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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Figures
Fig. 1. Embryonic expression level of pax1a-201 and pax1a-202. Expression level of pax1a-201 (red) and pax1a-202 (blue) were evaluated by RT-qPCR at 24, 48, 72 and 96 hpf. Expression level of pax1a-201 is roughly half of pax1a-202 at these stages.
Fig. 2. Developmental expression patterns of zebrafish pax1a gene. (A) Semi-quantitative RT-PCR indicates expression of pax1a from 1 cell to 18 hpf stages.
Expression of β-actin was used as a control. Quantitation of pax1a/α-actin band
intensity ratio from 1-cell to 18 hpf stages is shown. M, molecular weight marker; N, no template control. (B-Q) Signals for pax1a was detected by in situ hybridization in the developing pharyngeal pouches (pp) and sclerotome (s) from 18 to 120 hpf. Embryos in lateral or dorsal view are shown from 18 hpf (B, F), 24 hpf (C, G), 30 hpf (D, H), 36 hpf (E, I), 48 hpf (J, N), 72 hpf (K, O), 96 hpf (L, P) and 120 hpf (M, Q). Paraffin sections of a pax1a hybridized embryo shows pax1a signal in different pharyngeal pouches (pp) at 36 hpf (R, S), 48 hpf (T) and 96 hpf (U). sh, shield. Scale bar represents 100 µm.
Fig. 3. Developmental expression patterns of zebrafish pax1b gene. (A) Semi-quantitative RT-PCR indicates expression of pax1b from 1 cell to 18 hpf stages.
Expression of β-actin was used as a control. M, molecular weight marker; N, no template control. Quantitation of pax1a/α-actin band intensity ratio from 1-cell to 18 hpf stages is shown. (B-Q) pax1b is expressed in the developing pharyngeal pouches (pp) and sclerotome (s) from 18 to 120 hpf by in situ hybridization. Embryos in lateral view or dorsal view are shown from 18 hpf (B, F), 24 hpf (C, G), 30 hpf (D, H), 36 hpf (E, I), 48 hpf (J, N), 72 hpf (K, O), 96 hpf (L, P) and 120 hpf (M, Q). Paraffin sections of a pax1b hybridized embryo showed signals locates in different endodermal pouches during at 36 hpf (R), 48 hpf (S) and 96 hpf (T). sh, shield. Scale bars represent 100 µm.
Fig. 4. pax1b is expressed in pharyngeal pouches but not in the neural crest cells of pharyngeal arches. (A-C) Double fluorescence in situ hybridization revealed co-expression of pax1a (A, red) and pax1b (B, greed) in pharyngeal pouches 1-5 at 36 hpf.
Merged image (C) is shown. Lateral view of embryos is shown with anterior to the right. (D-F) Double fluorescence in situ hybridization indicated pax1b (D, red) is co-expressed with nkx2.3 (E, green) in pharyngeal pouches (pp) 2-6 (F, merge) at 36 hpf.
(G-H) pax1b (G, red) is also co-expressed with nkx2.7 (H, green) in pharyngeal pouches 3-6 (I, merge). (J-L) Expression of pax1b (J, red) in the pharyngeal pouches is adjacent to dlx2a (K, green), which is expressed in the neural crest cells of pharyngeal arches (pa). Relative localization is apparent in the merged image (L). Scale bars represent 100 µm.
Fig. 5. Comparison of pax1b expression level between wild type and Tg (pax1b:
eGFP) embryos. RT-qPCR was conducted. Compared to wild type embryos, a 1.5 fold increase of pax1b level was identified in heterozygous Tg (pax1b: eGFP) embroyos while a 0.4 fold reduction of pax1b level was detected inhomozygous Tg (pax1b:
eGFP) embroyos.
Fig. 6. Time lapse analyses of Tg(pax1b:eGFP) enhancer trap transgenic embryos reveals sequential segmental development of pharyngeal pouches.
Double fluorescence in situ hybridization indicated co-expression of egfp (A, D, green) with pax1b (B, E, red) in pharyngeal pouches (pp) 1-5 or 1-6 (C, F, merge) at 30 and 36 hpf. Time lapse analyses of Tg(pax1b:eGFP) transgenic embryos show morphogenesis of pharyngeal pouch 1-6 during 12 to 34 hpf stages. Lateral view of a Tg(pax1b:eGFP) embryo at 12 hpf (G), 12.5 hpf (H), 13 hpf (I), 14 hpf (J), 15 hpf (K), 16 hpf (L), 18 hpf (M), 20 hpf (N), 22 hpf (O), 24 hpf (P), 26 hpf (Q), 28 hpf (R), 30 hpf (S), 32 hpf (T) and 34 hpf (U) are shown. Scale bars represent 100 µm.
Fig. 7. Specificity evaluation of pax1a MO and pax1b MO. (A) Sequences of pax1a MO andpax1a 5mm MO as well as complementary pax1a mRNA sequence are shown . (B) Sequences of pax1b MO and pax1b 5mm MO as well as complementary pax1b mRNA sequence are shown. (C, E) eGFP green fluorescence is detected in embryos co-injected with pax1a-eGFP-pcDNA3 or pax1b-eGFP-pcDNA3 plasmid and respective pax1a or pax1b 5mm MO at 24 hpf. (D, F) No eGFP expression can be observed in the majority of embryos co-injected with pax1a-eGFP-pcDNA3 or pax1b-eGFP-pcDNA3 plasmid and respective pax1a or pax1b MO.
Fig. 8. Pax1a and Pax1b are redundant in regulating pharyngeal cartilage development. Mandibular pharyngeal arch 1 (Meckel’s cartilage (m) and
palatoquadrate (pq)), hyoid pharyngeal arch 2 (hyosymplectic (hs), interhyal (ih), ceratohyal (ch)) and five branchial arches (ceratobranchial (cb), hypobranchial (hb) and basibranchial (bb)) were identified in Alcian blue-stained wild type (A) and embryos injected with 0.75 ng each of 5mm pax1a MO and 5mm pax1b MO (B) at 96 hpf are shown. Mild pharyngeal cartilage defect (C, shortened cb 2-3, lack of cb 1 and 4 on one side and lack of cb1 on the other side) and two types of severe cartilage defects (D, straightened ch, shortened cb 2-3, lack of cb 1 and 4 on one side and lack of cb1-4 on the other side; E, reversed ch and lack of cb 1-4 on both sides) were detected in
respective embryos injected with 1.5 ng pax1a MO (MOa), 10 ng pax1b MO (MOb), or 0.75 ng of both pax1a MO and pax1b MO. The majority of embryos injected with suboptimal dose (0.75 ng) of both pax1a MO and pax1b MO exhibited severe
pharyngeal cartilage defects, while embryos injected with 1.5 ng pax1a MO or 10 ng pax1b MO showed less frequent severe phenotypes (F, G). Number of embryos for various treatment is shown under the x -axis. Brackets indicate lack of cb1-4. Scale bar represents 100 µm.
Fig. 9. pax1a- and pax1b-deficient embryos generated by CRISPR-Cas9
mutagenesis exhibit abnormality of hyoid cartilage and lack of ceratobranchial cartilages 1-4. Genomic structures of pax1a (A) and pax1b (B) include the target sites for pax1a sgRNA and pax1b sgRNA in exon 2 of each gene. Nucleotide sequence of wild type (WT) and sequences of pax1aas36 and pax1bas37 mutants containing 5-bp deletions (red dashed line) are shown (A, B). sgRNA sequence (yellow highlight) and protospacer adjacent motif (PAM, blue highlight) are indicated. (C, D) Sequences of WT and pax1aas36 or pax1bas37 mutants were compared. Positions of deleted sequences in pax1aas36 or pax1bas37 mutants are indicated by arrowheads. Normal patterning of pharyngeal arches was detected in Alcian blue-stained uninjected (E) or 1.5 ng pax1b MO-injected pax1a+/- incrossed embryos (F) at 96 hpf. About 21.1% of pax1a +/-incrossed embryos that had been injected with 1.5 ng pax1b MO failed to develop ceratobranchial cartilages (G, H). Similarly, normal pharyngeal cartilage development was identified in Alcian blue-stained uninjected pax1b-/- incrossed embryos (I) while loss of ceratobranchial cartilage and hyoid cartilage with inverted (J) or no (K) anterior-posterior polarity were detected in 0.75 ng pax1a MO-injected pax1b-/- incrossed embryos stained with Alcian blue at 96 hpf. A comparison of the percentage embryos with pharyngeal cartilage defects in uninjected and pax1a MO-injected pax1b-/- incross embryos is shown (L). A pax1a-/-; pax1b-/- double mutant embryo (N) exhibits a straight body, lack of swim bladder (arrowhead) and lack of gill cartilages (arrow), in contrast with wild type (M) at 96 hpf. Alcian blue-stained pax1a-/-; pax1b-/- double mutant embryo (P) has no ceratobranchial cartilage, in contrast with pax1a-/-; pax1b+/- mutant embryo (O) with normal pharyngeal cartilages at 96 hpf. Flat mount Alcian blue
staining of pharyngeal cartilage revealed loss of ceratobranchial (cb) cartilages 1-4 with retention of shorter cb cartilage 5 in pax1a-/-; pax1b-/- double mutants. Basibranchial (bb) cartilages 2-5 were also absent, with retention of bb cartilage 1. All hypobranchial (hb) cartilages and anterior hyomandibula (hm, arrowhead) were also absent in pax1a-/-; pax1b-/- double mutants (S) in contrast to wild type (Q) and pax1b-/- mutant embryos (R) at 96 hpf. Brackets indicate lack of cb1-4. Ch, ceratohyal; ih, interhyal; m, Meckel’s cartilage; op, opercular bone; pq, palatoquadrate; sy, symplectic; te, teeth. Scale bars represent 100 µm.
Fig. 10. Knockdown of pax1a and pax1b decreases expression of neural crest markers dlx2a or hand2 or endodermal pouches marker nkx2.3. Reduced dlx2a expression (C, D) is detected in 3-5 pharyngeal arches in pax1a; pax1b morphant embryos compare with WT (A) and 5mm control (B) at 30 hpf. Reduced (G) or absent (H) of hand2 expression are observed in 3-5 pharyngeal arches of pax1a; pax1b morphant embryos compare with WT (E) and 5mm control (F) at 30 hpf. Abnormal (K) and partial absent (L) nkx2.3 expression pattern is detected in pax1a; pax1b morphant at 36 hpf. Asterisks indicate reduced or absent expression. Affected embryos per total number of analyzed embryos are shown. Scale bars represent 100 µm.
Fig. 11. pax1a- and pax1b-deficient embryos display decrease or absence of dlx2a and hand2 expression in pharyngeal arches. Reduced expression of dlx2a (D) or hand2 (I) as well as absence of dlx2a (E) or hand2 (J) in the posterior pharyngeal arches (pa) was identified in 0.75 ng pax1a MO-injected pax1b-/- mutant embryos compared to uninjected pax1b-/- mutant (C, H), wild type (WT; A, F) or 0.75 ng pax1a MO-injected wild type (B, G) at 36 hpf. Similar defects in expression of dlx2a (L) or hand2 (N) in the posterior pharyngeal arches were also identified in pax1a-/-; pax1b-/- double mutants compared with sibling controls (i.e., pax1a+/-; pax1b-/- or pax1a+/+; pax1b-/-) (K, M) at 36 hpf. Asterisks indicate reduced or absent expression. Affected embryos per total number of analyzed embryos are shown. Scale bars represent 100 µm.
Fig. 12. Reduced dlx2a expression in pharyngeal arches 3-4 in pax1a; pax1b
morphants beginning at 22 hpf. Reduced dlx2a expression in pharyngeal arches 3-4 in pax1a; pax1b morphants beginning at 22 hpf. Similar dlx2a expression levels were detected in three migrating streams (S1, S2, S3) of cranial neural crest cells in uninjected (C), 0.75 ng pax1a MO-injected pax1b-/- mutant embryos (D), uninjected wild type (WT; A) or 0.75 ng pax1a MO-injected wild type (B) during 18-22 s stages.
Similar dlx2a expression levels in three migrating streams of cranial neural crest cells were observed in wild type and pax1a; pax1b morphants at 18 hpf (E, L) and 20 hpf (F, M). However, reduced dlx2a expression (asterisk) was identified in pharyngeal arches (pa) 3-4 of pax1a; pax1b morphants compared to wild type embryos at 22 hpf (G, N), 24 hpf (H, O), 26 hpf (I, P), 28 hpf (J, Q) and 30 hpf (K, R). Scale bars represent 100 µm.
Fig. 13. Similar levels of apoptosis and proliferation were observed in pax1a- and pax1b-deficient embryos. TUNEL assay was performed in wild type (WT) (A, B) and pax1a-/-; pax1b-/- double mutant (C, D) embryos that were hybridized with dlx2a antisense RNA at 26 hpf. Quantification of apoptotic cells in pharyngeal arches (pa) 3
Fig. 13. Similar levels of apoptosis and proliferation were observed in pax1a- and pax1b-deficient embryos. TUNEL assay was performed in wild type (WT) (A, B) and pax1a-/-; pax1b-/- double mutant (C, D) embryos that were hybridized with dlx2a antisense RNA at 26 hpf. Quantification of apoptotic cells in pharyngeal arches (pa) 3