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. Author manuscript; available in PMC: 2016 Apr 13.
Published in final edited form as: Genesis. 2015 Apr 13;53(0):270–277. doi: 10.1002/dvg.22850

prdm1a functions upstream of itga5 in zebrafish craniofacial development

Kristi LaMonica 1,2, Hai-lei Ding 1,3, Kristin Bruk Artinger 1,*
PMCID: PMC4411201  NIHMSID: NIHMS678394  PMID: 25810090

Abstract

Cranial neural crest cells are specified and migrate into the pharyngeal arches where they subsequently interact with the surrounding environment. Signaling and transcription factors, such as prdm1a regulate this interaction, but it remains unclear which specific factors are required for posterior pharyngeal arch development. Previous analysis suggests that prdm1a is required for posterior ceratobranchial cartilages in zebrafish and microarray analysis between wildtype and prdm1a mutants at 25 hours post fertilization demonstrated that integrin α5 (itga5) is differentially expressed in prdm1a mutants. Here, we further investigate the interaction between prdm1a and itga5 in zebrafish craniofacial development. In situ hybridization for itga5 demonstrates that expression of itga5 is decreased in prdm1a mutants between 18- 31 hpf and itga5 expression overlaps with prdm1a in the posterior arches, suggesting a temporal window for interaction. Double mutants for prdm1a;itga5 have an additive viscerocranium phenotype more similar to prdm1a mutants, suggesting that prdm1a acts upstream of itga5. Consistent with this, loss of posterior pharyngeal arch expression of dlx2a, ceratobranchial cartilage 2-5, and cell proliferation in prdm1a mutants can be rescued with itga5 mRNA injection. Taken together, these data suggest that prdm1a acts upstream of itga5 and are both necessary for posterior pharyngeal arch development in zebrafish.

Keywords: ceratobranchials, pharyngeal arches, and neural crest

Introduction

Neural crest cells (NCCs) are a transient cell population that arises at the junction between the neural and non-neural ectoderm. NCCs delaminate from the dorsal neural tube and migrate to distant sites where they differentiate into a variety of tissues. NCCs can be subdivided into 2 groups, cranial and trunk: Trunk NCCs gives rise to enteric neurons, pigment cells, and sensory nerves and glia, while cranial neural crest cells (cNCCs) give rise to neurons and glia of the cranial ganglia, connective tissue, in addition to cartilage and bone of the craniofacial skeleton (Le Douarin 1982; Schilling and Kimmel 1994; Graham 2003; Chai and Maxson 2006).

cNCCs begin delaminating and migrating at 12 hours post fertilization (hpf) in the zebrafish (Danio rerio); migrating in 3 distinct streams to ultimately populate the pharyngeal arches (PAs). The PAs are finger like projections in which cNCCs migrate into to form a mesenchyme with the mesoderm, surrounded by an outer layer of ectoderm and inner layer of endoderm (Graham 2003). These four tissue types interact through a wide variety of signaling pathways such as the FGF, Retinoic Acid, Wnt, and Endothelin families to direct differentiation of the PAs into the craniofacial skeleton (Clouthier et al. 2000; Bachler and Neubuser 2001; Clouthier and Schilling 2004; Eberhart et al. 2006; Kopinke et al. 2006; Nechiporuk et al. 2007; Blentic et al. 2008; Sperber and Dawid 2008). Posterior PA derivatives contribute to the gill slits in zebrafish and laryngeal cartilages in mammals.

The zinc finger containing transcription factor prdm1a plays an important role in posterior pharyngeal arch development in zebrafish and mice (Hernandez-Lagunas et al. 2005; Wilm and Solnica-Krezel 2005; Birkholz et al. 2009). prdm1a mutants in zebrafish have a loss of posterior ceratobranchial cartilages (CBs) 2-5 due to a reduction in cell proliferation (Birkholz et al. 2009). To further understand signaling downstream of prdm1a, we performed microarray analysis of prdm1a mutant embryos at 25 hpf compared to wildtype or heterozygous embryos. integrin α5 (itga5) expression is reduced in the pharyngeal arches of prdm1a mutants (Olesnicky et al. 2010). In addition, bioinformatics analysis suggests that the itga5 promoter contains a canonical prdm1a binding site (GAAAG), suggestive of a direct interaction.

Integrins are transmembrane receptors that bind to extracellular matrix as alpha and beta heterodimers to promote cell adhesion and migration. itga5 is expressed and functions in multiple tissue layers including mesoderm in both mice and zebrafish, and endoderm in zebrafish. Null itga5 mice are embryonic lethal due to mesodermal defects that result in a lack of posterior somites and paraxial mesoderm formation resulting in the inability of the embryo to turn (Yang et al. 1993). Zebrafish itga5 has been shown to function in the endoderm to pattern the PA2 neural crest during formation of the lateral hyoid cartilage (Crump et al. 2004), as well as interacting with Fgf signaling in the posterior cranial placodes (Bhat and Riley 2011), and has been shown to be required for somite boundaries and maintenance (Koshida et al. 2005; Julich et al. 2009). Here we demonstrate by double mutant and RNA rescue analysis that itga5 functions downstream of prdm1a.

Results

To determine the expression of itga5 specifically in the posterior pharyngeal arches of wildtype and prdm1a mutant embryos, we performed in situ hybridization (ISH) between 11- 31 hpf. Consistent with published data (Crump et al. 2004; Olesnicky et al. 2010), we observe expression of itga5 at 11-20 hpf at the neural plate border between the neural plate and the non-neural ectoderm that includes cNCCs and ectodermal placodal cells (Figure 1A–C). As development progresses, itga5 is strongly expressed in the pharyngeal arch region (Figure 1D, E). In prdm1a mutant embryos itga5 expression is decreased at 11, 13, 20 and 31 hpf (Figure 1A′, B′, C′, D′, E′) compared to wildtype embryos (Figure 1A, B, C, D, E). These data show that itga5 is expressed in the PAs, is reduced in prdm1a mutant embryos, and that its spatiotemporal expression pattern is similar to prdm1a in the posterior PAs (Birkholz et al. 2009). Interestingly, prdm1a expression is also decreased in itga5 mutant embryos at 24 hpf and 31 hpf (Supplemental Figure 1). To determine if both prdm1a and itga5 are expressed in the same tissue we utilized the Tg(prdm1a:GFP) transgenic fish line and performed double fluorescent ISH for DIG labeled itga5 and Fluorescein labeled GFP for prdm1a at 31 hpf. We observe expression in of itga5 and prdm1a in the same region of the posterior PAs (arrow in Figure 1F), but not in the fin bud and hatching gland where prdm1a is also expressed at this stage (Figure 1F, F′, F″). This localization suggests that prdm1a and itga5 expression overlaps in the posterior PAs and may function together to regulate posterior PA development.

Figure 1. itga5 is expressed in the posterior pharyngeal arch domain and is reduced in prdm1a mutants.

Figure 1

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itga5 mRNA expression by ISH in wild type and prdm1a mutant embryos. Lateral views, anterior is to the left. Wildtype expression of itga5 at 11 hpf (A), 13 hpf (B), 20 hpf (C.), and 31 hpf (D (lateral), E (dorsal). prdm1a mutant embryos have reduced expression of itga5 in the pharyngeal arches (arrows) at 11 hpf (A′), 13 hpf (B′.), 20 hpf (C′), and 31 hpf (D′ (lateral), E′ (dorsal) as observed in 25% of embryos that are homozygous mutant expected from a heterozygous cross. (F, F′F″) Confocal projected image of double Fluorescent ISH for itga5 and prdm1a representing a compressed stack at 10x magnification. Anterior is to the left. At 31 hpf prdm1a:gfp is expressed in the posterior PAs, hatching gland (hg), fin bud (fb) (F). itga5 is expressed in the PAs (F′). Both itga5 and prdm1a localize to the posterior PAs (yellow in F″). The apparent expression of itga5 that is punctate in the area of the hatching gland we consider to be background staining.

To determine if there is a genetic interaction between itga5 and prdm1a we performed a double mutant analysis. Heterozygotous itga5+/− and prdm1a+/− zebrafish were crossed to generate itga5−/−; prdm1a−/− embryos and assayed for the presence of posterior CBs with alcian blue. Based on the expected ratios, only 1 out of 16 embryos will be double mutant; we identified itga5−/−; prdm1a−/− double mutants, confirmed by genotyping followed by alcian blue processing at 5 days post fertilization (dpf). Single prdmla mutant embryos display an overall hypoplasia of the viscerocranium in addition to a loss of posterior CB cartilages 3-5, with only CB1 and sometimes CB2 present, consistent with our previous data (50% of time CB2 is present; Figure 2B, B′). As described previously, itga5 mutants display a viserocranium cartilage phenotype that is highly variable (Crump et al. 2004). In some embryos, we observe a hypoplastic foramen of the hyosymplectic derived from the hyoid arch (PA2) (50% of the time we observe a reduced or absent hypoplastic hyosymplectic; as seen in double mutants Figure 2D′, arrow). In addition, the posterior CBs 4-5 are absent in itga5 mutant embryos. In a subset of itga5 mutants we observe a fusion between CBs 1 and 2 and a subsequent loss of the remaining CBs often occurring on one side (12.5% have fused CB1 and 2; Figure 2C, C′). However, some genotyped itga5a mutant embryos display normal morphology of the hyoid arch (50% normal morphology; Figure 2C, C′). Analysis of double itga5−/−; prdm1a−/− mutant embryos is suggestive of an additive effect on the posterior PA and hyosympletic cartilage development. Double itga5−/−; prdm1a−/− mutant embryos lack CBs 2-5, similar to that of prdm1a mutants alone (Figure 2D, D′). In addition, there is an increase in the incidence of the reduction in size or absence of the foreman of the hyosymplectic cartilage (80% affected; with 40% reduced, 40% absent and 20% normal; Figure 2D, D′). These data show that loss of both prdm1a and itga5 leads to an additive phenotype that is more severe than loss of either gene alone, suggestive of a genetic interaction.

Figure 2. Double mutants for itga5 and prdm1a results in an additive viscerocranium phenotype.

Figure 2

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Ventral (A–D) and lateral (A′–D′) views, anterior is to the left. Paired images are from two different embryos. Flat mounts of alcian blue staining for cartilaginous elements at 5 dpf shows itga5 and prdm1a double mutants results in an additive effect. All phenotypes were confirmed by genotyping. (A, A′) Wild type embryos show the normal pattern of the viserocranium. prdm1a mutants are lacking cbs 3-5 (n=16; B, B′) and in itga5 mutants, a fusion between cbs 1-2 is observed followed by a loss of posterior ceratobranchials on the same side (C, C′) while the other side is lacking cb 5 (n=4 only confirmed by genotyping since we selected for prdm1a mutant phenotypes before genotyping). In some embryos, the foramen of the hyosymplectic is reduced (see arrow in D′). The double prdm1a−/−itga5−/− mutants are lacking all posterior CBs except CB1 (D, D′) and a reduced hyosymplectic (n=15 confirmed by genotyping from 4 different clutches). m-meckels, pq-palatoquadrate, hs-hyosymplectic, ch-ceratohyal, cb-ceratobranchials.

To determine if there is an epistatic relationship between itga5 and prdm1a, we injected itga5 mRNA into prdm1a morphants (or mutants) to determine whether itga5 mRNA injected into prdm1a morphants is sufficient to rescue the posterior CB phenotype. prdm1a morphants display phenotypes similar to prdm1a mutants, with loss of CB3-5 as well as a variable inversion of the ceratohyal cartilage at 5–6 dpf and a reduction of posterior arch expression of the postmigratory cNCC and PA marker dlx2a at 36 hpf, as we have shown previously (Figure 3B, F)(Birkholz et al. 2009). At doses of itga5a mRNA injected (between 50–112pg) into wildtype embryos, there were no obvious defects in viserocranium cartilage formation at 5 dpf or dlx2a expression at 36 hpf (Figure 3C, G). However, when 90 pg of itga5 mRNA was injected into prdm1a morphants and stained with alcian blue at 5 dpf, 79% of prdm1a morphant embryos injected with itga5 mRNA displayed 3 or more CBs at 6 dpf (Figure 3D, I), compared to prdm1a morphants (Figure 3B, I). We observed similar rescue with both 50 and 112 pg of itga5 mRNA injected into morphants as compared to itga5 mRNA injected embryos (data not shown). In addition, whereas most prdm1a morphants display only anterior PA expression of dlx2a, injection of itga5a mRNA rescues post-migratory NC expression of posterior dlx2a (Figure 3H, J), compared to uninjected controls (Figure 3F, J) and prdm1a morphants (Figure 3B, J) and mutants (data not shown). Quantification of the number of CBs and dlx2a expression is shown in Figure 3I, J.

Figure 3. Overexpression of itga5 mRNA in prdm1a morphants results in rescue of the ceratobrancial cartilages in prdm1a mutants.

Figure 3

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Anterior is to the left. (A–D) Ventral views of alcian staining for cartilaginous elements at 6 dpf showing that prdm1a morphants injected with itga5 mRNA rescues cbs (D, I) compared to prdm1a morphants (B, I), wild type, and itga5 mRNA (C, I). (n= WT=86/86, 3 or more cbs, prdm1a-MO= 6/14, 2 or less cbs (as shown in B) and 8/16, 3 or more cbs, itga5a mRNA= 49/49, 3 or more cbs (as shown in C), and prdm1a-MO rescued with itga5 mRNA =12/58, 2 or less cbs and 46/58, 3 or more cbs (as shown in D). dlx2a expression is rescued at 24 hpf with injection of itga5a mRNA in prdm1a morphants (H, J) to a level similar to wild type (E, J) (n= WT=18/18, 3 or more cbs (as shown in E), prdm1a-MO= 9/17, 3 or more cbs and 8/17, 2 or less cbs (as shown in F), itga5a mRNA= 7/7, 3 or more cbs (as shown in G), prdm1a-MO rescued with itga5 mRNA =13/16 (as shown in H). Quantification of the number of CBs and PAs in all conditions is shown in (F, J) from 3 different clutches. m-meckels, p-palatoquadrate, hs-hyosymplectic, ch-ceratohyal, cb-ceratobranchials.

In prdm1a mutants, there is a reduction of proliferation in the posterior arch region, and we hypothesize that a potential mechanism for the rescue of the prdm1a mutant phenotype with itga5 mRNA is through a recovery of cell proliferation. We stained prdm1a mutants, itga5 mRNA injected embryos and prdm1a mutants injected with itga5 mRNA and quantified the amount of proliferation as labeled by phosphohistone H3 immunofluorescence in the posterior arch region. We determined that proliferation was significantly increased in prdm1a mutants injected with itga5 mRNA (P<0.05) (Figure 4) compared to prdm1a mutants alone (Figure 4).

Figure 4. Overexpression of itga5 mRNA in prdm1a mutants recues cellular proliferation.

Figure 4

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Quantification of the number of phosphohistone H3 positive cells in all conditions, insets show representative images of whole mount embryos labeled with red phosphohistone H3 positive cells. Wild type embryos contain an average of 25 positive cells in the PA region similar to itga5 mRNA injected alone, while prdm1a mutants have significantly less cell proliferation with an average of 17 positive cells. prdm1a mutants injected with itga5 mRNA have a significantly higher rate of proliferation than what is observed in prdm1a mutants alone (p<0.05 by Student’s T-test, comparing mutant to prdm1a mutants injected with itga5 mRNA (as shown by the asterix on the figure); n= WT=9, prdm1a-mutant=8, itga5a mRNA=4, rescue=9). There is no significant difference between itga5 mRNA and wild type, rescue and wild type, and itga5 mRNA and rescue (p>0.05), showing the rescue is similar to the wild type and mRNA alone.

Our data demonstrate that itga5 is downstream of prdm1a and that injection of itga5 mRNA is sufficient to rescue the prdm1a mutant phenotype through an increase in cell proliferation. Canonically, itga5 acts as part of a heterodimer with itgb1 as a transmembrane receptor where itga5 interacts with fibronectin exclusively extracellularly (Harburger and Calderwood 2009). Intracellularly, the cytoplasmic tail of itgb1 is known to interact with α-actinin, vinculin, and paxillin for cell migration (Harburger and Calderwood 2009). Focal adhesion kinase (FAK) is also downstream of the integrin heterodimer and FAK plays known roles in both migration and proliferation through MAP kinase signaling via ERK (Fromigue et al. 2012). itgb1 expression via mRNA in situ hybridization is relatively unchanged in prdm1a mutants, which is not unexpected since itgb1 is known bind with other alpha subunits and not exclusively with itga5 (data not shown). The expression pattern of itgb1 is also more diffuse throughout the embryo, possibly reflecting localization of its other binding partners. Specifically, itga5 has been shown to play a role in inducing proliferation and differentiation in bone and in dental pulp stem cells (Hamidouche et al. 2009; Fromigue et al. 2012; Cui et al. 2014). itga5 knockdown using short hairpin RNAs in human dental pulp stem cells leads to a decrease in proliferation, and promotion of odontogenic differentiation (Cui et al. 2014). Itga5 activity is mediated through FAK, PI3K, and ERK, which have a known role in proliferation, suggesting a potential mechanism, although proliferation was not directly assayed in this study (Hamidouche et al. 2009). itga5 ultimately promotes an upregulation of osteoblast markers such as runx2, and increased osteogenesis (Hamidouche et al. 2009). In another study, Fromigue et al. show increased phospho-FAK and phosphorERK1/2 expression when itga5 peptide is overexpressed coupled with a two- fold increase in bone thickness, yet they did not observe an increase in BrdU incorporation (Fromigue et al. 2012). Interestingly, previous data have shown that itga5 mutants exhibit a hyoid arch defect, specifically a hypoplastic hyosymplectic (Crump et al. 2004) and variable posterior PA defects. We observe a more severe phenotype with the loss of both prdm1a and itga5, including loss of CB 2 which is derived from PA 3. Our data support a more posterior PA role for itga5, and this role is supported by experiments in the posterior cranial placode development (Bhat and Riley 2011). Furthermore, a role for prdm1a in the posterior cranial placodes has also been described, and localizes itga5 and prdm1a to the same region (Bhat and Riley 2011; Culbertson et al. 2011). Though these studies do not directly assay for proliferation, itga5 expression protects embryos from apoptosis as they observe a two-fold increase of apoptosis in itga5 morphants (Bhat and Riley 2011). These data suggest that itga5 acts via FGF8/MAP Kinase/PI3K signaling, and that this signaling pathway may be responsible for protecting against cell death (Bhat and Riley 2011). Since MAP Kinase family members do play a role in proliferation, it would be reasonable to hypothesize in the posterior pharyngeal arches, prdm1a through itga5 promotes proliferation, possibly via MAP kinase family since our previous data show no change in apoptosis (Birkholz et al. 2009; Bhat and Riley 2011). Here we show that itga5 expression is downregulated in prdm1a mutants, that prdm1a expression is also downregulated in itga5 mutants, and that both itga5 and prdm1a are both localized to the pharyngeal arches during craniofacial development. We further demonstrate that in double mutants, we observe a more severe phenotype and that the prdm1a mutant phenotype can be rescued with itga5 mRNA. We further show that proliferation is decreased in prdm1a mutants and can be rescued with the addition of itga5 mRNA. Taken together, our data show that there is an epistatic relationship between prdm1a and itga5 where itga5 expression can rescue the prdm1a mutant phenotype and a double mutant exhibits a more severe additive phenotype.

Methods

Zebrafish maintenance and lines

Zebrafish were cared for and maintained according to Westerfield’s Zebrafish Book (Westerfield 1993). The TAB and AB wildtype strains were used (Zirc) and mutant lines include prdm1am805 (narrowminded(nrd))(Artinger et al. 1999; Hernandez-Lagunas et al. 2005) and itga5b926 which were a generous gift from the Crump Lab (Crump et al. 2004); along with the tg[prdm1a::GFP] line (Elworthy et al. 2008). All embryos were staged following previously published standards for developmental staging (Kimmel et al. 1995) and mutants were genotyped using primers from the respective papers. All experiments utilizing zebrafish embryos conform to NIH regulatory standards of care and treatment and are approved by the UC Denver IACUC.

Morpholino and mRNA injections

Morpholino oligonucleotides (Gene Tools) were injected at the 1 cell stage with rhodamine-dextran (Sigma). prdm1a E2I2 splice site Morpholino was injected at 6 ng (Hernandez-Lagunas et al. 2005) and itga5 exon-intron 13 splice site Morpholino (Crump et al. 2004) was also injected at 6 ng. itga5 mRNA was amplified from 24 hpf cDNA isolated from whole embryos and cloned into pENTR/D-TOPO (Invitrogen) using the following primers: 5′-GGTTAAGGACGTGAACCATCTCTTCG and 3′-GGGGATAGACACGTTCGTCCA. The itga5 fragment was then put into pCS2+DEST using gateway cloning (Villefranc et al. 2007). itga5 mRNA was synthesized using the mMessage mMachine kit (Ambion). mRNA was injected at the 1-cell stage at 50 pg, 90 pg, 112 pg and 136 pg. prdm1a and itga5 mutant embryos were genotyped as described previously (Crump et al. 2004; Hernandez-Lagunas et al. 2005). Double mutant embryos were phenotyped for prdm1a mutant phenotypes, cut in half, and heads were used for staining and tails were genotyped.

In situ hybridization and Immunohistochemistry

Whole-mount RNA in situ hybridization (ISH) was performed as previously described (Thisse 1998) and visualized using the BM purple substrate (Roche). Fluorescent ISH was performed as previously described (Pineda et al. 2006; Powell et al. 2013). Briefly, a fluorescein conjugated antisense probe was synthesized from a full length pCS2+ GFP plasmid and used along with our antisense DIG conjugated probe for itga5 (Zirc). Antisense DIG conjugated probes were synthesized from full-length sequences out of the pCS2+ plasmid for the following genes: dlx2a (Akimenko et al. 1994), itga5 (ZIRC), sox10 (Olesnicky et al. 2010), and barx (Sperber and Dawid 2008). Immunohistochemistry was performed as previously described (Ungos et al. 2003; Birkholz et al. 2009; Johnson et al. 2011) and the phosphohistone H3 antibody (Upstate) was used at 1:500 and the Alexa 568 Goat anti Rabbit (Invitrogen) at 1:750. Embryos were imaged on a Leica confocal for subsequent counting of phosphohistone H3 positive cells. We outlined the posterior arch domain and counted as previously described (Birkholz et al. 2009). A student’s T-test was used to analyze significance between the groups.

Skeletal staining

Bone and cartilage staining was performed as previously described (Walker and Kimmel 2007; Johnson et al. 2011). Briefly, 5 dpf embryos were fixed for 1 hour at room temperature in 2% PFA, washed in 100 mM Tris pH 7.5/10 mM MgCl2 buffer, then incubated overnight in 0.04% alcian blue (Anatech Ltd)/80% ETOH/10 mM MgCl2. The next day, embryos were rehydrated through a series of ETOH washed and treated with 3% H2O2 to remove pigment then washed in 25% glycerol/0.1% KOH prior to alizarin red staining (0.5%, Sigma) for 30 minutes at room temperature and subsequent clearing and storage in 50% glycerol/0.1% KOH.

Supplementary Material

Supp FigureS1. Supplemental Figure 1.

prdm1a expression is downregulated in itga5 mutants. Anterior is to the left. (A–D) Lateral views, (A′–D′) dorsal views. prdm1a is expression is downregulated in the posterior pharyngeal arch domain of itga5 mutants at 24 hpf (B. B′) and at 31 hpf (D., D′)(approximately 25% of embryos per clutch) as compared to wild type embryos (A., A′, C, C′).

Acknowledgments

We thank past and present members of the Artinger lab especially Davalyn Powell, Morgan Singleton for excellent zebrafish care, Ana-Laura Hernandez for protocol troubleshooting, Leif Neitzel for dissection guidance, Adriana Estrada-Bernal for genotyping; Gage Crump for the itga5 zebrafish line and reagents. This work is supported by NIDCR post-doctoral fellowship F32DE021920 to K.L, and NIH grant R01DE017699 to K.B.A., and P30NS048154 to the UC Denver zebrafish core facility.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigureS1. Supplemental Figure 1.

prdm1a expression is downregulated in itga5 mutants. Anterior is to the left. (A–D) Lateral views, (A′–D′) dorsal views. prdm1a is expression is downregulated in the posterior pharyngeal arch domain of itga5 mutants at 24 hpf (B. B′) and at 31 hpf (D., D′)(approximately 25% of embryos per clutch) as compared to wild type embryos (A., A′, C, C′).

NIHMS678394-supplement-Supp_FigureS1.tif (12.4MB, tif)

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