DEVELOPMENTAL DYNAMICS 236:389–403, 2007 RESEARCH ARTICLE Migratory Patterns and Developmental Potential of Trunk Neural Crest Cells in the Axolotl Embryo Hans-Henning Epperlein,1* Mark A.J. Selleck,2 Daniel Meulemans,3 Levan Mchedlishvili,4 Robert Cerny,5 Lidia Sobkow,4 and Marianne Bronner-Fraser3 Using cell markers and grafting, we examined the timing of migration and developmental potential of trunk neural crest cells in axolotl. No obvious differences in pathway choice were noted for DiI-labeling at different lateral or medial positions of the trunk neural folds in neurulae, which contributed not only to neural crest but also to Rohon-Beard neurons. Labeling wild-type dorsal trunks at pre- and early-migratory stages revealed that individual neural crest cells migrate away from the neural tube along two main routes: first, dorsolaterally between the epidermis and somites and, later, ventromedially between the somites and neural tube/notochord. Dorsolaterally migrating crest primarily forms pigment cells, with those from anterior (but not mid or posterior) trunk neural folds also contributing glia and neurons to the lateral line. White mutants have impaired dorsolateral but normal ventromedial migration. At late migratory stages, most labeled cells move along the ventromedial pathway or into the dorsal fin. Contrasting with other anamniotes, axolotl has a minor neural crest contribution to the dorsal fin, most of which arises from the dermomyotome. Taken together, the results reveal stereotypic migration and differentiation of neural crest cells in axolotl that differ from other vertebrates in timing of entry onto the dorsolateral pathway and extent of contribution to some derivatives. Developmental Dynamics 236:389–403, 2007. © 2006 Wiley-Liss, Inc. Key words: DiI; fluorescent dextrans; GFP transgenic embryos; trunk neural crest; migration; dorsal fin; Rohon-Beard cells; lateral line; axolotl Accepted 31 October 2006 INTRODUCTION of the peripheral nervous system, pig- border between prospective neural The neural crest is a transient migra- ment and adrenomedullary cells, the plate and epidermis (Moury and Ja- tory population of cells found in all craniofacial skeleton and, in lower cobson, 1989; Selleck and Bronner- vertebrate embryos (Knecht and vertebrates, mesenchyme of the dor- Fraser, 1995). Because the neural Bronner-Fraser, 2002). These cells mi- sal fin (Hall and Ho¨rstadius, 1988; Le folds are contiguous with neural plate grate extensively along defined path- Douarin and Kalcheim, 1999). Neural and epidermis (Moury and Jacobson ways and give rise to diverse deriva- crest cells arise in a prospective neu- (1990), it is not clear where they “end” tives, including neurons and glial cells ral fold area of the ectoderm at the and neural tissue or epidermis begin. 1Department of Anatomy, TU Dresden, Dresden, Germany, 2Department of Cell and Neurobiology, University of Southern California Keck School of Medicine, Los Angeles, California 3Division of Biology and Beckman Institute, California Institute of Technology, Pasadena, California 4Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany 5Department of Zoology, Charles University Prague, Prague, Czech Republic Grant sponsor: DFG; Grant number: EP8/7-1; Grant sponsor: NIH; Grant number: NS051051; Grant sponsor: James H. Zumberge Research and Innovation Fund; Grant sponsor: Donald E. and Delia B. Baxter Foundation. *Correspondence to: Hans H. Epperlein, Department of Anatomy, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany. E-mail: [email protected] DOI 10.1002/dvdy.21039 Published online 19 December 2006 in Wiley InterScience (www.interscience.wiley.com). © 2006 Wiley-Liss, Inc. 390 EPPERLEIN ET AL. Furthermore, single cell lineage anal- al., 1988; Collazo et al., 1993). In the lyze the migratory patterns and deriv- ysis in several species suggests that a mouse, no timing differences exist be- atives of trunk neural crest cells single neural fold precursor can give tween entry onto the dorsolateral and emerging from the neural folds/tube rise not only to neural crest but also to ventromedial pathways (Serbedzija et (Fig. 1A–E). DiI injections were per- neural tube and epidermal derivatives al., 1990). Thus, species-specific differ- formed at different positions along the (Selleck and Bronner-Fraser, 1995). ences exist in some aspects of neural mediolateral axis (Fig. 1A,F) prior to Accordingly, Chibon (1966) concluded crest migration. There is reason to the onset of neural crest migration. that Rohon-Beard neurons, missing think that migratory behavior may Along the rostrocaudal axis, anterior, after neural fold ablation, were neural differ between axolotl (Ambystoma middle, or posterior levels of the dor- crest derived. mexicanum) and chick as well. In the somedial neural folds or neural tube A major unresolved question in neu- axolotl, some premigratory neural were injected (Fig. 1B–E;F). In the ral crest development concerns the re- crest cells, such as presumptive pig- trunk of early tailbud stage embryos, ment cells, appear to be committed to lationship between neural crest and the neural crest can be morphologi- neural tube with respect to the alloca- their fate prior to the onset of migra- cally identified as a wedge of cells that tion of different lineages and path- tion. Therefore, it is possible that sub- forms on the dorsal aspect of the neu- ways of migration. This question has sequent migration may follow differ- ral tube and expresses snail tran- been extensively studied in the chick ent rules. It is unknown whether the scripts (Fig. 1G). Later, an elevated embryo, which is amenable to culture premigratory crest is entirely “mo- neural crest string is present on top of and microsurgical manipulation in saic” and contains only unipotent cells combination with numerous cell- or a mixture of cells with different the neural tube (Fig. 1H). Once neural marking techniques for following neu- ranges of developmental potential. crest cells migrate, they move along ral crest migration (Le Douarin, 1969; Here, we address this issue by two primary pathways: a dorsolateral Le Douarin and Kalcheim, 1999; Ser- studying the migratory patterns and pathway underneath the epidermis bedzija et al., 1989; Elena de Bellard developmental potential of early dif- that is primarily thought to give rise and Bronner-Fraser, 2005). The mi- ferentiating trunk neural crest cells in to pigmented derivatives (melano- gratory behavior of avian neural crest the axolotl such as pigment cells, Ro- phores and xanthophores) and a ven- appears to be highly organized, such hon-Beard cells, or components of the tromedial pathway followed by pre- that the first cells to exit the neural lateral line. As a first step, we have cursors to sensory, sympathetic, tube migrate along a ventromedial performed a series of cell-marking and adrenomedullary, and glial cells. It is pathway through the somites, while grafting studies using dark wild-type sometimes difficult to discriminate be- later migrating cells are restricted to a and white mutant embryos, the latter tween laterally migrating neural dorsolateral pathway between the epi- having defective pigment cell migra- crest–derived pigment precursors, dermis and somites where they form tion (Dalton, 1953; Keller et al., 1982; epidermis, or neural tube on the one pigment cells (Le Douarin and Teillet, Lo¨fberg et al., 1985). The results show hand and between pigment precursors 1974; Guillory and Bronner-Fraser, that neural crest cells follow both a and components of the lateral line on 1986; Erickson et al., 1992; Serbedzija dorsolateral and a ventromedial mi- the other. To best illustrate this point, et al., 1989). In the chick, it has been gratory route similar to other verte- we examined dark instead of white proposed that melanocytes are speci- brates. However, the time course is mutant embryos since they have fied before entering the dorsolateral different than in other anamniotes or many more laterally migrating cells migratory route (Reedy et al., 1998) amniotes, since the dorsolateral route (Figs. 1I–L). DiI-labeled cells can be is taken first. We confirm that neural and that distinct neurogenic and mel- distinguished in transverse sections crest and Rohon Beard cells share a anogenic sublineages may diverge be- after one anterior (Fig. 1I) or three common lineage within the neural fore or soon after neural crest cell em- midtrunk injections (Fig. 1J) into the igration from the neural tube (Luo et folds and find that only few axolotl trunk neural fold from epidermal or al., 2003). neural crest cells contribute to the neural tube cells by means of their Despite the wealth of information dorsal fin, which also has a contribu- position (Fig. 1K). Pigment precursors on avian neural crest migration, it re- tion from the dermomyotome (Sobkow and components of the lateral line mains unknown whether ordered mi- et al., 2006). Furthermore, we show such as neurons (ganglionic cells), gratory behavior is common to all ver- that anterior but not mid- or posterior tebrate embryos, or whether it is trunk neural folds contribute to the glial cells, and lateral line nerves can avian specific. Some differences in mi- lateral line system. These results re- be distinguished through the use of gratory behavior from those observed veal interesting similarities and dif- specific markers. We found that glial in chick have been noted in amphibi- ferences in the migratory pathways cells are present only in the vicinity of ans like Xenopus as well