Pax1a and Pax1b regulate ceratobranchial cartilage formation by

expression of fgf3, tbx1 and edn1

Since pax1a- and pax1b-deficient mutants showed pharyngeal pouch

morphogenetic defects like medaka pax1-mutant embryos and Fgfs and Tbx1 are known to be essential for morphogenesis of pharyngeal pouches (Choe and Crump, 2014;

Crump et al., 2004a; Okada et al., 2016), we next compared expression of fgf3, tbx1 and edn1 (a downstream effector of Tbx1) in pax1a- and pax1b-deficient embryos and

control embryos. fgf3 is expressed in the pharyngeal endoderm (Crump et al., 2004b) and tbx1 expresses is detected in mesodermal core and endodermal epithelia of individual arches (Piotrowski and Nusslein-Volhard, 2000). Expression of edn1 is detected in the mesodermal cores, ectoderm and endoderm of pharyngeal arches, but not

in NC (Choudhry et al., 2011). In contrast to control embryos, reduced or absent expression of fgf3, tbx1 and edn1 in pharyngeal pouches 2-4 or 2-5 were detected in pax1a; pax1b morphants at 30 or 36 hpf (Fig. 15A-X). However, tbx1 expression in the

adjacent mesoderm and edn1 expression in the mesoderm core of arches 1-3 was not affected. At 36 hpf, a lack of fgf3, tbx1 and edn1 expression in pharyngeal pouches 2-5 was identified in pax1a MO-injected pax1b^-/- mutants compared to uninjected pax1b^-/- mutant controls, wild types or pax1a MO-injected wild types; meanwhile, tbx1 expression in the adjacent mesoderm and edn1 expression in the mesoderm core of arches 1-3 was not affected (Fig. 16 A-L). Similar defects in endodermal expression of fgf3, tbx1 and edn1 were detected in the pharyngeal pouches of pax1a^-/-; pax1b^-/- double mutants compared to sibling controls at 36 hpf (Fig. 16 M-R).

We next investigated whether Pax1a/b directly regulates fgf3 expression. Two conserved PAX1 binding elements were identified in two enhancer regions located 5′

upstream of fgf3 (-69,697 to -69,448 and -68,981 to -68,869) by the UCSC genome browser (Fig. 17A, B). Initially we evaluated whether Anti-PAX1 antibody from company can recognize endogenous Pax1a or Pax1b protein by Western blot. We isolated total protein from wild type, pax1a^-/- mutant, pax1b MO-injected pax1a -/-mutant, pax1b^-/- mutant, pax1a MO-injected pax1b^-/- mutant and pax1a^-/-; pax1b

-/-double mutant embryos at 36 hpf for SDS PAGE. Although ~40 kDa protein band which

is close to deduced amino acid number of Pax1a or Pax1b appeared when reacted with Anti-PAX1 antibody, they did not reflect expected protein level in different Pax1a and Pax1b deficiency embryos (Fig. 18 A). Since antibodies for zebrafish Pax1a and Pax1b

are not available, we performed chromatin immunoprecipitation by overexpressing pax1a-Myc and pax1b-Myc in 1-2 cell zygotes. Initial Western blot analysis confirmed a

protein band with molecular weight 52 kDa (5x Myc about 10 kDa + 38 kDa of Pax1a or 5x Myc about 10 kDa + 37 kDa of Pax1b), two protein bands between 38 and 24 kDa reacted with anti-Myc antibody for total protein isolated from embryos injected with pax1a-Myc and Myc plasmid (Fig. 18 B). We then injected pax1a-Myc or

pax1b-Myc plasmid into 1 cell zygotes and fixed developing embryos at 32-36 hpf. Isolated

chromatin was precipitated using anti-Myc antibody or anti-IgG antibody. Three protein bands including expected 52 kDa were only identified in supernatant using anti-IgG antibody but not anti-Myc antibody for immunoprecipitation by Western blot analysis;

this indicates Myc-tagged Pax1a or Myc-tagged Pax1b did express in embryos injected with pax1a-Myc or pax1b-Myc plasmid (Fig. 18 C). Subsequently we performed chromatin immunoprecipitation by overexpressing pax1b-Myc in 1-2 cell zygotes.

Injected embryos were fixed at 32-36 hpf, and isolated chromatin was precipitated using anti-Myc antibody or anti-IgG antibody. Significant enrichment of qPCR products was observed when using primer pairs to amplify PAX binding element (PBE) I and II, but

no enrichment was observed when using control primer pair for cdx1b exon II (Fig. 17 C-E). These results suggest that Pax1b can directly bind to PAX binding elements located in the 5′ upstream region of fgf3 to activate its transcription.

4. Discussion

In the present study, we used CRISPR-Cas9 mutagenesis and morpholino-mediated knockdown to show that zebrafish Pax1a and Pax1b redundantly regulate the

development of pharyngeal pouches and ceratobranchial cartilages. Like medaka pax1, zebrafish pax1a and pax1b are expressed in developing pouches. Time-lapse imaging of Tg(pax1b:eGFP) transgenic fish was used to further reveal that eGFP-postive

endodermal cells mark the location of newly developing pharyngeal pouches before they migrate laterally. Furthermore, unsegmented pharyngeal pouches 2-5 (with continuous expression of pax1a or pax1b and small outpocketings) were detected in zebrafish pax1a^-/-; pax1b^-/- double mutant embryos (Fig. 14), indicating Pax1a and Pax1b are essential for segmentation of pharyngeal pouches 2-5. We suspect that similar to medaka Pax1, zebrafish Pax1a and Pax1b may control pharyngeal pouch 2-5

segmentation via indirect downregulation of pax1a or pax1b transcription in the inter-pouch region. In pax1a^-/-; pax1b^-/- double mutant embryos, a lack of non-cell

autonomous negative regulation of pax1a or pax1b in the inter-pouch endoderm would result in unsegmented pharyngeal pouches 2-5 with continuous expression of pax1a or pax1b, much like the phenotype in medaka (Okada et al., 2016). However, in contrast to

the developmental defects in the undulated mouse vertebral column and abnormalities in the vertebral body and neural arches in medaka pax1 morphants, development of the

vertebral body was not affected in zebrafish pax1a- and pax1b-deficient embryos (Fig.

9). This difference may be due to the strong rostral to weak posterior expression pattern of pax1a (Fig. 2), in addition to the weak pax1b expression in cells within the

sclerotome (Fig. 3).

Also similar to the findings in medaka, this phenotype may be attributable to absent expression of fgf3 and tbx1 in the pharyngeal pouches (Fig. 16). Although fgf8a mutant and fgf3 morphant embryos possess normal pharyngeal pouches, pouch-forming endodermal cells fail to migrate laterally and distinct pouches are not formed in fgf8a and fgf3 double deficient embryos (Crump et al., 2004a). Based on these observations, it has been hypothesized that there is requirement for Fgf signaling from the lateral

mesoderm and ventral hindbrain during segmentation of the pharyngeal endoderm into pharyngeal pouches. In addition to Fgfs, Tbx1 is known to be essential for the

morphogenesis of pharyngeal pouches (Choe and Crump, 2014). Notably, while tbx1 mutant embryos fail to generate outpocketing in pouch-forming endoderm, Alcama expression is retained. Mesodermal Tbx1 is known to promote morphogenesis of pharyngeal pouches via modulation of wnt11r and fgf8a expression. Consequently, we conclude that like medaka Pax1, zebrafish Pax1a and Pax1b control morphogenesis of pharyngeal pouches via activation of fgf3 and tbx1. Interestingly, Alcama was also not detected in pax1a^-/-; pax1b^-/- double mutant embryos (Fig. 14), suggesting that zebrafish

Pax1a and Pax1b may regulate alcama expression as well.

Pharyngeal cartilage defects, including a lack of ceratobranchial cartilages 1-4 and minor defects in hyoid cartilage (loss of anterior hyomandibula, hyosymplectic and ceratohyal are not fused), were identified in zebrafish mutant embryos deficient in pax1a and pax1b (Fig. 9) as well as medaka pax1 mutant embryos (Okada et al., 2016).

In addition, zebrafish pax1a- and pax1b-deficient embryos share similar ceratobranchial cartilage defects with mutants or morphants of fgf3 or tbx1 (David et al., 2002; Herzog et al., 2004; Piotrowski et al., 2003). Downregulation of dlx2a in the cranial neural crest cells of stream III was detected in fgf3 morphants and mutants at 24 or 26 hpf,

indicating that Fgf3 has an early function of maintaining dlx2a expression for the

specification and survival of posterior neural crest cells. A reduction of dlx2a expression in the branchial arches was also identified in the tbx1 mutant embryo beginning at 16.5 hpf, suggesting that Tbx1 also participates in the specification and survival of posterior neural crest cells by modulating dlx2a expression. Zebrafish Dlx2a is essential for survival of neural crest cells that migrate to the pharyngeal arches (Sperber et al., 2008).

Additionally, the Dlx2 gene is known to play an important role in chondrogenesis as a regulator of cell-cell adhesion during mesenchymal condensation in the branchial arches of chick embryos (McKeown et al., 2005). In zebrafish pax1a- and pax1b-deficient embryos, reduced dlx2a expression in pharyngeal arches 3 and 4 was found at 22 hpf,

and absence of dlx2a expression in the same pharyngeal arches was observed beginning at 26 hpf (Fig. 11, 12). As a result, we propose that zebrafish Pax1a and Pax1b may modulate expression of fgf3 and tbx1 in pharyngeal pouches to maintain dlx2a

expression in stream III cranial neural crest cells, allowing for proper development of ceratobranchial cartilage. Although dlx2a was also shown to be necessary for neural crest cell survival within the first and second pharyngeal arches at 32 hpf in dlx2a morphants (Sperber et al., 2008), we detected similar numbers of TUNEL-stained neural crest cells in pharyngeal arches 3 and 4 when comparing pax1a^-/-; pax1b^-/- double

mutants and wild-type embryos at 26 hpf (Fig. 13). Nevertheless, this result does not preclude the possibility that apoptosis may occur in the cranial neural crest cells of stream III during migration to the pharyngeal arches 3-6 in pax1a- and pax1b-deficient embryos. Such a possibility will be explored in future studies.

Substantial reductions in expression of edn1 and hand2 were detected in the posterior pharyngeal arches 3-5 compared to anterior pharyngeal arches 1-2 of the tbx1 mutant at 25 or 34 hpf, suggesting that Edn1 and Hand2 are downstream effectors of Tbx1 (Piotrowski et al., 2003). In suc mutants, substantial decreases in hand2

expression were reported in the first and second arch as well as posterior arches (Miller et al., 2000). This observation suggests that Edn1 is non-autonomously required to maintain hand2 expression in cranial neural crest cells for the specification of ventral

pharyngeal cartilages. Furthermore, Edn1 signaling is required for the dorsal-ventral patterning of pharyngeal arches due to its regulation of hand2 and dlx genes (dlx2a, dlx5a, dlx3b, dlx6a), which are expressed in gradients along the axis (Walker et al.,

2006). Edn1 interacts with EdnrA/B to regulate hand2 expression in the ventral domain of early arches at 24 hpf, an action that is mediated by modulation of dlx5/dlx6

expression (Medeiros and Crump, 2012). In zebrafish pax1a- and pax1b-deficient embryos, a lack of tbx1 and edn1 expression was found in pharyngeal pouches 2-5, and hand2 expression was absent in pharyngeal arches 3-6 (Fig. 11, 16). Therefore, we

propose that zebrafish Pax1a and Pax1b may regulate expression of tbx1, which in turn modulates expression of effectors (edn1 and hand2) for proper development of

ceratobranchial cartilage. Besides the ceratobranchial cartilage defect, a loss of the anterior half of hyomandibula cartilage in the hyoid cartilage was observed in pax1a^-/-; pax1b^-/- double mutants (Fig. 9), similar to the phenotypes observed in integrinα5 and fgf8a mutants (Crump et al., 2004a; Crump et al., 2004b). Like these mutants, pax1a^-/-;

pax1b^-/- double mutants exhibited morphogenetic defects in pharyngeal pouch 1, likely meaning that integrinα5 signals could not be properly generated in the first pouch to

stabilize neural crest cells of the anterior hyomandibula in the adjacent hyoid cartilage.

In addition, we noticed that endogenous pax1b expression was increased in heterozygous but decreased in homozygous Tg(pax1b: eGFP) embryos compared to

wild type embryos (Fig 5). This observation is partly consistent with previous finding that the insertion of a Tol2 transposon-mediated gene trap construct interfere with endogenous transcript of trapped gene (Kawakami et al., 2004). We considered that the insertion of Tol2-Gal4-VP16; UAS: EGFP-Tol2 plasmid in the 3’UTR of pax1b gene may inhibit transcription of endogenous pax1b mRNA in homozygous Tg(pax1b:

eGFP) embryos. However, it is not clear why endogenous pax1b transcription was not

affected in heterozygous Tg(pax1b: eGFP) embryos as well. We also noticed that pax1a^-/-; pax1b^-/- double mutants lacked an inflated swim bladder at 96 hpf (Fig. 9I-L).

A previous study indicated that the inflation of swim bladder was affected in zebrafish embryos treated with a thyroid peroxidase inhibitor or knockdown of iodothyronine deiodinase, which is required to activate T4 into biologically active T3 form (Stinckens et al., 2016). Organogenesis of thymus and thyroid is conserved between mammals and zebrafish; thyroid gland is derived from precursors evaginated from the ventral floor of the pharynx. In contrast to mammalian thyroid gland is enclosed by connective tissue, thyroid follicles of teleosts are loosely distributed along the ventral aorta in the lower jaw region. In zebrafish larvae, T4 immunostaining showed that some non-follicular T4 domains were located ventral to the anterior basibranchial cartilage while later

appearing thyroid follicles were distributed along the ventral aorta (Wendl et al., 2002).

Therefore, we considered that lack of inflated swim bladder in pax1a^-/-; pax1b^-/- double

mutant embryos may be attributed to failure of development or function of thyroid follicular cells. To answer this speculation, we can compare expression level of hhex, nk2.1a and pax2.1 which are required for differentiation or growth of thyroid follicular

cells and analyze level of T4 and T3 in pax1a^-/-; pax1b^-/- double mutants and wild type embryos (Elsalini et al., 2003; Wendl et al., 2002). Interestingly, mouse Pax1 is also expressed in the third pharyngeal endodermal pouch, from which thymus epithelium is derived, and it is later expressed in the adult thymus, where it is required for thymocyte maturation into T lymphocytes (Wallin et al., 1996). In pax1 medaka mutants, thymus-specific expression of foxN1 was also lost (Okada et al., 2016). We can also evaluate foxn1 gene expression in pax1a^-/-; pax1b^-/- double mutant embryos to understand whether thymus development is also affected.

5. Conclusion

In conclusion, zebrafish Pax1a and Pax1b have overlapping function in the modulation of proper morphogenesis of pharyngeal pouches and ceratobranchial cartilage formation (Fig. 19). Zebrafish Pax1a and Pax1b act as an upstream integrator, modulating expression of fgf3 and tbx1 in pharyngeal pouches. Fgf3 signaling and Tbx1 are known to regulate dlx2a expression in the neural crest cells, as an essential action for neural crest differentiation and chondrogenesis. Tbx1 is also known to regulate expression of edn1, which then interacts with EdnrA/B in neural crest cells to modulate expression of downstream genes essential for neural crest differentiation and cartilage formation (i.e., hand2, dlx3b, dlx5a, and dlx6a). In zebrafish pax1a- and pax1b-deficient embryos, downregulation of fgf3, tbx1 and edn1 in the pharyngeal pouches as well as an absence of dlx2a and hand2 expression in the developing posterior pharyngeal arches result in a lack of ceratobranchial cartilage formation. Together with previous findings, our data provide a more complete understanding of the molecular circuitries controlling pharyngeal cartilage development from pharyngeal endodermal pouches in zebrafish.

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