*Corresponding author: Lennart Olsson, Institute of Systematic Zoology and Evolutionary Biology, University of Jena, Erbert-strasse 1, D-07743 Jena, Germany, phone: ++49-3641-949160; fax: ++49-3641-949162; e-mail: [email protected] is now renewed interest in comparative embry-ological studies, as part of broader attention to the rela-tionship between evolution and development, or “evo-devo.” Many recent textbooks give overviews of thisfield (Arthur, 1997; Gerhart and Kirschner, 1997; Hall,1998; Carroll et al., 2001; Wilkins, 2002). Most con-temporary work in developmental biology involveseither studies of a single organism or comparisonsamong a small number of so-called model species, andoften the main interests are early embryonic patterningand conserved genes and functions. It is also important,however, to extend comparative studies to embraceother species, as well as the variation in developmentalprocesses and mechanisms that underlies the evolutionof novelties. This paper presents data on cranial neural crest devel-opment in the Mexican axolotl (Ambystoma mexi-canum). This is perhaps the most well studied salaman-der species embryologically, and it has often been usedas a model for salamanders in general. The Mexican ax-olotl remains an important organism for studies whosemain focus is on the evolution of development. Thepresent study is part of a larger project on the evolutionof head development in lungfishes and amphibians, in-cluding both frogs and salamanders (e.g., Olsson et al.,2000). We emphasize neural crest development becausethe cranial neural crest has played an important role inthe evolution of cranial patterning (Hunt et al., 1991;Cranial neural crest emergence and migration in the Mexican axolotl(Ambystoma mexicanum) Pierre Falck1, James Hanken2, Lennart Olsson3,*Received June 25, 2002. Revised version received September 6, 2002. Accepted September 6, 2002. 1Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden2Museum of Comparative Zoology, Harvard University, Cambridge, USA3Institute of Systematic Zoology and Evolutionary Biology, University of Jena, GermanySummaryThe timing and pattern of cranial neural crest cell emergence and migration in the Mexican axolotl, Ambystoma mexicanum, are as-sessed using scanning electron microscopy (SEM). Cranial neural crest cells emerge and begin to migrate at the time of neural fold clo-sure and soon form three distinct streams. The most anterior (mandibular) stream emerges first, at the level of the mesencephalon. Cellsin this stream migrate rostroventrally around the optic vesicle. The second (hyoid) and third (branchial) streams emerge in close succes-sion at the level of the rhombencephalon and extend ventrolaterally. Cells forming the hyoid stream migrate rostral to the otic vesicle,whereas the branchial stream divides into two parallel streams, which migrate caudal to the otic vesicle. At later stages (stage 26 on-wards) the cranial neural crest cells disperse into the adjacent mesoderm and can no longer be followed by dissection and SEM. The pat-tern of cranial neural crest emergence and migration, and division into migratory streams is similar to that in other amphibians and inthe Australian lungfish (Neoceratodus forsteri). Emergence of crest cells from the neural tube, relative to the time of neural tube clo-sure, occurs relatively late in comparison to anurans, but much earlier than in the Australian lungfish. These results establish a morpho-logical foundation for studies in progress on the further development and fate of cranial neural crest cells in the Mexican axolotl, as wellas for studies of the role of cranial neural crest in cranial patterning.Key words: head development, cell migration, pattern formation, salamander0944-2006/02/105/03-195 $ 15.00/0Zoology 105 (2002): 195–202© by Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/zoologydehydrated in an ethanol series (50%, 70%, 90%, 95%and absolute ethanol) and transferred into liquid CO2ina critical point dryer. The dried embryos were mounted,sputter-coated with gold/palladium, and examined in aPhilips CM10 scanning electron microscope. Digitalpictures were obtained as TIFF files. Scanning electronmicrographs were assembled into montages, each con-taining 4–24 separate images.ResultsCranial neural crest cell migrationAt stage 17, cranial neural crest cells have not yet differ-entiated. Epidermis covers the neural fold and no crestcells are visible (data not shown). The mandibular neu-ral crest stream appears initially at stage 18, atop theneural folds at the level of the future mesencephalon. Itscells begin to migrate rostrally and ventrolaterally be-tween the epidermis and the neural folds (Fig. 1A). Atstage 19, the mandibular neural crest stream continuesto migrate rostroventrally. A second neural-crest streamappears on the neural tube, at the level of the rhomben-cephalon, and its cells begin to migrate ventrolaterallybetween epidermis and the neural folds (Fig. 1B).Neural folds fuse at stage 20, forming the neural tube.The mandibular neural crest stream continues to mi-grate rostroventrally. The second neural crest stream issubdividing to form two distinct streams, an anteriorhyoid stream and a posterior branchial stream, both ofwhich migrate ventrolaterally (Fig. 2A, B). By stage21, the mandibular neural crest stream surrounds theoptic vesicle. The hyoid and branchial streams continueto migrate ventrolaterally between the epidermis andneural tube (Fig. 3A). Stage 22 resembles stage 21, al-though the hyoid stream has begun to extend onto thesurface of the mesoderm. Crest-free zones on the neuraltube between the mandibular, hyoid and branchialstreams are more pronounced than in earlier stages(Fig. 3B).By stage 23, the once thick layer of mandibular neuralcrest cells on the dorsal part of the neural tube is thin-ning (Figs. 4A, C). Hyoid and branchial neural creststreams continue to migrate ventrally between the epi-dermis and mesoderm. The branchial neural creststream begins to divide into two parallel streams(Fig. 4B).At stage 24, the once single branchial neural creststream is clearly divided into two parallel streams(Figs. 5A, B). The hyoid neural crest stream migratesventrally faster than either of the two branchial streamsand begins to overtake them. On the dorsal part of theneural tube, the mandibular and hyoid streams remainconnected by a patch of cells, although only a few cellsHanken and