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. 2011;6(9):e24443.
doi: 10.1371/journal.pone.0024443. Epub 2011 Sep 9.

Chondrogenic and gliogenic subpopulations of neural crest play distinct roles during the assembly of epibranchial ganglia

Affiliations

Chondrogenic and gliogenic subpopulations of neural crest play distinct roles during the assembly of epibranchial ganglia

Maya D Culbertson et al. PLoS One. 2011.

Abstract

In vertebrates, the sensory neurons of the epibranchial (EB) ganglia transmit somatosensory signals from the periphery to the CNS. These ganglia are formed during embryogenesis by the convergence and condensation of two distinct populations of precursors: placode-derived neuroblasts and neural crest- (NC) derived glial precursors. In addition to the gliogenic crest, chondrogenic NC migrates into the pharyngeal arches, which lie in close proximity to the EB placodes and ganglia. Here, we examine the respective roles of these two distinct NC-derived populations during development of the EB ganglia using zebrafish morphant and mutants that lack one or both of these NC populations. Our analyses of mutant and morphant zebrafish that exhibit deficiencies in chondrogenic NC at early stages reveal a distinct requirement for this NC subpopulation during early EB ganglion assembly and segmentation. Furthermore, restoration of wildtype chondrogenic NC in one of these mutants, prdm1a, is sufficient to restore ganglion formation, indicating a specific requirement of the chondrogenic NC for EB ganglia assembly. By contrast, analysis of the sox10 mutant, which lacks gliogenic NC, reveals that the initial assembly of ganglia is not affected. However, during later stages of development, EB ganglia are dispersed in the sox10 mutant, suggesting that glia are required to maintain normal EB ganglion morphology. These results highlight novel roles for two subpopulations of NC cells in the formation and maintenance of EB ganglia: chondrogenic NC promotes the early-stage formation of the developing EB ganglia while glial NC is required for the late-stage maintenance of ganglion morphology.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Neural crest cells migrate to abut epibranchial placodes.
(A) Lateral view of Tg(pax2a:EGFP)w37/+ embryo at the six somite stage showing fluorescein uncaged in rhombomere 5 (outlined region). Placode precursor field expressing EGFP is visible below the uncaged region (arrowheads). (B) Lateral view of the same embryo at 24 hpf. Fluorescein-labeled NC cells derived from rhombomere 5 (green) are visible in two regions (brackets) ventral to the otic vesicle (asterisk). (C and D) Transverse section through embryo at level indicated by dotted line in B. Placode is visible as thickened ectoderm under transmitted light and by α-Pax2 staining in red (C and D, thick arrows). In D, fluorescein-labeled cells are just medial to, but excluded from, the placode. (E and F) In situ hybridization at the level of the vagal placode shows neural crest-derived glial precursors adjacent to the neural tube expressing foxd3; in F, a group of cells corresponding to the fluorescein-labeled region in D expressing the chondrogenic crest marker dlx2a. Placodes in E and F indicated by black arrows. Scale bar = 50 µm.
Figure 2
Figure 2. Disruptions in neural crest cause late defects in epibranchial ganglion morphology without affecting placode specification.
(A–E) Ventral view of head skeleton visualized with anti-Collagen2a antibody at 4 dpf. (A) The following structures are visible in wildtype: Meckel's cartilage, palatoquadrate, ceratohyal and ceratobranchial arches. Sox10 mutant (B) exhibits normal morphology. Radical disruption of structure is apparent in foxd3 mutants (C) and disc1 morphants (E). prdm1a mutant (D) displays grossly normal anterior jaw structures and deficits in the posterior arches; in this example, an unpaired ceratobranchial vestige can be seen (arrowhead). (F–J) Anti-Pax2 immunofluorescence at 24 hpf. Wildtype (F) shows facial placode and unsegmented glossopharyngeal/vagal placode field (K–O) phox2b expression at 48 hpf. Wildtype (K) displays normal segregation of facial,glossopharyngeal, and vagal ganglia. Defects are apparent in foxd3 (M) and prdm1a (N) mutants and disc1 morphant (O). sox10 mutant (K) display morphology and segmentation comparable to wildtype siblings (F). P–T: Ganglion morphology visualized by anti-Elavl3/4 antibody staining at 4 dpf. (K) Wildtype control shows glossopharyngeal and vagal ganglia. Posterior lateral line, statoacoustic and the fused trigeminal, anterior lateral line and facial ganglia can also be seen. Defects are apparent in sox10 (Q), foxd3 (R), prdm1a (S) mutants and disc1 morphant (T). Arrow in (S) shows an example of fusion often seen in prdm1a mutants. Abbreviations: mk = Meckel's cartilage; pq = palatoquadrate; ch = ceratohyal; cb = ceratobranchial arches; OV = otic vesicle; F = facial ganglion; Tg/aLL/F = trigeminal/anterior lateral line/facial ganglion complex; SA = statoacoustic ganglion; pLLg = posterior lateral line ganglion; G = glossopharyngeal ganglion; V = vagal ganglia. Scale bar in (A) = 100 µm. Scale bars in (F), (K) and (P) = 50 µm.
Figure 3
Figure 3. Wildtype and sox10−/− NC is capable of rescuing cranial ganglion formation in prdm1anl3 mutant embryos.
(A–F) EB ganglion rescue mediated by transplantation of wildtype NC into prdm1anl3 mutant embryos expressing Tg(NeuroD:EGFP). (A, B) Left (control) and right (transplanted) lateral views of prdm1anl3 mutant head with rhodamine-positive wildtype NC donor cells at 3 dpf. Tg(neuroD:EGFP)nl1 expression reveals an absence of identifiable cranial ganglion structures on the non-transplanted side (A) while transplanted side shows restoration of these structures, including identifiable glossopharyngeal and vagal ganglia. (C, D) Distribution of rhodamine-positive donor NC cells shows an absence of fluorescent label in the cranial ganglion field of the non-transplanted side (C). In the transplant side (D), rhodamine-positive donor cells are present in parallel columns extending ventrally from the ganglion field (arrows) and in a single line perpendicular and immediately dorsal to columns (star), likely corresponding to the ceratobranchials and the rostral basicranial commisure, respectively. (E and F) Merged channel high-magnification views of control (E) and rescue (F) sides with DAPI counterstain (blue). Note DAPI-labeled branchial arches containing rhodamine-positive donor cells (brackets). (G–L) Partial EB ganglion rescue following transplantation of sox10−/− NC cells into prdm1anl3 host embryos. (G,H) Tg(NeuroD:EGFP) expression in prdm1anl3 embryo showing absence of small EB ganglia in control side (G) and a partial rescue of ganglion formation in ventral vagal region of transplanted side (arrowhead). (I,J) Distribution of rhodamine-labeled donor cells in control side (I) and rescue side (J). (K,L) Merged channel high-magnification views of control (K) and rescue (L) sides with DAPI counterstain. Rescued side shows juxtaposition of rescued ganglion with the rhodamine-positive arches (brackets). Margins of statoacoustic ganglia are indicated by dashed white lines. Abbreviations: Tg/aLL/F = trigeminal/anterior lateral line/facial ganglion complex; SA = statoacoustic ganglion; pLLg = posterior lateral line ganglion; G = glossopharyngeal ganglion; V = vagal ganglia. Scale bar in (A) = 50 µm.

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