View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Available online at www.sciencedirect.com R Developmental Biology 263 (2003) 176–190 www.elsevier.com/locate/ydbio Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve Vivian M. Lee,1 John W. Sechrist,1 Simone Luetolf, and Marianne Bronner-Fraser* Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA Received for publication 11 March 2003, revised 28 July 2003, accepted 28 July 2003 Abstract The chick ciliary ganglion is a neural crest-derived parasympathetic ganglion that innervates the eye. Here, we examine its axial level of origin and developmental relationship to other ganglia and nerves of the head. Using small, focal injections of DiI, we show that neural crest cells arising from both the caudal half of the midbrain and the rostral hindbrain contribute to the ciliary as well as the trigeminal ganglion. Precursors to both ganglia have overlapping migration patterns, moving first ventrolaterally and then rostrally toward the optic vesicle. At the level of the midbrain/forebrain junction, precursors to the ciliary ganglion separate from the main migratory stream, turn ventromedially, and condense in the vicinity of the rostral aorta and Rathke’s pouch. Ciliary neuroblasts first exit the cell cycle at early E2, prior to and during ganglionic condensation, and neurogenesis continues through E5.5. By E3, markers of neuronal differentiation begin to appear in this population. By labeling the ectoderm with DiI, we discovered a new placode, caudal to the eye and possibly contiguous to the trigeminal placode, that contributes a few early differentiating neurons to the ciliary ganglion, oculomotor nerve, and connecting branches to the ophthalmic nerve. These results suggest for the first time a dual neural crest and placodal contribution to the ciliary ganglion and associated nerves. © 2003 Elsevier Inc. All rights reserved. Keywords: Chick; DiI; Migration; Neuronal differentiation; Neurogenesis; Ophthalmic nerve; Trigeminal ganglion Introduction neurons and glia of the cranial ganglia (Le Douarin and Kalcheim, 1999). Neural crest cells emerge from the neural tube shortly Cranial ganglia arise both from neural crest cells and after its closure and subsequently undergo extensive migra- from a second ectodermal population, cranial neurogenic tions. A variety of techniques have been used to map the placodes, which are thickenings in the ectoderm, from contributions of neural crest cells arising at different axial which cells delaminate and migrate into the head mesen- levels to different derivatives. For example, cranial neural chyme much like neural crest cells. Some cranial sensory crest cells arising from rhombomere (r) 1 and r2 populate ganglia, such as the acoustic ganglion, are entirely derived the trigeminal ganglia and mandibular processes of the first from placodes; others, such as the trigeminal ganglion, arch, whereas r4 neural crest contributes to the second receive a dual contribution from the neural crest and pla- (hyoid) arch (Lumsden et al., 1991; Sechrist et al., 1993). codes (Hamburger, 1961). The parasympathetic ciliary gan- Following migration, cranial neural crest cells differentiate glion has been reported to be entirely neural crest-derived into a plethora of cell types, including the skeletal elements (D’Amico-Martel and Noden, 1983). This ganglion pro- of the face, muscle connective tissue, pigment cells, and vides cholinergic innervation to the eye. Previous studies have examined the origin of the avian ciliary ganglion from the neural crest by ablating the neural folds to remove neural crest precursors at selected cranial * Corresponding author. Fax: ϩ1-626-395-7717 E-mail address: [email protected] (M. Bronner-Fraser). axial levels (Hammond and Yntema, 1958) or by grafting 1 These authors contributed equally to this work. quail or 3H-thymidine-labeled chick neural folds or tube 0012-1606/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.07.004 V.M. Lee et al. / Developmental Biology 263 (2003) 176–190 177 Fig. 1. DiI-labeled migratory neural crest cells shown in whole-mount and transverse sections. (A) An embryo injected at the caudal midbrain level at the 8-somite stage and collected at 20 somites (18 h later, 2.25 days total). (A) In dorsal whole-mount view, streams of DiIϩ cells move ventrolaterally (arrowhead) and rostrally (arrow) in the longitudinal plane. By the 20-somite stage, some migratory cells have approached the level of the caudal eye/forebrain (FB). (B) A transverse section showing the primary injection site and ventrolateral migratory stream (arrowhead). (C) Rostral migrating cells (arrow) dorsal to caudal eye level and lateral to the floor of the rostral midbrain (rMB) represent trigeminal/ciliary neural crest; the latter has not yet moved ventromedially. The position of these neural crest cells (arrow) overlaps that of placode-derived ophthalmic nerve cells. Dash lines in (A) indicate plane of section for (B) and (C). segments orthotopically into host chick embryos to deter- both ciliary and trigeminal ganglia initially overlap while mine the axial levels from which ciliary ganglionic precur- migrating, but those destined to contribute to the ophthalmic sors arise (D’Amico-Martel and Noden, 1983; Narayanan portion of the trigeminal ganglion and the ciliary ganglion and Narayanan, 1978; Noden, 1975, 1978). These studies subsequently turn rostrally and move toward the eye. The have provided somewhat contradictory results, with some ciliary precursors move inward at the level of the midbrain/ investigators concluding that the ciliary ganglion solely forebrain junction and condense on the developing oculo- arose from midbrain neural crest and others suggesting a motor nerve in the vicinity of the rostral aorta and Rathke’s possible rostral hindbrain contribution as well. Since the pouch. Some ciliary neuroblasts exit the cell cycle prior to ablations and grafts incorporated rather large areas of tissue, and during ganglionic condensation with differentiation oc- the precise axial level(s) from which ciliary precursors arose curring postmitotically. In addition to a neural crest contri- was not well determined. In addition, there has been little bution, vital dye labeling of the ectoderm reveals a previ- information about the dynamic aspects of migration of cil- ously unknown contribution of a ventrolateral postoptic iary precursors to their target sites, the time of origin and ectodermal placode to the ciliary ganglion and oculomotor differentiation of the first ciliary neurons, and whether there nerve. Large sensory neurons have been described in the is any placodal contribution to the ciliary ganglion. ciliary ganglion and in association with cranial nerves in- Refined techniques that allow labeling of small popula- cluding the oculomotor nerve for nearly 100 years (Borto- tions of neural crest precursor cells are now readily avail- lami et al., 1977; Rogers, 1957; Tozer, 1912), but their able. Use of the vital dye, DiI, has significantly enhanced origin has remained elusive until now. our ability to track and identify both trunk and cranial neural crest populations (Serbedzija et al., 1989). Migration of placodal cells can be followed as well by briefly bathing the embryos with DiI after dorsal neural tube closure at the 11- Materials and methods to 12-somite stage (Stark et al., 1997). Here, we use small focal injections of DiI combined with Embryos birth-dating and molecular markers to precisely determine the axial level of origin of ciliary precursors. We also follow Fertilized chicken eggs (White Leghorn and Rhode Is- their migratory trajectories as a function of time and deter- land Red) were obtained from commercial sources and mine their early differentiation sequence. Our results show incubated at 38°C until having 7–15 somites [stages 9–11, that not only caudal midbrain, but also rostral hindbrain (Hamburger and Hamilton, 1951)]. A window was cut in the neural crest contributes to the ciliary ganglion. Precursors to egg shell, and a 1:20 mixture of India ink and Ringer’s 178 V.M. Lee et al. / Developmental Biology 263 (2003) 176–190 Table 1 Injection sites and distribution of DiI-labeled neural crest cells in vicinity of ciliary ganglion Injection site & embryo # DiI labeling Distribution of DiI ϩ cells Oph N Op V Ciliary gang Oc N Caudal forebrain 19s3E4.5 ϩϪϪ Ϫ 27s3E5.5 ϩϪϪ ϩϪ Rostral midbrain 38s3E4.5 ϩ Ϫ ϩϪ ϩ 49s3E4.5 ϩ Ϫ ϩϪ ϩ Mid midbrain 59s3E5.5 ϩ Ϫ ϩϩ ϩ 69s3E4.5 ϩ ϩ ϩϪ ϩ Caudal midbrain 79s3E5.5 ϩ ϩ ϩϩ ϩ 89s3E5.5 ϩ ϩ ϩϩ ϩ 98s3E4.5 ϩ ϩ ϩϩ ϩ 10 9s3E4.5 ϩϩϩϩϩϩ Midbrain–hindbrain Jct. 11 8s3E5.5 ϩϩϩ ϩ 12 8s3E3.5 ϩ ϩ ϩϩ ϩ 13 8s3E5.5 ϩ ϩ ϩϩ ϩ 14 9s3E3.5 ϩϩϩ ϩ 15 9s3E4.5 ϩϩϩϩϩϩ Rostral hindbrain (r1) 16 9s3E5.5 ϩϩϩ Ϫ 17 8s3E3 ϩ ϩ ϩϪ Ϫ 18 8s3E4.5 ϩ ϩ ϩϪ ϩϪ 19 9s3E5.5 ϩϩϩ ϩϪ r1/r2 Jct. 20 8s3E3.5 ϩϩϩϩ ϩϪ Rostral hindbrain (r2) 21 8s3E4 ϪϩϪ Ϫ Rostral hindbrain (rostral r3) 22 8s3E3.5 ϪϪϪ Ϫ Mid-hindbrain (caudal r3) 23 8s3E4 ϪϪϪ Ϫ Mid-hindbrain r4 24 9s3E4 ϪϪϪ Ϫ Mid-hindbrain r5 25 9s3E3.5 ϪϪϪ Ϫ ϩϩ, many labeled cells. ϩ, some labeled cells. ϩϪ, few, scattered labeled cells. Ϫ, no labeled cells. Ciliary gang, ciliary ganglion. Oc N, oculomotor nerve. Oph N, ophthalmic nerve. Op V, ophthalmic ganglion. solution was injected beneath the blastoderm to visualize and back-loaded into glass micropipettes. A small amount the embryo. of dye was injected by air pressure into the dorsal neural tube surface at selected locations (Table 1). Eggs were Focal DiI injections sealed and returned to incubator until the time of harvest (E2–E5.5). Chicken embryos between 7–10 somites (stages 9–10) were used for focal injections.