Ascidian neural crest-like cells: phylogenetic distribution, relationship to larval complexity, and pigment cell fate

Migratory neural crest-like cells, which express the cell surface antigen HNK-1 and develop into pigment cells, have recently been identified in the ascidian Ecteinascidia turbinata. Here we use HNK-1 expression as a marker to determine whether neural crest-like cells are responsible for pigment development in diverse ascidian species. We surveyed HNK-1 expression and tyrosinase activity in 12 ascidian species, including those with different adult organizations, developmental modes, and larval sizes and complexities. We observed HNK-1 positive cells in every species, although the timing of HNK-1 expression varied according to the extent of larval complexity. HNK-1 expression was initiated during the late tailbud stage in species in which adult features are formed precociously in large complex larvae. In contrast, HNK-1 positive cells did not appear until the swimming tadpole or juvenile stage in species with small simple larvae in which most adult features appear after metamorphosis. Double labeling experiments indicated that HNK-1 and tyrosinase are expressed in the same subset of pigment-forming mesenchymal cells in species with complex or simple larvae. In addition, the absence of HNK-1 and tyrosinase expression in albino morphs of the colonial ascidian Botryllus schlosseri suggested that the major fate of neural crest-like cells is to become pigment cells. The results suggest that ascidian neural crest-like cells and vertebrate neural crest cells had a common origin during chordate evolution and that their primitive function was to generate body pigmentation.     

The larval ocelli of Golfingia misakiana (Sipuncula, Golfingiidae) and of a pelagosphera of another unidentified species

 The inverse cerebral ocelli of the pelagosphera larva of Golfingia misakiana and of another unidentified larva are composed of two or three sensory cells and one supportive pigmented cell. The sensory cells bear an array of microvilli as well as a single cilium with poor undulation of its membrane; the photoreceptive organelles are regarded as the rhabdomeric type. A striking feature of these cells is the cores, which extend within the microvilli from the tip into the midregion of the cell. It is suggested that these structures are identical with the submicrovillar cisternae found in the cerebral inverse eyes of larvae of Polychaeta. The findings allow the conclusion that in the pelagosphera of the Sipuncula, contrary to the teleplanic veliger larvae of Gastropoda, a lengthy pelagic cycle is not correlated with the development of a ciliary photoreceptor. Additionally, it is assumed that the pigment cup ocelli in larvae of Sipuncula are homologous with the cerebral inverted pigment cup ocelli of larvae of Polychaeta. Accepted: 19 March 1997     

Structure of eyes in trombidioid mites (Acariformes: Trombidioidea)

The eyes or ocelli of trombidioid mite larvae of Euschoengastia rotundata, Hirszutiella zachvatkini and Camerotrombidium pexatum, and larvae and adults of Platytrombidium fasciatum were studied by means of transmission electron microscopy. These species together with larvae of Odontacarus efferus, Ericotrombidium hasgelum, Walchia chinensis and adult E. rotundata and H. zachvatkini were also studied under scanning electron microscope. The eyes of larvae are not inverted and characterized by an epicuticular lamellar lens. The group of phoreceptor cells with rhabdomeres arranged typically of Chelicerata is underlaid by a pigment cup. The eyes of adult mites are inverted, perikarions of photoreceptor cells are situated between the lens and rhabdomeres; tapetum occupies the space between the pigment cup and rhabdomeres. Sensitivity of eyes to light is similar to that of primary eyes of spiders dwelling on soil surface.     

Ultrastructure of the larval eye of the scorpionfly Panorpa dubia (mecoptera: Panorpidae) with implications for the evolutionary origin of holometabolous larvae

The evolutionary origin of holometabolous larvae is a long‐standing and controversial issue. The Mecoptera are unique in Holometabola for their larvae possessing a pair of compound eyes instead of stemmata. The ultrastructure of the larval eyes of the scorpionfly Panorpa dubia Chou and Wang, 1981 was investigated using transmission electron microscopy. Each ommatidium possesses a cornea, a tetrapartite eucone crystalline cone, eight retinula cells, two primary pigment cells, and an undetermined number of secondary pigment cells. The rhabdomeres of the eight retinula cells form a centrally‐fused, tiered rhabdom of four distal and four proximal retinula cells. The rhabdomeres of the four distal retinula cells extend distally into a funnel shape around the basal surface of the crystalline cone. Based on the similarity of the larval eyes of Panorpidae to the eyes of the hemimetabolous insects and the difference from the stemmata of the holometabolous larvae, the evolutionary origin of the holometabolous larvae is briefly discussed. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca²⁺–, Mg²⁺–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.     

Statocyte and Ocellar Pigment Cell in Embryos and Larvae of the Ascidian, Styela plicata (Lesueur)

The processes of formation of two pigmented cells, the statocyte and the ocellar pigment cell, in the cerebral vesicle of larvae of the ascidian Styela plicata were investigated in whole mount specimens and serial paraffin sections by light microscopy. The pigmentations of the two cells became visible simultaneously in embryos at the stage of tail elongation, 5–6 hr after fertilization. The pigmented cells were at first located side by side in the dorsal wall of the neurocoel. Growth of the pigment mass in the ocellus ceased at about 6.5 hr, while that in the statocyte continued through the hatching period (9–10 hr) up to the swimming stage. The pigment mass in the statocyte consisted of two blocks which joined together during their growth. The statocyte migrated from the dorsal to the ventral wall of the cerebral vesicle by the swimming stage. In swimming larvae, the more ventral of the two pigment blocks of the statocyte formed an inverted pigment cup and a cluster of protuberances projected into it from the ventral wall of the cerebral vesicle. Phylogenetically, the sensory organs in the cerebral vesicle of Styela plicata seem intermediate between those in Pyuridae and Botryllinae with respect of their structure and process of differentiation.     

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti-arrestin antibody

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti-arrestin (anti-Ci-Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro-posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero-posteriorly at the midline of the posterior sensory vesicle (SV). The Ci-arr gene was expressed throughout the SV at the embryonic mid-tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci-Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti-Ci-Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step-down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive.     

Relations between pigmentation of the cornea and the number of epidermal melanophores in Rana temporaria L. larvae

The dynamics of the external cornea pigmentation in Rana temporaria L. larvae at the 22d developmental stage have been studied under conditions favourable for various course of certain morphological reactions in the pigment system. The cornea together with the surrounding skin is transferred on the dorsal surface of the larva body, and the piece of the dorsal surface skin is put instead of the cornea removed. When using the reciprocal transplantation method and preserving the organism's integrity (without disturbing melanocyte-stimulating source--namely, the hypophysis, and melatonine sources--namely, the pineal gland and the lateral eyes) the corneal pigmentation is observed on the background of perfect morphological reactions in the pigment system, while the larvae are maintained on the dark and light substrates, that is at various density of the pigment cells (120 larvae have been used). The pigmentation dynamics have been studied from the 6th up to the 20th day in total preparations. The epidermal melanophores density is estimated in 4 areas of each preparation. The melanin amount is estimated by means of the electron paramagnetic resonance-spectrometry according to the contents of free radicals expressed in relative units. A direct proportional dependence between the significantly higher melanin contents (1.5-fold) and a significantly quicker (1.5-fold) process of the corneal pigmentation is revealed, that agrees with an increasing number of the pigment cells per one unit of the body surface in the larvae maintained on the dark substrate. In the larvae maintained on the light substrate, the dependence is of a reverse character. It is probable that the factors forcing the pigmented cells, at cultivation the neural crest cells in vitro to reject from each other, affect the pigmentation of the larval cornea in vivo. If it is the case, the processes specific for the embryonal period, transgress during the cornea pigmentation at the larval stages of development.     

Evolution and development of brain sensory organs in molgulid ascidians

The ascidian tadpole larva has two brain sensory organs containing melanocytes: the otolith, a gravity receptor, and the ocellus, part of a photoreceptor. One or both of these sensory organs are absent in molgulid ascidians. We show here that developmental changes leading to the loss of sensory pigment cells occur by different mechanisms in closely related molgulid species. Sensory pigment cells are formed through a bilateral determination pathway in which two or more precursor cells are specified as an equivalence group on each side of the embryo. The precursor cells subsequently converge at the midline after neurulation and undergo cell interactions that decide the fates of the otolith and ocellus. Molgula occidentalis and M. oculata, which exhibit a tadpole larva with an otolith but lacking an ocellus, have conserved the bilateral pigment cell determination pathway. Programmed cell death (PCD) is superimposed on this pathway late in development to eliminate the ocellus precursor and supernumerary pigment cells, which do not differentiate into either an otolith or ocellus. In contrast to molgulids with tadpole larvae, no pigment cell precursors are specified on either side of the M. occulta embryo, which forms a tailless (anural) larva lacking both sensory organs, suggesting that the bilateral pigment cell determination pathway has been lost. The bilateral pigment cell determination pathway and superimposed PCD can be restored in hybrids obtained by fertilizing M. occulta eggs with M. oculata sperm, indicating control by a zygotic process. We conclude that PCD plays an important role in the evolution and development of brain sensory organs in molgulid ascidians.     

The ontogeny of facultative superposition optics in a shrimp eye: hatching through metamorphosis

Summary Compound eyes of larval and first postlarval grass shrimp (Palaemonetes pugio Holthuis) were studied with light and electron microscopy following adaptation to darkness or bright light. Larvae have well-developed apposition eyes, including 3 main types of accessory screening and reflecting pigments and a fourth class of putatively reflective granules recently described in adult shrimps. Rhabdoms contain orthogonally layered microvilli, and by the last larval stage, 8 retinular cells. Ocular accessory pigments in both light- and dark-adapted larvae are distributed much like those of light-adapted adults, but the distal mass of reflecting pigment is concentrated dorsally in larvae and ventrally in adults. Since larvae swim upside-down, reflecting pigment is oriented downward in all developmental stages and may function for countershading. Light and dark adaptational migrations of all 3 major accessory pigments commence abruptly at metamorphosis to the first postlarva. Upon dark adaptation in postlarvae, superposition optics remain impossible because (1) distal screening pigment migrates only slightly, (2) no clear zone has developed, and (3) the crystalline cones remain circular in cross section. Nevertheless, a slight improvement in photon catch is expected due to extensive redistributions of reflecting pigment and retinular cell screening pigment granules.

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Fine structure of photoreceptors in larval trematodes

Summary The fine structure of photoreceptors is described in miracidia of Fasciola hepatica, Heronimus chelydrae, Allocreadium lobatum, and Spirorchis sp., and in a spirorchiid cercaria. All have in common eyespots consisting of pigment cells with chambers occupied by rhabdomeres consisting of retinular cell dendrites with numerous microvilli. Photoreceptors of the miracidia show a bilateral asymmetry which is most pronounced in H. chelydrae with a pair of well separated eyespots unequal in size. The smaller right one consists of a pigment cell and two rhabdomeres; the larger left eyespot has an anterior pigment cell with two rhabdomeres and a posterior cell containing one rhabdomere. Photoreceptors in the other species of miracidia also have five rhabdomeres but contain only two pigment cells which are closely apposed. Each contains a pair of lateral rhabdomeres and a fifth one occupies a posteromedian extension of the left pigment cell. In the number of rhabdomeres, their relationship to pigment cells and the resulting asymmetry, photoreceptors are more alike in the distantly related species of miracidia studied than they are in ocellate cercariae or even in the miracidium and cercaria of the same species or two closely related ones. From the asymmetry of photoreceptors in larvae of certain flatworms other than digenetic trematodes, it seems that eyespots of miracidia have retained an ancestral pattern whereas the diversity of photoreceptors in cercariae reflects the varied phototactic behavior of those larvae which complete their life cycles by all the means known for cercariae with a free-swimming period. In both miracidia and cercariae, photoreceptors show an anterior-posterior organization that would seem to be concerned with orientation of the larvae with respect to light.Supported in part by a David Ross Fellowship of the Purdue Research Foundation and in part by U.S.P.H.S. Grants 1T1 GM 1392 01 and 2T1 Al 106 07. We express thanks to Dr. Keith Dixon for aid in obtaining and processing miracidia of Fasciola hepatica; to Prof. Clark P. Read for his valuable comments and suggestions; and to Profs. Charles W. Philpott and Richard H. White for advice concerning electron microscopy.     

Neural crest cell behavior in white and dark embryos of Ambystoma mexicanum: Epidermal inhibition of pigment cell migration in the white axolotl

Melanophores in larvae of the white (dd) strain of the Mexican axolotl (Ambystoma mexicanum) are confined to the dorsal midline of the trunk and dorsal posterior part of the head, whereas those in dark larvae (D_-) are distributed over the flank as well. Our results show that this phenotype of white larvae is the result of the failure of the melanophores or their neural crest precursor cells to migrate laterally due to an inhibition of or a failure in the support of their migration in the subepidermal space by the overlying epidermis. Correlated light and scanning electron microscopy of dissected larvae showed melanophores occupying the subepidermal space on the flank of dark larvae, whereas these cells were restricted to the dorsal midline of white larvae. Grafting experiments in which patches of epidermis, the underlying mesoderm, or both, were exchanged between dark and white embryos suggested that white epidermis alone can prevent the integration of pigment cells on the flank of dark larvae and, conversely, that grafts of dark epidermis alone can support their migration on the flank of white larvae. Mesoderm, when grafted alone, could not be shown to have similar effects.     

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts.

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the 'standard' stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.     

Characterization of Photorhabdus luminescens Growth for the Rearing of the Beneficial Nematode Heterorhabditis bacteriophora

Culturing the bioluminescent bacterium Photorhabdus luminescens in nutrient broth (NB) is used to recover phase I cells. These phase I cells were highly luminescent for up to 7 h in this media and the luminosity could also be seen with the naked eye after a 15 min eye adjustment period in a dark room. Red pigmentation is a known trait of phase I cells and was visually distinct within the culture media. The color shade of the red pigment varied on nutrient agar and in NB suggesting that the concentration of the pigment produced is dependent upon density of phase I cells within a specified area. The specific growth rate (μ) and doubling time (g) was determined during the logarithmic growth phase to be 0.36 h⁻¹ and 2.1 h, respectively in NB medium. The nematode-bacterium suspension was injected into larvae of Galleria mellonella to test for entomopathogencity. Within 24 h post-injection insect mortality was seen along with dark red pigmentation and extremely high luminosity indicating infection with P. luminescens.     

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with a quartet of micromeres

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with micromeres was examined. An animal cap consisting of mesomere-descendants was isolated from a 32-cell stage embryo, and recombined with a quartet of micromeres isolated from a 16-cell stage embryo. The recombined embryos were completely depleted of the progenitors of an archenteron, pigment cells, blastocoelar cells and muscle cells. Secondary mesenchyme-like cells (induced SMC) were released from the archenteron derived from the animal cap cells in the recombined embryos. Some induced SMC differentiated into pigment cells, confirming previous data for another echinoid species. Moreover, three different kinds of mesodermal cells-blastocoelar, muscle and coelomic pouch cells-were formed in the recombined larvae. Experiments using a fluorescent probe confirmed that the pigment, blastocoelar, muscle cells and cells in part of the coelomic pouches in the recombined larvae were derived from the animal cap mesomeres. These results indicated that the animal cap mesomere had the potential to differentiate through cell fate regulation into four mesodermal cell types-pigment, blastocoelar, muscle and coelomic pouch cells-.     

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti‐arrestin antibody

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti‐arrestin (anti–Ci‐Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro‐posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero‐posteriorly at the midline of the posterior sensory vesicle (SV). The Ci‐arr gene was expressed throughout the SV at the embryonic mid‐tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci‐Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti–Ci‐Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step‐down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Host-parasite relationship of Angiostrongylus cantonensis in Lymnaea palustris. II. Histopathology.

Larvae of Angiostrongylus cantonensis cause histopathologic changes in Lymnaea palustris, being characterized by mechanical damage to cells and nonspecific cellular responses to the larvae. Amoebocytes, fibroblasts, and pigment cells accumulate around larvae, encapsulating them in nodules. The intensity of the response is variable.     

A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: time-lapse cinemicrographic analysis of pigment cell movement in vivo and in culture

The pattern of migration and motile activity of developing pigment cells of the Mexican axolotl, Ambystoma mexicanum, were analyzed by time-lapse cinemicrography in vivo and in culture. In vivo, melanocytes of dark (D/-) larvae migrate from dorsal to ventral in a highly directional manner. They are elongated and aligned parallel to the direction of migration. Nearly all protrusive activity occurs at their ventral, leading edges. Translocation occurs at a mean rate of 0.7 micron/min and involves alternate or simultaneous advance of the leading and trailing edges of the cell. Indirect evidence suggests that cytoplasmic flow is common. Directional migration occurs in apparent absence of contact between melanocytes. In white (d/d) larvae, protrusive activity is infrequent and the melanocytes move slowly or not at all. Explanted neural crest cells of dark and white larvae attach, spread, and differentiate into melanophores and xanthophores in culture. Individual cultured cells are unbiased in direction of protrusive activity and path of migration. Centrifugal spreading occurs by contacting inhibition of movement. Distribution of protrusive activity, polarity, and contact behavior changes with developmental age in vivo and in culture in ways that may be important in establishing the pigment pattern.     

Pigment pattern formation in the larval salamander Ambystoma maculatum

As part of an ongoing comparative study of pigment patterns and their formation in embryos and larvae of ambystomatid salamanders, Ambystoma maculatum from two differnt populations, one in the northern (New York) and one in the central (Tennessee) United States, were investigated. Scanning electron microscopy was used to study early neural crest development. Light microscopy in combination with markers for the two pigment cell types (xanthophores and melanophores) made it possible to follow pigment cell migration before the pigment cells were fully differentiated. A bilateral pigment pattern consisting of two horizontal melanophore stripes surrounding an interstripe area populated by xanthophores formed in the larvae. In both populations, some variation was present in the form of a continuum ranging from clear horizontal stripes to extreme cases with a random pattern. Unlike the other ambystomatids that have been investigated, the neural crest cells in A. maculatum do not form aggregates and no vertical bars are formed. Instead, both the pattern and its formation are very similar to what has been reported for salamandrids. If pattern formation mechanisms can act as developmental constraints we would expect the A. maculatum pattern to be the primitive condition in the Ambystomatidae, using the Salamandridae as the outgroup. There is no strong support for this when aggregate formation is used as a character and mapped onto phylogenies for the group. The aggregate formation mechanism, and the pigment pattern that it leads to, have most likely been secondarily lost in A. maculatum. © 1993 Wiley-Liss, Inc.