Epibranchial ganglia orchestrate the development of the cranial neurogenic crest

Abstract

The wiring of the nervous system arises from extensive directional migration of neuronal cell bodies and growth of processes that, somehow, end up forming functional circuits. Thus far, this feat of biological engineering appears to rely on sequences of pathfinding decisions upon local cues, each with little relationship to the anatomical and physiological outcome. Here, we uncover a straightforward cellular mechanism for circuit building whereby a neuronal type directs the development of its future partners. We show that visceral afferents of the head (that innervate taste buds) provide a scaffold for the establishment of visceral efferents (that innervate salivatory glands and blood vessels). In embryological terms, sensory neurons derived from an epibranchial placode--that we show to develop largely independently from the neural crest--guide the directional outgrowth of hindbrain visceral motoneurons and control the formation of neural crest-derived parasympathetic ganglia.

Fig. 1.

Viscerosensory and visceromotor neurons project in the absence of parasympathetic ganglia. (Upper Left) Neurofilament stain of the facial nerve complex at E11.5 and (Right) schematic at E13.5. (Lower) Phenotype of the head parasympathetic ganglia and branches of the geniculate ganglion in the indicated genetic backgrounds (Rosa^locDTA stands for Rosa^{lox-stop-lox-DTA}). The schematics depict the structures in whose precursors the gene has been inactivated or where DTA was expressed as hollowed-out shapes (and do not represent the phenotype). (A–E) Transverse sections through the head of E13.5 (A–D) or E12.5 (E) embryos stained by immunohistochemistry for Phox2b (A–C and E) or Phox2a (d) to detect the Spg and lingual ganglia (the submandibular proper is in another plane of section). An orange asterisk indicates their absence. (A′–E′), Neurofilament stain of the facial nerve and its branches at E11.5. The GSPN and CT are indicated by an upward or downward black arrowhead, respectively, and their absence by a black asterisk. The main branch of the facial nerve is indicated by a green arrowhead. (A″, D″, E″), Flat-mounted E11.5 hindbrain retrogradelly labeled with DiI placed in the second branchial arch (Inset). V, VII, VIII, IX, and X: trigeminal, geniculate, statoacoustic, petrosal, and nodose ganglia. VIIm_b, branchial motoneurons of the facial nucleus; VIIm_v, visceral motoneurons of the salivatory nucleus; CT, corda tympani; GSPN, greater superficial petrosal nerve; nVII, facial nerve; SCG, superior cervical ganglion, S/Lg, submandibular and lingual ganglia; Spg, sphenopalatine ganglion; TS, tractus solitarius. (Scale bar, 200 μm.)

Fig. 2.

Early ablation of neural crest does not affect the formation of the geniculate ganglion and reveals an alternate source of glial cells. (A and B) Whole-mount neurofilament staining of the facial nerve branches in wild-type and Wnt1::Cre; Rosa^{lox-stop-lox-DTA} embryos. The GSPN and CT are indicated by black arrowheads, respectively upward and downward. In that particular mutant the GSPN appears duplicated. The main branch of the facial nerve is indicated by a green arrowhead. (C–J)Transverse sections of wild type and Wnt1::Cre; Rosa^{lox-stop-lox-DTA} embryos at the indicated stages, at the level of the geniculate ganglion (C, D, and G–J) or trunk (E and F), hybridized with a Sox10 probe (blue), followed by Phox2b immunohistochemistry (orange). The black arrowheads indicate the geniculate ganglion. The black asterisks in F show the absence, in Wnt1::Cre; Rosa^{lox-stop-lox-DTA} embryos, of Phox2b⁺/Sox10 ⁺ enteric neurons. nt, neural tube; O, otic vesicle; IV, fourth ventricle; VII, geniculate ganglion. [Scale bar: (C–F), 100 μm; (G–I): 200 μm.]

Fig. 3.

The geniculate ganglion organizes the parasympathetic innervation of the head. For each genotype, the schematic indicates which structures Phox2b has been deleted from as hollowed out shapes (and do not represent the phenotype). (A–E) Transverse sections through the head of E13.5 embryos stained by immunohistochemistry for Phox2b (A, B, D, and E) or Phox2a (C) to detect the Spg and lingual (Lg) ganglia. Their absence is indicated by an orange asterisk. (A′–E′) Lateral view of the geniculate ganglion and its branches on whole-mount neurofilament stains of E11.5 embryos. The GSPN and CT are indicated by an upward and downward black arrowhead, respectively, and their absence by a black asterisk. A green arrowhead points to the main branch of the facial nerve. (A″, B″, C″, and E″) Retrograde DiI labeling of motor neurons and sensory projections from the second branchial arch in the indicated genetic background. VII, geniculate ganglion; VIIm_b, branchial motoneurons of the facial nucleus; VIIm_v, visceral motoneurons of the salivatory nucleus; CT, corda tympani; GSPN, greater superficial petrosal nerve; Lg, lingual ganglia, Spg: sphenopalatine ganglion; TS, tractus solitarius. (Scale bar, 200 μm.)