Expression of cxcl12a in endodermal pouches. (A) Temporal evaluation of cxcl12a transcriptional activity at 18-somite stage. (B) Spatiotemporal dynamics of cxcl12a expression within the developmental window ranging from 24 hpf to 72 hpf. Triangular markers delineate regions manifesting cxcl12a transcripts in the pharyngeal area. (C) Concomitant fluorescence in situ hybridization of cxcl12a at 36 hpf employing Tg(nkx2.3:mCherry) transgenic embryos. Probes used for staining include cxcl12a-Fluorescence probe (green) and mCherry-DNP (red) probes. Scale bar, 50 μm.

Depletion of cxcl12a impairs craniofacial cartilage development. (A) Phenotypic assessment of embryos at designated developmental stages, noting discernible craniofacial aberrations and pericardial edema in cxcl12a mutant embryos. The triangle annotates the zebrafish mandibular region. Scale bars set at 200 mm. (B) Alcian blue histochemical staining of craniofacial cartilage structures at 5 dpf and corresponding in situ hybridization images of col2a1a. Anatomical components denoted are: ep, ethmoid plate; tc, trabeculae cranii; mk, Meckel’s cartilage; bh, basihyal; ch, ceratohyal; pq, palatoquadrate; hs, hyosymplectic; cb, ceratobranchial. The triangle designates the mandibular sector. Scale bars: 100 µm.

NCC specification and migration. (A) Comparative in situ hybridization analysis of wild type and cxcl12a mutant embryos administered either control MO or cxcl12b MO, harvested at indicated time points and probed for foxd3, dlx2a and crestin. (B)In vivo imaging of the pharyngeal compartment of wild type and cxcl12a mutants injected with either control MO or cxcl12b MO, set against a Tg(sox10:EGFP) reporter background. Pharyngeal arches are numerically annotated. PA denotes pharyngeal arch. Scale bars are set at 50 μm.

Cxcl12a function in CNCC proliferation after migration. (A) WT and cxcl12a mutant embryos subjected to control MO or cxcl12b MO administration were harvested at 36 hpf and interrogated via in situ hybridization employing hand2 and sox10 specific probes. Embryonic lateral views are presented with anterior orientation to the left. (B–C)In vivo live confocal micrographs of Tg(nkx2.3:mCherry;fli1:GFP) or Tg(nkx2.3:mCherry;sox10:EGFP) transgenic embryos injected with control MO, cxcl12a MO, or a combination of cxcl12a and cxcl12b MO at 24 hpf. Scale bar, 50 μm. (E,F) Apoptotic cell assessment in pharyngeal domains via TUNEL assays in cxcl12a mutants and wild-type siblings, injected with either control MO or cxcl12b MO against a Tg(sox10:EGFP) background. Pharyngeal arches (PA) are indicated. Scale bars represent 50 μm. Quantitative analysis of TUNEL-positive and GFP-positive cells within the pharyngeal arch was performed from nine independent embryos (F). Error bars depict standard deviation (SD). NS signifies statistical non-significance as per Student’s t-test. (G,H) Bromodeoxyuridine (BrdU) incorporation assays reveal attenuated proliferative activity within CNCC populations in cxcl12a mutants subjected to control MO or cxcl12b MO treatment. Harvested embryos expressing sox10:EGFP were immunostained with anti-BrdU (red) and anti-GFP (green) antibodies. Pharyngeal regions were visualized through confocal microscopy (G). Scale bars are set at 50 μm. Quantification of BrdU-positive and GFP-positive cells within the pharyngeal arch was achieved from nine independent embryos (H). Error bars represent standard deviation (SD). Statistical significance denoted by ***, p < 0.001 as determined by Student’s t-test.

cxcl12a mutants exhibit reduced pharyngeal pouch cells. (A) Expression levels of nkx2.3 and cv2 expression were assayed in wild-type or cxcl12a mutants post-injection with either control MO or cxcl12b MO. (B–C) Confocal microscopy elucidates images showing pharyngeal pouch cellular architecture in wild-type and cxcl12a, or combined cxcl12a and cxcl12b knockdown embryos within a Tg(nkx2.3:mCherry)(B) or Tg(sox17:GFP)(C) background at 36 hpf. Scale bar, 50 μm. (D) Quantification of mCherry-positive cells within the Tg(nkx2.3-mCherry) transgenic line was executed from a sample size of nine embryos. Error bars are indicative of standard deviation (SD). Statistical significance denoted by *, p < 0.05; **, p < 0.01, as gauged by Student’s t-test.