Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel’s cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-β) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-β interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-β signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.


Introduction
Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Craniofacial anomalies, excluding cleft lip and palate, occur in 1 out of every 1600 births in the United States [1]. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate [2,3]. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral (D/V) axis. For example, Auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling [4][5][6] and Alagille syndrome is caused by defects in Jagged (Jag)-Notch signaling [7,8]. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory [9][10][11].
Craniofacial development in vertebrate organisms is a highly conserved process and involves interactions between multiple signaling pathways. The zebrafish Danio rerio is a model vertebrate organism particularly suited to the study of early developmental events. Craniofacial development begins shortly after the end of gastrulation with the specification and subsequent migration of CNCCs from the dorsal part of the neural tube. The CNCCs migrate ventro-laterally from the dorso-medial neural tube to one of seven pharyngeal arches. Once in the arches, the CNCCs receive and participate in multiple signaling events [12]. Lateral ectoderm and medial endoderm cells together form pouches that cup the CNCCs and deliver various paracrine signals important for both the survival and the patterning of the adjacent CNCCs [13][14][15]. The zebrafish jaw structures derived from the first arch CNCCs are the dorsal palatoquadrate (PQ) and ventral Meckel's (M) cartilages [16,17].
BMP and Jag-Notch signaling regulate ventral and dorsal CNCC patterning, respectively [9][10][11]. Edn1 signaling is required for ventral CNCC patterning downstream of BMP signaling [10,18]. The combined action of these signaling pathways sub-define regions of CNCCs along the D/V axis within the first pharyngeal arch. These sub-defined territories are evident in the layered expression patterns of the distal-less (dlx) family of transcription factors [19]. Specifically, loss of Edn1 signaling results in the loss or reduction of ventral and intermediate structures such as Meckel's cartilage (M) and the jaw joint. Additionally, the loss of Edn1 signaling has been shown to result in the dorsalization of ventral structures [18,[20][21][22]. In mice, mutants for either edn1 -/or ednra -/display loss of expression for dlx3-6, and hand2 [21,22]. Similarly, edn1 -/mutant zebrafish lose ventral and intermediate expression of dlx3-6 and hand2 [18]. Overexpression of Bmp4 expands ventral territory via upregulation of edn1 and the corresponding downstream network of transcription factors including hand2 and several dlx genes [10]. Conversely, disruption of later stage Bmp signaling by overexpressing a dominant negative Bmp receptor after CNCC induction results in the loss of ventral edn1 and dlx6a expression, intermediate-ventral dlx3b expression, and the ventral expansion of the normally dorsal restricted jag1b [9]. In zebrafish, jag2 and jag1b are expressed in the CNCCs and pharyngeal pouch endoderm [11,23] and antisense knockdown of them results in reductions in dorsal cartilages [24]. Zebrafish jag1b mutants display dorso-posterior defects in PQ formation; antisense morpholino knockdowns of notch2 yield a similar phenotype consistent with a traditional Jag-Notch signaling requirement for dorsal development. Loss of Jagged-Notch signaling results in the dorsal expansion of ventral and intermediate specifying dlx3b and dlx5a markers. Misexpression of jag1b results in the dorsalization of ventral structures, particularly with the transformation of M into a more PQ like structure. Jag1b is normally restricted to the dorsal-most CNCC within the first arch, however ubiquitous overexpression of jag1b results in the loss of ventrally expressed hand2 and edn1 as well as restriction of ventral-intermediate markers dlx3b, dlx5a, and dlx6a to the most ventral territory of the arch [11]. Taken together, these interactions yield a model in which BMP signaling patterns ventral and intermediate territory at least partly through induction of Edn1 signaling, while Jag-Notch signaling patterns dorsal territory, and mutual antagonism stabilizes patterning along the D/V axis [25]. These combined interactions yield spatial restrictions of a nested network of transcription factors into dorsal, intermediate, and ventral territories that prefigure the future lower jaw, joint, and upper jaw cartilages.
Wdr68/Dcaf7 (hereafter referred to as Wdr68) is a highly conserved 342 amino acid length member of the WD40 repeat domain family of proteins that are generally known to function as scaffolding proteins [31][32][33]. Vertebrate Wdr68 interacts with several protein kinases including several members of the Dual-specificity tyrosine phosphorylation-regulated kinase (Dyrk) gene family [34][35][36][37][38]. Dyrk1a maps to the Down Syndrome (DS) critical region of human chromosome 21 and overexpression of Dyrk1a is a major contributor to the neurodevelopmental defects present in DS patients [39,40]. Dyrk1a and Dyrk1b possess RNApII-CTD kinase activity important for the expression of several genes [41]. High-throughput protein interaction assays also suggest that Wdr68 interacts with the pSmad2/3 components of TGF-β signaling [42]. The subcellular localization of Wdr68 is likely regulated. When expressed alone, Wdr68 is found in both the cytoplasm and nucleus. However, co-expression with the RNApII-CTD kinase Dyrk1a induces nuclear translocation of Wdr68 [35,37,43]. Wdr68 is expressed widely in early development and regulates ventral cartilage formation in the zebrafish [36,43]. Specifically, wdr68 is required for expression of edn1 and genes downstream of Edn1 such as bapx1, hand2, and several dlx genes [36]. wdr68 is also required for the expression of the spaw, lft1, and lft2 genes [34] that are downstream of BMP signaling in the zebrafish [44,45].
Together, these previous findings suggest Wdr68 may modulate TGF-β/BMP signaling to downstream targets such as the Edn1-Jag1b network for D/V patterning. The wdr68 hi3812 allele contains a retroviral insertion within the first exon after codon 44 and is therefore considered a null allele [36,46]. Here we report that the temperature-dependent severity of the cartilage defects present in wdr68 hi3812 homozygotes is a consequence of differential perdurance of maternally derived Wdr68. We then exploited the temperature-dependence, along with a Tg (hsp70l:GFP-Wdr68) zebrafish line, to define a temporal window between the 17-somites and prim-5 stages during which wdr68 activity is required for craniofacial development. Notably, this window overlaps substantially with the known onset of edn1 expression and activity during craniofacial development. We also report that edn1 expression can partially rescue dlx6a expression in wdr68 mutants, consistent with it functioning downstream of wdr68. However, edn1 expression was unable to restore hand2 expression in wdr68 mutants. We also found ventral expansion of the expression of the normally dorsal-restricted jag1b, hey1, and grem2 genes in wdr68 mutants. We further report pharmacogenetic interactions between BMP signaling and wdr68. Specifically, the BMP antagonist dorsomorphin (DM) exacerbates the wdr68 cartilage and edn1 expression defects while the BMP agonist isoliquiritigenin (ISL) partially rescues the wdr68 cartilage and edn1 expression defects. Using CRISPR/Cas9 generated mouse C2C12 sublines lacking wdr68, we found no significant changes in pSmad1/5 levels, pYap1 levels, or BRE-Luciferase responsiveness to BMP ligands. We also found little to no defects in pSmad1/5 levels in zebrafish wdr68 mutants. However, co-challenge with TGF-β and BMP ligands revealed significant enhancement of TGF-β interference with BRE-Luciferase responsiveness to BMP ligands in C2C12 sublines lacking wdr68. Suggesting conservation of this mechanism in vivo, we also report that treating wdr68 mutants with the TGF-β inhibitor SB431542 also partially rescued the craniofacial and edn1 expression defects. Further analysis of dorsal CNCC markers revealed that expression of dlx1a and dlx2a was severely reduced in anterior dorsal and ventral 1 st arch CNCC territory in wdr68 mutants. While ISL treatment failed to restore dlx2a expression, SB431542 partially rescued dlx2a in wdr68 mutants. Together, the findings reported here reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a. These findings also suggest interactions between Wdr68 and TGF-β signaling that will require further investigation.

Zebrafish husbandry
This project was approved and conducted under the approved Cal State LA IACUC protocol (14-2 Renewal 11-3) and the zebrafish animals (TAB14 and AB Ã backgrounds) used in it were reared in strict accordance with IACUC guidelines. Zebrafish embryos were raised at 24˚C, 28.5˚C, or 32˚C as specified in specific experiments.

Alcian blue staining
Alcian blue staining was performed as previously described [34,43]. Briefly, after fixation with 4% PFA, embryos were washed in Phosphate Buffered Saline + 0.1% Tween-20 (PBST) and dehydrated through 50% PBST/ 50% Methanol (MeOH) while rocked for 5 minutes. Samples were put into 100% Methanol and stored on ice for 30 minutes. Methanol was then removed and samples stained in 0.1% Alcian Blue overnight while rocking. The next day samples were rinsed twice with 100% Ethanol (EtOH) and then rehydrated through 50% PBST/ 50% MeOH into PBST. Samples were digested in 0.05% Trypsin in saturated Sodium Borate (Na 2 B 4 O 7 ) for 3 hours at 37˚C. Samples were bleached overnight in a solution of 3% H 2 O 2 ; 1% KOH at 4˚C. The next day samples were washed in PBS + 1.0% Tween-20 for 5 min and then re-suspended in 80% glycerol; 0.1% Tween-20. Animals were scored for the presence of the M and PQ cartilages.
followed by three washes with PBST. Embryos were then washed twice with PBSTTD (PBST + 0.1% Triton X-100 + 1% DMSO) for 5 min. Embryos and primary antibodies were preblocked for 30 min at RT in Block solution (PBSTTD + 0.5% Boehringer Mannheim Block solution + 10% Lamb serum) and then incubated in primary antibody overnight at 4˚C. The next day embryos were washed twice quickly and five times for 10 minutes each in PBSTD (PBST + 1% DMSO) and then re-blocked for 30 minutes before exposure to blocked secondary antibody for 2 hours at RT. Then, embryos were washed twice quickly and five times for 10 minutes each in PBSTD followed by imaging on a Zeiss Apotome microscope.

Confocal imaging
The Tg(sox10:mCherryCAAX) animals were kindly provided by the Crump Lab [47]. Confocal multi-TIFF stacks were captured using an Olympus IX81 confocal microscope on agarosemounted embryos and analyzed using FIJI volume viewer.

Heat Shock Rescues
Tg(hsp70l:GFP-wdr68) csu9 animals were crossed to wildtype TAB14 fish. Embryos were collected at the one cell stage and injected with either 3812-2/4 morpholino, blocking translation of endogenous Wdr68, or with the negative control 3812-1 morpholino. Embryos were then allowed to develop in a 28.5˚C incubator. Heat shocks were performed at 39˚C for 30min at each of the stages of interest. Heat shocks were conducted at 5-somites, 12-somites, 15-somites, 17-somites, 20-somites, 25-somites, prim-5, prim-12 and prim-20 stages. After 30 minutes of heat shock, animals were returned to 28.5˚C, and allowed to develop until the formation of the swim bladder was observed in control animals [approximately 5 days post fertilization (dpf)]. Animals were then fixed for 1 hour at room temperature or overnight at 4˚C in 4% PFA in PBST, and cartilages scored via Alcian Blue Staining. SP6, T7, or T3 Transcription Kits per manufacturer's instructions (Ambion), with the adaptation of using digoxigenin-labeled-UTP. ISH was performed as previously described [34,53] with the following modifications. The jag1b, hey1, grem2, and dlx1a probes were used at 0.2ng/ μL in hybridization buffer (hyb) overnight at 65˚C. The edn1, hand2, dlx6a, and dlx2a probes were used at 0.5ng/μL in hyb buffer overnight at 70˚C. Once development was complete, embryos were washed in PBST, bleached in 10% H 2 O 2 ; 5% formamide in PBST for 20 min. Finally embryos were washed in PBST and cleared in 80% glycerol containing 0.1% Tween-20 in dH 2 O for imaging.

Embryo Genotyping
Embryos were separated into individual Eppendorf tubes, excess water removed, and then digested in Proteinase K at a final concentration of 100μg/ml for 2 hours at 55˚C and were vortexed every 15 minutes during the incubation. Samples were then diluted two-fold with dH 2 O and vortexed for 1 minute. Proteinase K was then inactivated by heating samples to 96˚C for 15 minutes. Samples were then centrifuged at top speed for 1 minute to pellet cellular debris. 2μL of the solution was then used as template in a PCR reaction using the previously described primers 3812-c; 3812-3; and LTR-f1 [36].

Drug Treatments of Embryos
Adult wdr68 hi3812/+ zebrafish were crossed to obtain groups of embryos that were placed in a 24˚C incubator for later dorsomorphin (DM) treatment or a 32˚C incubator for later isoliquiritigenin (ISL) or SB431542 (SB) treatment. Dechorionated embryos were exposed to 0.1% DMSO control or DM/ISL/SB starting at the 14-to 15-somites stage and processed at 5 dpf for Alcian staining or at the 20-24 somites stages for ISH analysis. Various drugs were kept on the embryos until fixation.

Western blots on zebrafish embryo or C2C12 cell extracts
Embryos were ground on ice in a 1.5 mL microcentrifuge tube using a plastic pestle in 50μL ice-cold RIPA buffer (50mM Tris pH 8, 150mM NaCl, 1% Igepal CA630, 0.5% NaDeoxycholate, 0.1% SDS) with 1x Protease Inhibitor Cocktail (PIC). The samples were vortexed for 1 minute and then centrifuged for 1 minute at room temperature at high speed. The samples were maintained on ice for 1 hour and every 10 minutes were vortexed for 1 minute. After the 1-hour period ended, the tubes were centrifuged to pellet debris and supernatants quantified by Bradford assay prior to being subjected to western blot analysis as described further below. C2C12 cell extracts were made from 10cm plates of cells grown in GM or Differentiation Medium (DMEM + 2% Horse serum + 100ug/mL pen/strep), rinsed twice with ice-cold PBS, and then incubated in 0.5mL ice-cold RIPA buffer + 1x PIC for 15 min at 4˚C. Cells were then scraped from the plate, incubated on ice for 1 hour, centrifuged at 10,000g at 4˚C and supernatants quantified by Bradford assay prior to being subjected to western blot analysis as follows. Equal amounts of protein samples were boiled for 5 minutes at 95˚C, ran on 8-16% SDS-PAGE gels, and then transferred onto PVDF membrane (Thermo Scientific). The PVDF membrane was blocked overnight at 4˚C with 5% non-fat dry milk in PBST (PBS+0.01% Tween-20) with 0.02% NaN 3 . The following day the blocking buffer was removed and blocked primary antibody was added. The membrane was then rinsed 3X for 5 minutes with PBST and then blocked secondary antibody was added. After the incubation period the membrane was rinsed 3X for 5 minutes with PBST. In most cases, the same blot was stripped and re-probed for relevant controls (beta-tubulin, total Yap).

Statistical Analysis
Pairwise comparisons were performed using the Student's T-test. Experiments containing 3 or more conditions were subjected to one-way ANOVA and post-hoc Tukey HSD tests.

Results
wdr68 activity is required during a window between the 17 somites and prim-5 stages The wdr68 hi3812/hi3812 mutant was originally described as presenting a range of jaw defects that varied from only mild joint fusions to near complete losses of the palatoquadrate (PQ) and Meckel's (M) cartilages [36]. The initial characterization was completed using embryos raised at the standard 28.5˚C. We have since found that rearing the animals at 24˚C yielded mutant animals that mostly present only the mild M-PQ joint fusion defects ( Fig 1B red arrowhead, compare to 1A). Specifically at 24˚C, 79% of mutants presented only mild M-PQ fusions versus 21% displaying severe loss of the M and/or PQ cartilage. In contrast, rearing embryos at 32˚C yielded mutant animals that mostly present severe losses of the M and PQ cartilages ( Fig  1C compare to 1A, 1B). Specifically at 32˚C, 6% of mutants presented only mild M-PQ fusions versus 94% displaying severe loss of the M and/or PQ cartilage. Because the wdr68 hi3812 allele is a retroviral insertion within exon-1 near a splicing junction [36], and insertional mutations can cause temperature-dependent splicing defects [56], we examined animals reared at 24˚C versus 32˚C by RT-PCR in an effort to detect potential alternative splicing products but found no evidence to support such a mechanism (data not shown).
We next used immunofluorescence to determine whether Wdr68 protein might exhibit temperature-dependent differences in perdurance (Fig 1D and S1A-S1D Fig). We expected to find Wdr68 protein in very early stage wdr68 hi3812/hi3812 mutants because we previously reported that wdr68 transcripts are maternally supplied [36]. Consistent with that expectation, 100% of shield-stage embryos obtained from wdr68 hi3812/+ in-crosses that were analyzed by immunofluorescence were positive for Wdr68 expression when raised at either 24˚C or 32˚C. Random genotyping identified 2/16 wdr68 hi3812/hi3812 mutants confirming that the appropriate breeding cross had been made. However, by the 17-somites stage embryos raised at 32˚C were strikingly different than those raised at 24˚C (Fig 1D, 17s red versus blue). While no embryos raised at 24˚C displayed severe loss of Wdr68 expression, a near-mendelian ratio (20%) of embryos raised at 32˚C lacked Wdr68 expression. Genotyping confirmed that 5/5 embryos raised at 32˚C and lacking Wdr68 expression by the 17-somites stage were wdr68 hi3812/hi3812 mutants. Likewise, 9/9 embryos raised at 32˚C and possessing Wdr68 expression were wildtype or wdr68 hi3812/+ (hereafter summarized as +/ Ã ). Random genotyping of the embryos raised at 24˚C that all had Wdr68 expression identified 3/10 wdr68 hi3812/hi3812 mutants confirming that the appropriate breeding cross had been made. By the prim-25 stage, both embryos raised at 24˚C and 32˚C yielded near-mendelian ratios (24.1% and 23.8%, respectively) of embryos lacking Wdr68 expression. Thus, a critical function for M and PQ formation is performed during late somitogenesis by Wdr68 that is cut short in embryos raised at 32˚C.
To further delineate the temporal window during which Wdr68 functions to mediate M and PQ formation, we generated Tg(hsp70l:GFP-wdr68) zebrafish lines (S1E-S1G Fig). The lines were first characterized by heat shocking them at 39˚C for various periods of time followed by fluorescence imaging (S1E and S1F Fig) and then harvested for western blot analysis using anti-Wdr68 antibody (S1G Fig). We found that a 30-minute heat shock was sufficient to yield fusion protein expression that lasted for at least 7 more hours (S1F and S1G Fig, lane 7). We then injected embryos from outcrosses of the Tg(hsp70l:GFP-wdr68) adults with a previously described antisense wdr68-MO [36] and subjected them to 30-minute heat shocks at various developmental stages to determine how late in development restored Wdr68 activity could still rescue craniofacial development ( Fig 1E). Overexpression of GFP-Wdr68 in otherwise wildtype embryos did not yield any observable defects (S2A and S2B Fig). Over 75% of nontransgenic wdr68-MO injected sibling embryos displayed jaw defects in spite of receiving heatshocks at all developmental time-points examined indicating that heat shock alone was unable of rescue ( Fig 1E, red line non-Tg). These embryos were also indistinguishable from both transgenic and non-transgenic wdr68-MO injected embryos that did not receive any heat shock ( Fig 1E, -HS  Thus, restoring Wdr68 function as late as the prim-5 stage is sufficient to rescue craniofacial development. Together these findings indicate that Wdr68 function is important for craniofacial development during a window between the 17-somites and prim-5 stages.

edn1 functions downstream of wdr68
We previously showed that edn1 expression depends on wdr68 activity and that expression of downstream targets of Edn1 signaling, such as dlx6a and hand2, are also reduced or absent in wdr68 hi3812/hi3812 mutants [36]. The simplest model for these observations is that edn1 functions downstream of wdr68 and that the primary function of wdr68 in lower jaw development is, either directly or indirectly, the induction of edn1 expression. A simple prediction of this model is that restoring edn1 expression in a wdr68 hi3812/hi3812 mutant should also restore the expression of edn1 target genes, such as dlx6a and hand2. To test this model, we generated a plasmid construct for expressing edn1, injected embryos from crosses of wdr68 hi3812/+ adults with various combinations of mRNAs or plasmids, and then processed the embryos raised at 32˚C for either alcian blue staining of cartilages (Fig 2A-2E) or in situ hybridization (ISH) using dlx6a probe to assess potential rescue (Fig 2F-2Q). The negative control mRNA EF1a had no effect on wildtype siblings and failed to rescue wdr68 hi3812/hi3812 mutants (Fig 2A and  2B). In contrast a significantly higher fraction of the embryos injected with Edn1 mRNA displayed partially restored M-like cartilages in wdr68 hi3812/hi3812 mutants (Fig 2C and 2E; compare Edn1 vs EF1a, p<0.001). However, Edn1-injected wdr68 hi3812/hi3812 mutants largely failed to form normal PQ-like cartilages. As expected, wdr68 hi3812/hi3812 mutants injected with Flag-Wdr68 mRNA displayed nearly-normal M and PQ cartilages (Fig 2D and 2E; compare FW vs EF1a, p<0.001). As control for ISH analysis, wildtype sibling embryos injected with plasmids expressing GFP and dsRed (G/R) displayed normal expression levels of dlx6a in the pharyngeal arches (Fig 2F and 2G, red arrowhead). Likewise, wdr68 hi3812/hi3812 mutants injected with the G/R plasmid mix displayed the expected loss of 1 st arch dlx6a expression (Fig 2H and 2I, red arrowhead). Wildtype sibling embryos injected with plasmids expressing GFP and Edn1 (G/Edn1) were slightly smaller than normal but displayed near-normal expression levels of dlx6a in the pharyngeal arches (Fig 2J and 2K, red arrowhead). In contrast, wdr68 hi3812/hi3812 mutants injected with the G/Edn1 plasmid mix partially restored dlx6a expression (compare Fig 2L and  2M with 2H and 2I). Genotyping confirmed that 0/12 mutants injected with the G/R plasmid mix had restored dlx6a expression (Fig 2H and 2I, 0% rescue), while 9/14 mutants injected with the G/Edn1 plasmid mix had restored dlx6a expression (Fig 2L and 2M, 64% rescue). Notably, the restoration of dlx6a expression appeared largely restricted to the ventral-most region of the 1 st arch in G/Edn1-injected wdr68 hi3812/hi3812 mutants (compare Fig 2M to 2K). A significant caveat to these findings is that, at least in our hands, plasmid-based Edn1 is very broadly expressed in these experiments and thus the partial rescue may be the result of a nonlocal ectopic Edn1 source (S3E and S3F Fig).
To further explore the epistatic relationships between edn1 and wdr68, we examined dlx6a expression in Tg(hsp70l:GFP-Wdr68) embryos injected with antisense morpholino knockdown of edn1 (Et1-MO) (Fig 2N-2Q). Wildtype sibling control embryos displayed normal dlx6a expression (Fig 2N, red arrowhead). As expected, wildtype sibling embryos injected with Et1-MO displayed severe loss of dlx6a expression in all pharyngeal arches (Fig 2O, red arrowhead). Heat shock induction of GFP-Wdr68 overexpression did not alter dlx6a expression in control embryos (Fig 2P) and failed to rescue dlx6a expression in Et1-MO animals (Fig 2Q). We also found that GFP-Wdr68 overexpression did not affect edn1 expression (S3A and S3B  Fig). Expression of dlx6a in Et1-MO;wdr68 hi3812/hi3812 mutants was severely reduced in all pharyngeal arches similar to that observed in embryos only injected with Et1-MO (S3C and S3D  Fig; Fig 2O). In contrast, alcian blue stained cartilages readily detected a PQ-like cartilage in Et1-MO animals that was absent in Et1-MO;wdr68 hi3812/hi3812 mutants (S3G and S3H Fig).
Thus, edn1 appears to function downstream of wdr68 to facilitate ventral cartilage development and dlx6a expression.
To further explore the regulatory relationships between wdr68 and edn1, we also examined hand2 expression (S5I- S5P Fig). Expression of hand2 is both edn1-dependent and BMPdependent [9]. Wildtype sibling embryos injected with the G/R plasmid mix displayed normal expression levels of hand2 (S3I and S3J Fig, red arrowhead). Likewise, wdr68 hi3812/hi3812 mutants injected with the G/R plasmid mix displayed the expected loss of 1 st arch hand2 expression (S3K and S3L Fig, red arrowhead). Wildtype sibling embryos injected with the G/ Edn1 plasmid mix displayed near-normal expression levels of hand2 (S3M and S3N Fig, red  arrowhead). In contrast to that observed for dlx6a, wdr68 hi3812/hi3812 mutants injected with the G/Edn1 plasmid mix failed to rescue hand2 expression (compare S3O and S3P Fig with S3K  and S3L Fig). Genotyping confirmed that 0/6 mutants injected with the G/Edn1 plasmid mix had restored hand2 expression (S3O and S3P Fig, 0% rescue). Thus, while edn1 is downstream of wdr68, more complex regulatory interactions exist for at least some edn1 target genes, such as hand2, that require other wdr68-dependent functions for expression.
wdr68 restricts jagged-notch signaling to first arch dorsal territory Signaling interactions between dorsally-restricted jag1b and notch2 are important for the induction of similarly dorsally-restricted hey1 and grem2 expression [11]. Because edn1 expression is known to be important for proper restriction of jag1b, hey1, and grem2 expression from ventral territory, we examined their expression in embryos raised at 32˚C from crosses of wdr68 hi3812/+ adults. ISH revealed expansion of jag1b into the ventral territory of the first arch structure in wdr68 hi3812/hi3812 mutants (compare Fig 3A to arrowhead in Fig 3B). Genotyping confirmed that 5/5 animals displaying the phenotype shown in Fig 3A were wildtype (+/ Ã ), while 6/6 animals displaying the phenotype depicted in Fig 3B were wdr68 hi3812/hi3812 mutant animals. Expression of the transcription factor hey1 depends on Jag-Notch signaling [11]. We also found expansion of hey1 expression into the ventral territory of the first arch structure (compare Fig 3C to arrowhead in Fig 3D). Genotyping confirmed that 5/5 animals displaying the phenotype shown in Fig 3C were wildtype (+/ Ã ), while 5/5 animals displaying the phenotype depicted in Fig 3D were wdr68 hi3812/hi3812 mutant animals. Expression of the normally dorsally-restricted BMP signaling antagonist grem2 is also dependent on Jag-Notch signaling [10]. Mirroring our findings with jag1b and hey1, we found expansion of grem2 expression into the ventral territory of the first arch structure (compare Fig 3E to arrowhead in Fig 3F). Genotyping confirmed that 5/5 animals displaying the phenotype shown in Fig 3E were wildtype (+/ Ã ), while 4/4 animals displaying the phenotype depicted in Fig 3F were wdr68 hi3812/hi3812 mutant animals. Thus, we found that wdr68 is required to restrict Jagged-Notch signaling events from ventral territory.
A BMP antagonist exacerbates the severity of craniofacial defects and loss of edn1 expression in wdr68 mutants BMP signaling is required for edn1 expression and ectopic BMP signaling can induce ectopic edn1 expression [9,10]. Thus, edn1 is a downstream target of BMP signaling. We previously showed that edn1 expression is also downstream of wdr68 function [36] (and Fig 2). ISH analysis failed to detect any defects in BMP4 expression in wdr68 mutants (S4 Fig), suggesting wdr68 functions downstream or in parallel to BMP signaling. A simple model combining these observations is that wdr68 facilitates BMP induction of edn1. If true, then treating wdr68 hi3812/hi3812 mutants with a BMP antagonist will exacerbate loss of M formation and edn1 expression. To test this, we treated embryos, from matings of wdr68 hi3812/+ adult zebrafish, raised at 24˚C with 10μM Dorsomorphin (DM) starting at the 14-15 somites stage. We chose to start treatments at this relatively late stage in order to avoid perturbing the very early developmental roles of BMP signaling. When grown at 24˚C, wdr68 hi3812/hi3812 embryos displayed only mild M-PQ joint fusions (see Fig 1B). We found that DMSO-treated wildtype (+/ Ã ) embryos exhibited normal cartilage formation while wdr68 hi3812/hi3812 embryos exhibited M-PQ joint fusions (compare Fig  4A to 4B). Genotypic analysis of embryo tails separated from heads prior to Alcian blue staining confirmed that 6/6 embryos like that shown in Fig 4B were wdr68 hi3812/hi3812 mutants. DMtreated wildtype (+/ Ã ) embryos appeared the same as DMSO-treated wildtypes (compare Fig 4C  to 4A). However, DM-treated wdr68 hi3812/hi3812 mutants lost M and also had severely reduced PQ cartilages while vehicle treated mutants displayed only joint fusions between M and PQ (compare Fig 4D to 4B). Genotypic analysis confirmed that 6/6 embryos like that shown in Fig  4D were wdr68 hi3812/hi3812 mutants. We also quantified the fraction of mutants in each treatment exhibiting the mild M-PQ fusion and found that 23.2% of DMSO-treated animals displayed the M-PQ fusion versus only 4.2% of the DM-treated animals (Fig 4E, blue bars, p<0.046). Conversely, we found that only 3.7% of DMSO-treated animals displayed severe losses of M and PQ versus 20.0% of DM-treated animals (Fig 4E, orange bars, p<0.040).
To test whether partial inhibition of BMP signaling in mild wdr68 hi3812/hi3812 embryos would yield a synergistic reduction of edn1 expression, we again treated embryos raised at 24˚C with DM starting at the 14-15 somites stage. We then used ISH to observe expression of edn1 at the 20 somites stage. In embryos treated with DMSO, we found normal expression of Fig 3. Wdr68 restricts expression of jag1b, hey1, and grem2 from ventral territory. ISH analysis on embryos raised at 32˚C. A) normal expression of jag1b in dorsal territory at the prim-25 stage, B) expansion of jag1b into ventral territory in wdr68 hi3812/hi3812 mutants, C) normal expression of hey1 in dorsal territory at the prim-25 stage, edn1 in wildtype (+/ Ã ) siblings (Fig 4F, red arrowhead) and reduced but still barely detectable expression of edn1 in wdr68 hi3812/hi3812 mutants (Fig 4G, red arrowhead). Genotypic analysis confirmed that 7/7 embryos in Fig 4F were wildtype (+/ Ã ) and 5/5 embryos shown in Fig 4G were wdr68 hi3812/hi3812 mutants. In embryos treated with DM, we found mildly reduced expression of edn1 in wildtype (+/ Ã ) siblings (Fig 4H, red arrowhead)  In the DM-treated wdr68 hi3812/hi3812 mutants, we were unable to detect edn1 expression (Fig 4I, red  arrowhead). Genotypic analysis confirmed that 8/8 embryos shown in Fig 4H were wildtype (+/ Ã ) and that 5/5 embryos shown in Fig 4I were wdr68 hi3812/hi3812 mutants.

A BMP agonist can partially rescue Meckel's cartilage formation and edn1 expression in wdr68 mutants
If wdr68 facilitates BMP induction of edn1, then treating wdr68 mutants with a BMP agonist should at least partially rescue M cartilage formation and edn1 expression. To test this, we treated embryos raised at 32˚C with the BMP signaling agonist isoliquiritigenin (ISL) starting at the 14-15 somites stages [57]. Embryos were raised at 32˚C so that mutants would exhibit severe loss of the M cartilage from which potential rescue of M cartilage formation could be most clearly assessed (see Fig 1C). Alcian blue stained zebrafish cartilages were dissected and flat mounted on slides for additional clarity. At 32˚C, we found that DMSO-treated wildtype zebrafish developed the M cartilage as expected (Fig 5A, red arrowhead). Also at 32˚C, we found that most DMSO-treated mutants exhibited complete loss of M and reduction of PQ as previously described (Fig 5B, red arrowhead). ISL-treated wildtype embryos were indistinguishable from DMSO-treated wildtype (compare Fig 5C to 5A). In contrast, ISL-treated mutants raised at 32˚C exhibited a partial restoration of the M cartilage (compare Fig 5D to 5B, red arrowheads). However, ISL treatment did not appear to significantly restore formation of the PQ (compare Fig 5D to 5A and 5C). We quantified the fraction of mutants in each treatment exhibiting a discernible M-like cartilage element and found that 59% of ISL-treated animals displayed an M-like cartilage versus only 17% of DMSO-treated animals (Fig 5E, p<0.006).
To determine whether enhanced BMP signaling in severe wdr68 hi3812/hi3812 embryos would partially rescue edn1 expression, we again treated embryos raised at 32˚C with 5μM ISL starting at the 14-15 somites stages. We then used ISH to observe expression of edn1 at the 20 somites stage. In embryos treated with DMSO, we found normal expression of edn1 in wildtype siblings (Fig 5F) but were unable to detect edn1 expression in wdr68 hi3812/hi3812 mutants (Fig 5G). Genotypic analysis confirmed that 10/10 embryos phenotypically like that shown in Fig 5F were wildtype (+/ Ã ) and 5/5 embryos phenotypically like that shown in Fig 5G were wdr68 hi3812/hi3812 mutants. Strikingly in embryos treated with ISL, no embryos displayed a severe reduction of edn1 expression. Genotyping was performed to unequivocally identify wildtypes and mutants. Wildtype siblings displayed normal expression of edn1 (compare Fig  5H to 5F). The known BMP agonist role for ISL suggested we might see overexpression of edn1 in these animals but that was not observed (Vrijens et al., 2013). Instead, the ISL-treated embryos appeared to yield no phenotypic mutants with respect to edn1 expression level (compare Fig 5I to 5G). Consistent with that notion, random genotypic analysis revealed that 2/9 embryos with normal edn1 expression levels were indeed wdr68 hi3812/hi3812 mutants (Fig 5I). D) expansion of hey1 into ventral territory in wdr68 hi3812/hi3812 mutants, E) normal expression of grem2 in dorsal territory at the prim-25 stage, F) expansion of grem2 into ventral territory in wdr68 hi3812/hi3812 mutants. Thus, ISL treatment rescued edn1 expression in wdr68 hi3812/hi3812 mutants to near-normal levels.

Canonical BMP signaling is not impaired in cells lacking Wdr68 expression
To further examine whether Wdr68 might directly impact BMP signaling, we used CRISPR/ Cas9 gene targeting technology to generate loss-of-function deletions in the Wdr68/Dcaf7 locus in mouse C2C12 cells. Western blot analysis and DNA sequencing confirmed the generation of two independently isolated mutant sublines, Δwdr68-5 and Δwdr68-9 (Fig 6A and S4B  Fig) as well as a non-target control (NT1) subline. The transcriptional co-activator Yap is reported to be important for BMP signaling in mammalian cells [58,59]. In flies, the ortholog of Wdr68, Riquiqui (Riq), is also reported to positively regulate the Yap ortholog yorkie through its interaction with the kinase Minibrain (Mnb) that negatively regulates the Hippo signaling pathway kinase Warts (Wts) [60]. Therefore, we examined both total Yap and pYap levels in the control, Δwdr68-5, and Δwdr68-9 sublines but found no significant differences between them (Fig 6A). To further characterize the sublines, we examined the levels of pSmad1/5 after a one-hour treatment of the cells with 0, 1, 10, or 100ng/mL BMP4 and found no significant reproducible differences between the sublines (Fig 6B). To determine whether Wdr68/Dcaf7 might generally facilitate BMP signaling in a functional assay, we transfected the control and deletion sublines with the BRE-Luc reporter plasmid [55] along with a SV40-Renilla plasmid, and generated a dose-response curve to BMP4 ligand (Fig 6C). All relative light unit responses were normalized to the vehicle-treated control subline (Fig 6C, leftmost column). Overall, no significant decreases in fold-induction were found in the Δwdr68-5 and Δwdr68-9 sublines relative to the control subline (Fig 6C grey bars versus black bars). Immunofluorescence analysis also revealed little consistent difference in pSmad1/5 levels between wildtype and wdr68 hi3812/hi3812 mutant zebrafish embryos (Fig 6E and 6F). As expected, ISL treatment did consistently increase pSmad1/5 signal (compare Fig 6H to 6G). Thus, Wdr68/ Dcaf7 does not appear to directly modulate BMP signaling pathway activity.
The TGF-β inhibitor SB431542 can partially rescue the M cartilage defects and edn1 expression in wdr68 mutants If enhanced TGF-β interference with BMP signaling contributes to the wdr68 mutant phenotype in zebrafish, then inhibiting TGF-β pathway function should at least partially restore cartilage development in wdr68 mutants. SB431542 is a well-characterized inhibitor of the TGF-β family receptor kinases Alk4/5 [61], that is also known to inhibit TGF-β signaling activity in the zebrafish [62][63][64]. To avoid perturbing earlier developmental roles of TGF-β family signaling, embryos were treated with 10μM SB431542 starting at the 14-to 15-somites stages. Embryos were raised at 32˚C so that mutants would exhibit severe loss of the M cartilage from which potential rescue of M cartilage formation could be most clearly assessed (Fig 1C). At 32˚C, we found that DMSO-treated wildtype zebrafish developed the M cartilage as expected (Fig 7A, red arrowhead). Also at 32˚C, we found that most DMSO-treated mutants exhibited complete loss of M and reduction of PQ as previously described (Fig 7B, red arrowhead). 10μM SB431542-treated wildtype embryos were indistinguishable from DMSO-treated wildtypes (compare Fig 7C to 7A). In contrast, SB431542-treated mutants raised at 32˚C exhibited a partial restoration of the M and PQ cartilages (compare Fig 7D to 7B, red arrowheads). Quantitation of the changes in phenotypic severity revealed a significant shift in the number of mutants displaying severe versus mild phenotypes. Under DMSO treatment, mild wdr68 hi3812/hi3812 embryos were 8.6% of the total sample population whereas severe wdr68 hi3812/hi3812 embryos were 14.3% of the total population (Fig 7E, left). Under SB431542 treatment, mild wdr68 hi3812/hi3812 embryos were 19.2% of the total population whereas severe mutants were 8.0% of the total population (Fig 7E, right). Thus, SB431542 treatment decreased the fraction of severe mutants relative to the DMSO control (Fig 7E, red bars, p < 0.012).
The TGF-β inhibitor SB431542 can partially rescue dorsal dlx2a expression in wdr68 mutants A molecular explanation for the absence of the PQ in wdr68 hi3812/hi3812 mutants is still lacking. Interestingly, SB431542-treated wdr68 hi3812/hi3812 mutants not only displayed partial rescue of the ventral M cartilage, but also a consistent partial rescue of the dorsal PQ cartilage (Fig 7D). This was in contrast to either ectopic expression of edn1 (Fig 2C), or ISL-treatment (Fig 5D), both of which failed to restore the dorsal PQ cartilage in wdr68 hi3812/hi3812 mutants. It has been previously reported that simultaneous antisense knockdown of dlx1a and dlx2a causes loss of the PQ while retaining the M and PTP [65]. The reported dlx1a/2a-MO phenotype bears similarities to the residual defects we observed for both ectopic edn1 ( Fig 2C) and ISL-treatment ( Fig 5D) in wdr68 hi3812/hi3812 mutants. Therefore, we re-examined the expression of the dlx1a and dlx2a genes at the prim-12 stage in wdr68 hi3812/hi3812 mutant embryos raised at 32˚C (Fig  8). In wildtype sibling embryos, we found robust expression of dlx2a in both dorsal and ventral CNCCs of the 1 st and 2 nd arches (Fig 8A and 8B). In contrast, we found severely reduced dlx2a expression in the anterior portion of the 1 st arch in wdr68 hi3812/hi3812 mutants (Fig 8C and 8D, compare red underline between 8D and 8B). Consistent with the relatively minor 2 nd archderived cartilage defects in wdr68 hi3812/hi3812 mutants (Figs 1C, 2B, 5B and 7B), expression of dlx2a in the 2 nd arch was relatively unchanged in wdr68 hi3812/hi3812 mutants (Fig 8D compare blue underline to 8B). To determine whether inhibiting TGF-β signaling in severe wdr68 hi3812/hi3812 embryos would partially rescue dlx2a expression, we again treated embryos raised at 32˚C with 10μM SB431542 starting at the 14-to 15-somites stages. While SB431542 treatment yielded no discernible changes in wildtype siblings, we found partial rescue of anterior dorsal and ventral 1 st arch expression of dlx2a in wdr68 hi3812/hi3812 mutants (Fig 8E and 8F, compare red underline to Fig 8C and 8D). Analysis of dlx1a expression revealed a similar loss control and did not differ substantially between lanes. C) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids and induced with 0, 1, 10, or 100ng/mL BMP4 in GM. No significant differences were found between control and deletion sublines. Representative experiment shown from at least 3 independent trials. D) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids, induced with 10ng/mL BMP4, and then challenged with 0, 0.1, 1.0, or 10ng/mL TGF-®1. At 10ng/mL TGF-®1 interference with BRE-Luc activity was significantly greater in the Δwdr68-5 and Δwdr68-9 sublines relative to NT1 controls (* = p < 0.002). Representative experiment shown from at least 3 independent trials. E-H) Immunofluorescence detection of pSmad1/5 in prim-12 stage zebrafish embryos raised at 32˚C. E) wildtype sibling embryo. F) wdr68 hi3812/hi3812 mutant embryo. G) DMSO-treated wildtype sibling. H) ISL-treated wildtype sibling.

Temperature dependence of the wdr68 mutant phenotype
Here we report that modest differences in maternally-derived protein stability, caused by incubation at different temperatures, can yield dramatic shifts in phenotypic severity even for mutants that are otherwise expected to be null mutations (Fig 1A-1D). This finding suggests that conducting forward genetic screens at temperatures other than the field standard of 28.5˚C may reveal additional mutable loci that have as yet gone undetected.
wdr68 functions upstream of edn1 during a period between the 17-somites and prim-5 stages to induce and restrict dorsal-specifying genes from ventral territory We identified a developmental period between the 17-somites and prim-5 stages during which wdr68 function is particularly important for craniofacial development (Fig 1D and 1E). Notably, this window contains the point at which edn1 expression begins, around the 18-somites stage [18], and overlaps substantially with the period during which Edn1 signaling is important for craniofacial patterning [9,10]. Specifically, Alexander et al., 2011 found that restoring Edn1 expression as late as the prim-5 stage was able to partially rescue the defects caused by dnBmpr1 expression. In contrast, restoring Edn1 expression around the prim-25 stage was too late, similar to our findings with Wdr68. We found that ectopic expression of edn1 in wdr68 mutants was able to partially restore the 1 st arch expression of a canonical edn1-dependent target gene, dlx6a, consistent with the model that wdr68 functions upstream of edn1 (Fig 2). However, we also found that ectopic edn1 expression was insufficient to restore 1 st arch hand2 expression in the absence of wdr68 (S3 Fig). The expression of hand2 depends on both edn1!dlx6a and BMP signaling [9]. Thus, interference with BMP signaling to hand2 in a wdr68 mutant might prevent edn1!dlx6a induction of hand2. As an attempt to test this model, we treated wdr68 mutants with ISL to enhance BMP signaling and edn1 expression simultaneously, but the treatment failed to restore hand2 expression (S4C and S4D Fig). While that result may simply reflect a shortcoming of the experimental procedure (e.g. insufficient levels of BMP signaling, edn1!dlx6a expression, or both), it may also indicate a distinct wdr68-dependent requirement for hand2 expression. Nonetheless, and consistent with the known role for edn1 expression, we found that wdr68 is required to properly restrict jag1b, hey1, and grem2 from ventral territory (Fig 3). The simplest explanation for these ventral expansions of dorsal territory markers is the failure of edn1 expression in wdr68 mutants. The expansion of dorsal-specifying markers into ventral territory (Fig 3) is often accompanied by transformation of ventral cartilages into dorsal cartilages [9,10]. Curiously, such transformations are not observed in wdr68 mutants, which instead display losses of both dorsal and ventral cartilage elements. Our findings on the wdr68-dependence of dlx1a and dlx2a expression suggests that jag1b-mediated transformation of CNCCs may require the dlx1a/2a expression program.
The losses of dlx1a/2a expression (Fig 8) combined with the observed reductions of Tg (sox10:mCherryCAAX) label in wdr68 mutants (S5 Fig) raises the possibility that a subset of the CNCCs in wdr68 mutants may be lost due to apoptosis. This is an intriguing possibility that will require further investigation. However, the detection of dorsal-specifying markers such as jag1b and hey1 in both dorsal and ventral territory (Fig 3) indicates that a population of CNCCs remains within the 1 st arch of wdr68 mutants at least until about the prim-25 stage. The ultimate fate of the mis-patterned cells remains unclear.
wdr68 function does not directly modulate BMP signaling but may interfere with TGF-β signaling Although a BMP antagonist and agonist yielded expected exacerbations and rescues of the defects in wdr68 mutants, respectively (Figs 4 and 5), we found no strong evidence of a direct impact on BMP signaling pathway function (Fig 6). Notably, while analysis of the edn1 promoter sequence revealed the presence of DNA sequence elements associated with BMP responsiveness (unpublished observations), it is unclear whether Smad1/5 complexes directly regulate the edn1 promoter.
Interestingly, we found that TGF-β interference with BMP signaling was enhanced in mouse C2C12 cells lacking Wdr68 and that inhibition of TGF-β signaling partially restored jaw cartilage development, edn1, and dlx2a expression in zebrafish wdr68 mutants (Figs 6D, 7 and 8). Nonetheless, is remains unclear whether these wdr68-TGF-β pathway interactions are direct or indirect.
The ISL-mediated rescue of edn1 expression (Fig 5I) was more robust than the SB431542-mediated rescue of edn1 expression (Fig 7I). Yet, ISL-mediated rescue of the jaw cartilages was less complete (Fig 5D) than the SB431542-mediated rescue of the jaw cartilages ( Fig 7D). This discrepancy can be explained by the potency of edn1 plus the absence of dlx1a and dlx2a expression in wdr68 mutants. First, fairly low levels of edn1 expression can still mediate M formation (Fig 4G). Second, SB431542 treatment was able to also rescue dlx2a ( Fig  8E and 8F) while ISL treatment did not (S4E and S4F Fig). Thus, the combined actions of a relatively potent edn1 signaling molecule and simultaneous restoration of edn1 and dlx2a in SB431521-treated embryos likely underlies the relatively well formed jaw cartilages. That said, the mechanism by which SB431542 restores jaw development in wdr68 mutants is still poorly understood and will require further investigation.
Wdr68 is a scaffolding protein and a large number of interacting factors have been identified by various high-and low-throughput approaches. Interestingly, Wdr68 has been reported to physically interact with Smad2, Smad3, and Ski [42,66]. Wdr68 also interacts with the kinases Dyrk1a and Dyrk1b [38], that are reported to interact with Smad3 [42] and Ski [67], respectively. Thus, Wdr68 may organize a multi-subunit complex capable of modulating TGFβ pathway function. Together, the interactions of these factors potentially impact numerous disease states including cleft lip/palate, auriculocondylar syndrome, Alagille syndrome, and Down syndrome. Future investigations aimed at testing these potential roles are needed.