrally by the parasphenoid (aort, figures 4d, 6b and 7a). A dorsally directed canal leaves the dorsal margin of the aorta to open on the lateral wall of the braincase above the parasphenoid. There is no indication of either a posterior dorsal fontanelle or otoccipital fissure.

Otic region
The otic region is the widest part of the braincase ( figure 6a,b). Much of its dorsal surface is unossified to accommodate the large anterior dorsal fontanelle, which extends from the posterior margin of the hyoid facet to above the orbits (adf, figures 3c, 4b and 9b ). Lateral to the fontanelle is an excavation for the fossa bridgei (fb, figures 8b and 9), which extends posteriorly to the post-temporal fossa and anteriorly to the  postorbital process. The lateral margin of the fossa bridgei is strongly concave, wrapping around the hyomandibular facet. The medial margin is convex, with multiple small projections giving an irregular edge. A total of five canals connect with the fossa bridgei. Two canals issue from its anteromedial corner. The more ventral, for the spiracular canal, enters the top of the postorbital process and travels along its entire length, opening just posterior to the lateral commissure on the ventral surface of the braincase (spic, figures 5b, 6b and 9e ). On the left side, the lateral commissure is broken, and consequently this canal appears to open midway along its length. The more dorsal of the two anteromedial canals opens into the posterolateral corner of the orbit, just below the orbital roof, and transmitted the otic nerve (otn, figures 4a, 6b,d and 8b). An additional canal leaves more posteriorly from the medial margin and opens into the roof of the hyomandibular facet and may have transmitted the recurrent lateralis branch of the facial nerve (frla, figures 6b and 8b). Two narrow canals exit the anterolateral corner to open on the side of braincase just below the skull roof, and probably housed branches of the otic nerve (otn, figures 5b,d, 8b and 9b,e). Dorsal to the fossa bridgei is a separate small cavity, which also lies within the perichondrium of the braincase. Owing to specimen preservation, the chamber is only fully observable on the right side. The chamber has a medial connection to the orbit, opening into the dorsolateral corner of the posterior wall (soph2, figure 4b). An additional small canal leaves the lateral margin of the chamber to open onto the lateral surface of the braincase dorsal to the otic nerve canals (ca, figure 5b). The articular facet for the hyomandibular is elongate and horizontally oriented (hmf, figures 5b,d and 6b), and a deep dilatator fossa lies dorsal to the hyoid articulation (dil, figures 5b,d and 6b). Ventral to the facet, the lateral wall of the braincase narrows towards the midline, and this face is pierced by a number of canals. The most posterior of these is the lenticular opening for the vagus nerve (X, figures 5b,d and 6b), positioned ventral to the posterior margin of the hyoid facet. A shallow depression between the vagus nerve and hyoid facet is the supratemporal fossa (stf, figures 5d and 6b), delimited anteriorly by a vertical strut. The glossopharyngeal nerve ( probably its supratemporal branch) exits the braincase via a small canal at the base of this strut (IXst, figures 5b,d and 6b), adjacent to an elliptical pit. This pit appears to be at least partly the result of mechanical preparation, as it punches through the perichondrium to reach the saccular chamber on one side, but comparison of both sides shows that the perichondral lining of the endocavity is complete, and there is no vestibular fontanelle. The original morphology of the pit is unclear, but it may represent an articular facet for the first suprapharyngobranchial (sup.ph, figure 5). A shallow groove for the jugular marks the lateral face of the braincase anterior to this facet, and can be traced to a foramen in the lateral commissure ( jug, figures 5b,d and 6b). A large opening on the ventral margin of the jugular groove marks the exit of a stout canal, which originates on the lateral face of the sacculus near its anterior margin. This canal may be the main trunk of the glossopharyngeal nerve (IX, figures 5b,d and 6b). More anteriorly, a large opening for the hyomandibular branch of the facial nerve (VIIfhm, figures 5b,d and 6b) pierces the roof of the jugular groove. The jugular vein and main trunk of the facial nerve continue through the postorbital process into the orbit.

Orbitotemporal region
The posterior margin of the orbitotemporal region is formed by the lateral commissure and postorbital process ( por, figures 4b, 5b,d and 6b,d). The lateral commissure is reduced to a thin splint laterally, and the extent to which it is covered by the ascending process of the parasphenoid is difficult to discern. The orbital walls are pierced by a complex anastomosing series of canals, some of which are difficult to identify.    Rendering (e,g) and interpretive drawing (f,h) of the basisphenoid and parasphenoid circulatory system in left lateral (e,f ) and ventral (g,h) view. Endocast in grey, canals in red. Scale bar is 50 mm. aa, anterior ampulla; anas, anastomosing canal; aort, dorsal aorta; asc, anterior semicircular canal; bhc, buccohypophyseal canal; can.bsp, unidentified canal in basisphenoid; cc, crus commune; epsa, efferent pseudobranchial; fb, fossa bridgei; foca, occipital artery; focn, occipital nerve; fm, foramen magnum; frla, recurrent lateralis branches of the facial nerve; ha, horizontal ampulla; hsc, horizontal semicircular canal; ica, internal carotid; lcc, lateral cranial canal; mcv, middle cerebral vein; myo, myodome; not, notochord; opa, ophthalmic artery; optl, optic lobe; otn, otic nerve; pa, posterior ampulla; pal, palatine artery; psc, posterior semicircular canal; pv, pituitary vein; sacc, sacculus; spic, spiracular canal; ssu, sinus superior; tel, telencephalon; utr, utriculus; v?, unknown vein; III/Vprof oculomotor nerve and profundus branch of the trigeminal nerve; IV, trochlear nerve; V, trigeminal nerve; VI, abducens nerve; VII, facial nerve; VIIfhm, hyomandibular branch of the facial nerve; IX, glossopharyngeal nerve; IXst, supratemporal branch of the glossopharyngeal nerve; X, vagus nerve. rsos.royalsocietypublishing.org R. Soc. open sci. 5: 180497 contact the visceral surface of the skull roof (soph, figures 4b and 6d). The two canals in the posterolateral corner of the orbit have different points of origin: the more ventral, for the otic nerve (otn, figures 4b, 6d, 8b and 9b,e), connects to the fossa bridgei; and the more dorsal, which may also carry branches of the superficial ophthalmic (soph2, figures 4b and 6d), to the chamber that lies above the fossa bridgei. Of the two canals in the posteromedial corner of the orbit, the more anterior and dorsal carries the trochlear nerve from the optic lobes (IV, figures 6d, 8b and 9b). The more posterior originates from the dorsal part of the midbrain, above and some way posterior to the trochlear nerve, but its identity is unclear (v?, figures 6d, 8b and 9e). Two canals open on the medial wall of the orbit via a single foramen. These canals have a single root on the forebrain on the right of the specimen and a double root on the left, and transmitted the oculomotor and profundus nerves into the orbit (III and Vprof, figures 6d and 8b). The final set of canals, approximately midway up the posterior orbital wall, is centred about the trigeminofacialis chamber (tfc, figure 4b), which is continuous posteroventrally with the jugular canal. Two canals exit from the same point on the anterior face of the utriculus before separating into separate canals. These canals enter the orbit separately, but both are continuous within the trigeminofacialis chamber. The more dorsal transmits the trigeminal nerve (V, figures 4b, 6d, 8b and 9e) and the more ventral the facial nerve (VII, figures 4b, 6d and 8b,f ). Two grooves extend from the dorsal margin of the chamber. The groove on the posterior margin of the orbit is short, with no canal at its dorsal limit. It terminates some way ventral and medial to any of the canals that connect to the skull roof, but may have transmitted the superficial ophthalmic nerves, which entered the orbit via the underlying chamber. The groove on the medial orbital wall is deeper, and a canal at its dorsal margin connects to the cranial cavity via two closely positioned openings. This probably transmitted the internal carotid artery (ica, figures 4b, 6d and 8d). A ventral connection links the trigeminofacialis chamber and the median posterior myodome (myo figures 4b, 6d and 8b,f,h) 4b, 6d and 8b,f,h). There is no obvious opening for the palatine branch of the facial nerve into the orbit. The braincase is broken midway along the orbit, and the skull roof has partially collapsed. A single opening on the midline represents where the braincase is broken through the endocranial cavity of the forebrain (endo, figures 4b and 6d); neither the exit of the optic (II) nor olfactory (I) nerve is preserved. The basisphenoid is well ossified, contributing to the medial walls and floor of the orbit, although the interorbital septum is formed entirely by the parasphenoid ( psp, figures 4b, 5d, 6d and 7a). The basipterygoid process, which has both a dermal and endoskeletal component, is robust. The internal carotid opens into the posterior margin of the basisphenoid via a foramen anterior to the posterior myodome. The subocular shelf is narrow and formed entirely by the parasphenoid.

Parasphenoid
The parasphenoid extends the full length of the preserved braincase, although a fragment is missing in the middle where the specimen is broken between the midpoint of the basiocciput and the postorbital process. The posterior margin of the parasphenoid is weathered, exposing the basioccipital, but a midline aortic notch appears to have been absent (figures 4 and 6). Although housed entirely within the basiocciput posteriorly, most of the floor of the aortic canal is formed by the parasphenoid (aort, figure 7a). As the parasphenoid is broken midway along the basiocciput, the nature of the bifurcation into the lateral aortae is unknown. A narrow median keel on the ventral surface of the parasphenoid (ke, figures 5b,d and 6b) is flanked by paired shallow grooves (gr, figures 5b and 6b); the depth of the left groove has been exaggerated during specimen preparation. As the dorsal and lateral aortae are enclosed within the braincase, the purpose of these grooves is unclear. The parasphenoid extends up the lateral walls of the braincase to the level of the foramen magnum, effectively cloaking the basiocciput. The ascending processes of the parasphenoid are narrow and strut-like, although it is difficult to assess how far they extend dorsally up the postorbital process. The basipterygoid processes are robust, projecting dorsally as well as laterally (bpt, figures 4b, 5b,d and 6b,d), and the ophthalmic artery pierces the orbital floor posteromedial to the process (opa, figures 4b, 5d and 6d,e). An opening at the anterior limit of the basipterygoid process marks the exit of the palatine nerve and artery from the basisphenoid ( pal, figures 4b, 5b,d and 6b,d,e). A V-shaped ridge is present on the ventral surface of the parasphenoid, and the efferent pseudobranchial artery exits posterior to where the two arms of the ridge converge on the midline (epsa, figures 5d and 6b,e). Anteriorly, the buccohypophyseal canal exits the parapshenoid on the midline (bhc, figure 6b). The internal carotid foramen is not preserved, and presumably entered the parasphenoid through the missing section. A broad parasphenoid keel extends dorsally between the orbits, forming the interorbital septum ( psp, figures 4b, 5d, 6d and 7a). No teeth are present on the parasphenoid.

Endocast
The regions of the endocranial cavity corresponding to the midbrain, hindbrain and bony labyrinth are almost completely preserved, although only a short portion corresponding to the narrow forebrain is present in the specimen (tel, figure 8). As the internal walls of the braincase are sometimes poorly mineralized (figure 7a), contrast between the braincase and infilling sediment is low, with the exact boundaries of some regions of the endocranial cavity difficult to discern.

Midbrain
The area of the optic lobes (midbrain) is large, representing the widest part of the endocast (optl, figure 8b). Much of the dorsal surface is unfinished, indicating the position of the extensive anterior dorsal fontanelle (adf, figure 8b). Two closely associated canals leave from the anterolateral face of the midbrain, entering the orbit via a single (on the right of the specimen) or double (on the left) opening. These probably transmitted the oculomotor and profundus nerves (III/Vprof, figure 8b). The trochlear nerve leaves the cranial cavity posteriorly and dorsally (IV, figures 8b and 9b).

Hindbrain
There is no clear division between the midbrain and hindbrain, although the portion of the hindbrain positioned behind the crus commune sits far ventral relative to the rest of the endocavity, perhaps indicating that the cerebellum projected into the fourth ventricle. Cerebellar auricles cannot be identified. Several canals exit the cranial cavity in the anterior part of the hindbrain. Dorsally, a stout canal transmits an unidentified vessel into the dorsal part of the orbit (v?, figure 8b). The middle cerebral vein exits the cranial cavity some way ventral to this (mcv, figure 8b), and the internal carotid enters more ventrally still (ica, figure 8b). The trigeminal and facial nerves exit from the angle between the midbrain and utricular recess (V and VII, figure 8b). Posterior to the labyrinth, the vagus nerve leaves the ventral margin of the hindbrain through a large, anteroposteriorly elongate canal (X, figure 8b). The region corresponding to the spinal cord is by far the narrowest portion of the endocast, and from its lateral margins issue a dorsal, bifurcating canal for the occipital nerves (focn, figures 8b and 9b,e ), and a ventral, single canal for the occipital artery (foca, figures 8b and 9b,e ).

Labyrinth
The walls of the bony labyrinth are fairly well ossified, except in the region of the sinus superior and anterior ampulla. The three semicircular canals are narrow, and all are fully enclosed in bone (asc, hsc, psc, figures 8b and 9b,e). Both the anterior and posterior canals are anteroposteriorly short, and the horizontal canal is almost isoclinal in its curvature (figure 9c). The orientation of the anterior ampulla is unclear (aa, figure 8b). The utriculus is dorsoventrally tall, and there is a slight suggestion of division into dorsal and ventral components (utr, figure 8b). A small saccular notch is present (sac, figure 8b). Although incompletely ossified, the sinus superior is long (ssu, figure 8d), and the crus commune projects above the roof of the hindbrain and slightly towards the midline (cc, figures 8d and 9b). The lateral cranial canal is very well developed (lcc, figures 8b and 9b,e ). As well as anterior and posterior connections to the cranial cavity, it has a dorsal extension that almost reaches the underside of the skull roof. At least a single canal, probably for the endolymphatic ducts (end.d, figure 7a), connects the cranial cavity to the median sinus positioned between the lateral cranial canals.

Circulatory system
The buccohypophyseal canal is elongate, leaving the anteriormost point of the posterior myodome and continuing anteroventrally (bhc, figure 8). A narrow canal branches from its lateral margin level with the exit of the internal carotids into the orbit (can.bsp: unidentified canal in the region of the basisphenoid, figure 8f,h). Further anteriorly, the buccohypophyseal canal bends dorsally in an 's' shape, before opening onto the ventral surface of the parasphenoid. The path of the internal carotid artery is incompletely known, largely due to the loss of part of the parasphenoid. It presumably entered the parasphenoid posterior to the basipterygoid process (?ica, figure 6e) and continued via an anterodorsally directed canal. This canal opens onto the orbital floor posteromedial to the basipterygoid processes (ica, figures 6e and 8f,h), and enters the cranial cavity through a foramen in a dorsally directed groove medial to the trigeminofacialis chamber (ica, figure 6d,e). The efferent pseudobranchial pierces the parasphenoid beneath the basipterygoid process (epsa, figures 5d, 6d and 8f,h) and is transmitted through a vertical canal, exiting onto the floor of the orbital as the ophthalmic artery (opa, figures 4b, 5b,d, 6d,e and 8f,h). The internal carotid and efferent pseudobranchial communicate via a short anastomosing canal (anas, figures 6e and 8f ), and are also linked via the parabasal canal ( pbc, figure 6e), which seems to have housed the palatine nerve and artery. The path of the palatine into the orbit is not known, but it appears to have entered the parabasal canal through the same opening as the internal carotid, and opens anteriorly on the lateral margin of the parasphenoid ( pal, figures 4b, 5b,d, 6b,d,e and 8f,h). The internal carotids and efferent pseudobranchials do not anastomose with their antimeres, but a narrow transverse canal connects the two ophthalmic arteries.

Body size
The neurocranium of Dapedium is inclined at approximately 508 from the horizontal in articulated specimens, and body size typically ranges from around 15 to 35 cm [18]. Skull length accounts for approximately 18% of body length in Dapedium, but accounts for a much smaller proportion (approx. 10%) in the largest known dapediids. Body length of a large Sargondon tomicus is estimated about 1 m [12], with absolute head length of a medium-sized specimen approximately 6 cm and of the large specimen approximately 10 cm [65].
The Although represented only by an isolated neurocranium, this specimen bears conspicuous similarities to dapediids, in particular the braincase described for Dapedium sp. [27,55]: short aortic canal, parasphenoid 'wings' cloaking the basiocciput, median occipital crest punctuated by a large ligamentous pit, parasphenoid extending dorsally between the orbits, fossa bridgei continuous posteriorly with the post-temporal fossa, median sinus between the lateral cranial canals, canals for endolymphatic ducts connecting the cranial cavity to the median sinus and absence of differentiated braincase ossifications. Similar likenesses are found with the endocast of Dapedium sp. [40], including a large lateral cranial canal extending dorsal to the endocranial roof, narrow semicircular canals and a lenticular utriculus. The majority of actinopterygian material from Schesaplana has been identified as either Birgeria or Lepidotes [66] although unpublished elements in the collections at PIMUZ have been referred to the giant dapediid taxon Sargodon (A. Latimer 2016, personal observation), known from the Late Triassic (Norian) of Italy [12]. The limited anatomical overlap between Sargodon and the braincase described here makes comparisons difficult: Sargodon is known from articulated but laterally compressed fossils, with no part of the braincase, and only small portions of the skull roof, described. Differences are evident in some of the few comparable regions, for example the parietals and dermopterotic of Sargodon are quadrate, and those of the specimen described here are markedly rectangular. Furthermore, the cranium of even the largest Sargodon specimen known is only around 10 cm in length. By contrast, the braincase described here is approximately 17 cm, and is incomplete anterior to the orbit, suggesting a total cranial length of at least 20 cm. Consequently, we feel justified in erecting a new genus and species for this specimen: Scopulipiscis saxciput. It is possible that undescribed cranial elements at PIMUZ belong to Scopulipiscis saxciput rather than Sargodon, as their identification was most probably based on size.
In the strict consensus tree of our phylogenetic analysis (figure 10), Scopulipiscis saxciput is resolved in a polytomy with all included Dapedium species, Heterostrophus phillipsi, Sargodon tomicus, Dandya ovalis, Hemicalypterus weiri, Paradapedium egertoni, Tetragonolepis oldhami and Tetragonolepis semicincta. This clade is supported by seven homoplastic characters: c.58, one or two suborbitals; c.156, braincase ossifications not differentiated; c.247, complete series of dorsal ridge scales; c.258, caudal fin not forked; c.269, suborbitals extend ventral to orbit; c.275, hem-like median fins; c.276, long-based anal fin. The monophyly of Dapedium is not upheld, and the genus is recovered as paraphyletic in both the Adams consensus tree and agreement subtree (electronic supplementary material, figure S1). Additional characters found in dapediid taxa to the exclusion of immediate outgroups, but which cannot be optimized due to uncertainty over the primitive condition in the clade, include: c.270, coronoids contribute to lateral dentition field; c.273, dorsal extension of parasphenoid between orbits; c.274, ligament pit on posterior face of braincase; c.278, ossified centra absent.
The failure to resolve Dapedium as monophyletic is hardly surprising, given that placement of material in this genus traditionally largely relied on the presence of a deep-bodied morphology, and differentiation of species on characters that could covary in individual specimens [12]. Doubts over the monophyly of the genus have been raised before [28], and it is possible that detailed investigation of Sinemurian material from Dorset may find further objections. Only four dapediid taxa are excluded in the agreement subtree (electronic supplementary material, figure S1a), suggesting that only some of the uncertainty stems from conflicting endoskeletal and dermal character sets. The monophyly of dapediids, in contrast, seems well supported, albeit with a different set of characters to those identified in previous phylogenetic analyses [14,37].

Placement of dapediids
Dapediids (along with Hulettia) form a clade of stem holosteans, sharing with other holosteans the following five homoplastic characters: c.89, two infradentaries; c.112, anterodorsal process of suboperculum; c.135, anterodorsal myodome single; c.173, parasphenoid with multifid anterior margin; c.178, aortic notch in parasphenoid absent. While dapediids have been recovered as total group holosteans before, they are more typically resolved as the sister group to ginglymodians [36,37]. A stem holostean position was recovered by Ló pez-Arbarello & Sferco [10] on the basis of two homoplastic characters (four or more supraorbitals and a presupracleithrum), neither of which are optimized as supporting this relationship in the current analysis. A position deep on the holostean stem goes some way to explaining rsos.royalsocietypublishing.org R. Soc. open sci. 5: 180497 the peculiar combination of apparent holostean and teleost features that have influenced past hypotheses of phylogenetic relationships.
The position of Hulettia as sister taxon to dapediids, with the support of three homoplastic characters (c.247, basal fulcra on dorsal fin; c.249, scutes anterior to anal fin; c.284, preoperculum shorter than operculum), is unexpected. We note that Hulettia shows large numbers of character reversals (for the full list of character optimizations see electronic supplementary material, Supplementary Data), suggesting that this position may not be robust. However, Hulettia has previously been associated with Dapedium [32]. Macrepistius is resolved as sister to all other holosteans, supported by one unambiguous (c.10, olfactory nerve pierces premaxilla) and five homoplastic characters (c.14, single median dermal bone capping snout; c.50, tube-like canal bearing anterior arm of antorbital; c.61, circumorbital ring of supraorbitals and infraorbitals closes the orbit; v.107, paired vomer; c.161, sphenotic with small dermal component). The holostean crown is united by just three homoplastic characters (c.175, parasphenoid teeth small; c.176, parasphenoid not pierced by internal carotid; c.272, parasphenoid wings around basiocciput absent), indicating that features previously identified as uniting the living radiations may in fact be more widely distributed across the total group.

Placement of pycnodonts
Pycnodonts are recovered as stem neopterygians in our analysis. The monophyly of pycnodonts is robustly supported by one unambiguous (c.282, prearticular symphysis) and thirteen homoplastic characters. Discoserra is recovered as the immediate outgroup of pycnodonts, with a clade comprising Bobasatrania þ Ebenequa falling as the sister taxon. While this broader clade is supported by five homoplastic characters, we do not regard it as robust: at least some of the characters optimized as supporting this group (e.g. c.276, long-based anal fin) probably reflect anatomical similarities related to a deep-bodied morphology rather than a close phylogenetic relationship. Furthermore, Platysomus is resolved as sister taxon to an unlikely clade comprising cladistians and chondrosteans. These incongruent placements highlight that further work is needed to resolve the relationships of other radiations of deep-bodied Palaeozoic fishes.
A limited understanding of the internal skeleton of pycnodonts-and those taxa recovered as closely related-may also be a contributing factor. Despite an extensive fossil record stretching from the Late Triassic to the Eocene, few pycnodont neurocrania are described: the most comprehensive description of an early occurring pycnodont braincase is based on a specimen that now appears to be lost (Mesturus sp. [67]). Derived pycnodont neurocrania are known from well-preserved Cretaceous specimens of Neoproscinetes penlavi and Iemanja palma from Brazil, but are poorly ossified [50,68]. The earliest described pycnodonts are named from articulated but two-dimensional remains from the Norian of Italy [69], with isolated dental elements found in Norian and Rhaetian deposits of Belgium and Luxembourg [70]. As such, we consider our results relating to pycnodonts as preliminary: only a small proportion of the vast diversity of pycnodonts are included, and only one for which the braincase is known in detail. In particular, detailed description of primitive pycnodonts (for example, material from the Early Norian of Italy mentioned as possessing lateral gulars [71]), as well as three-dimensional neurocranial material, may help clarify the position of the group.

Dapediid diversity
Ray-finned fishes with an adaptation to durophagy were well represented in the Triassic [21,72], although dapediids are thought to have made up a fairly small proportion of this diversity. Although many durophages were wiped out during the End-Triassic extinction, dapediids survived and went on to radiate in the Jurassic, presumably filling vacated ecospace [73]. The discovery of a new, exceptionally large [74] dapediid in the Late Triassic indicates that dapediids may have been more diverse before the End-Triassic extinction than previously thought. Although dental elements are not preserved for Scopulipiscis saxciput, similarities in braincase anatomy to Dapedium seem likely to perform a functional role in strengthening the skull during durophagy, including the pronounced midline occipital crest, the deep insertion point for a longitudinal ligament, and the extension of the parasphenoid between the orbits and around the basioccipital. Many of these specializations are seen in other durophagous actinopterygians, including Carboniferous eurynotiforms [75] and Eocene gymnodonts [76].