Evolution and development of pigment cells: at the crossroads of the discipline.

The following is a summary of the current state of comparative biology with respect to pigmentation. Recent results from molecular analyses of genes involved in pigmentation in lower vertebrates are compared with similar data from mouse and man. Particular emphasis has been placed on evolutionary and developmental aspects of pigmentation. Recent advances in molecular biology of lower vertebrate pigmentation allow for the comparison of orthologous molecules across a wider range of species than ever before; some of these results are summarized and used to highlight the current state of pigmentation from a comparative perspective. A more cellular, organismal approach is also explored to highlight some important lessons from comparative biology. Lastly, large-scale evolutionary questions are put into a framework that highlights both the differences and similarities between mammals/birds and other vertebrates. It is the opinion of the authors that important, long-standing questions in these areas can now be addressed in ways that have not been possible before. Thus, the discipline is at an exciting crossroads where developmental and evolutionary data can be used to create a unified view of pigment cells and pigments across many species.

What insights into vertebrate pigmentation has the axolotl model system provided?

Amphibians have been judiciously exploited by developmental biologists for many years for studying basic developmental mechanisms in vertebrates. In this review, the contributions that have been made by urodeles, in particular the axolotl (Ambystoma mexicanum), to the study of pigment cell biology are elaborated. Pigment cell differentiation is described, and the wild-type pigment phenotype is contrasted to pigment mutants such as albino, axanthic, melanoid, and white. Methods used for studying pigmentation, including recently developed molecular biological tools, are included to illustrate the significance of the axolotl as a model system for studying vertebrate pigmentation.

Effects of melanocyte-stimulating hormone on wild-type and white axolotl neural crest cells.

The goals of the current research were twofold: to study the effects of melanocyte-stimulating hormone (MSH) on undifferentiated axolotl (Ambystoma mexicanum) neural crest cells and to determine whether wild-type or white mutant axolotl neural crest cells respond differently to MSH or to either of two agonists of the MSH signal transduction pathway (cholera toxin or N6,O2-dibutyryl adenosine 3',5'-monophosphate (dbcAMP). We found that MSH induces melanophore differentiation in axolotl neural crest cells in a dose-dependent manner; however, white, but not wild-type, neural crest cells are inhibited by the highest concentration of MSH (6 x 10(-6) M) tested. The effects of cholera toxin and dbcAMP indicate that the differentiation of melanophores from white neural crest cells may be more cAMP sensitive than wild-type neural crest cells. These results suggest that MSH is likely to play a regulatory role in the initial pigment pattern formation of wild-type and white axolotls.

The identification and partial cloning by PCR of the gene for tyrosinase-related protein-1 in the Mexican axolotl.

The tyrosinase gene family is currently composed of three members, tyrosinase and two tyrosinase-related proteins, TRP-1 and TRP-2. These three gene products have all been found to act in the synthesis of melanin pigments with the enzyme tyrosinase catalyzing the initial rate-limiting steps. Thus far these genes have primarily been analyzed in higher vertebrates. We have used degenerate PCR primers to isolate a large fragment of an axolotl tyrosinase-related protein. Sequence analysis of the entire 1,057-bp fragment isolated indicates a high degree of similarity to the mouse TRP-1, the product of the brown locus. Phylogenetic analysis supports the conclusion that the fragment isolated corresponds to the axolotl TRP-1 homolog. This is the first TRP-1 gene to be identified in an amphibian species.

Inhibition of neural crest cell differentiation by embryo ectodermal extract.

The white mutation in Mexican axolotls has long been thought to be a defect associated with the embryonic extracellular environment, but not with embryonic neural crest cells. Thus it was believed that pigment cells in white axolotls disappear from the skin during early development, not because they are intrinsically defective but because they have no choice but to move into an unfavorable environment. We present evidence to suggest that: (1) white neural crest cells are in fact intrinsically different from dark (wild-type) cells, and (2) an inhibitor is produced in white embryonic ectoderm that actively suppresses the migration, differentiation, and survival of pigment cells in this animal. How these observations fit into the existing body of literature on the white mutant and a model for how the white phenotype might develop are discussed.

Pigments and ultrastructures of pigment cells in xanthic sailfin mollies (Poecilia latipinna).

Electron micrographs of skin from xanthic (gold) sailfin mollies revealed numerous xanthophores, as well as scattered melanophores. The melanophores were seen to contain premelanosomes in various stages of development. This is consistent with the fact that xanthic mollies have been shown to be tyrosinase positive. Melanosomes in xanthic mollies appear to develop by one of two pathways: 1) from an endoplasmic reticulum-derived vesicle which develops an internal lamellar framework, and 2) by fusion of multiple Golgi-derived vesicles which lack an internal lamellar framework. Analysis of the pigments in the skin of the xanthic mollies identified four colorless pteridine pigments (xanthopterin, isoxanthopterin, neopterin, and pterin) and a carotenoid with an absorbance spectrum similar to beta-carotene. It appears that, unlike some other poeciliid fishes, sailfin mollies do not use pteridine pigments for orange coloration. Rather, they appear to rely primarily on carotenoids.

Ultrastructural analysis of iridophore organellogenesis in a lizard, Sceloporus graciosus (Reptilia: Phrynosomatidae).

Transmission electron microscopy (TEM) data from the ultrastructure of lizard skin iridophores (reflective dermal chromatophores) are used to illustrate the organellogenesis of small rectangular reflecting platelets, which are the color-generating components of these cells. During the development of reflecting platelets, crystals are deposited within double-membraned vesicles from electron-dense material located within the vesicles. The crystals are initially small but expanded lengthwise eventually to fill the vesicle that contains them. The inner membrane then tightly surrounds the crystal whereas the outer membrane is much more loosely associated with the inner-membrane-bound crystal. These observations allow discussion of the possible origin of the precursor double-membraned vesicles from endoplasmic reticulum (ER) and Golgi-derived vesicles. A model is proposed that incorporates our findings and other published reports to explain the origin of the precursor double-membraned vesicles via three alternative pathways.

Effects of fetal bovine serum and serum-free conditions on white and dark axolotl neural crest explants.

Neural crest cells from both white mutant and dark (wildtype) axolotls (Ambystoma mexicanum) were cultured in increasing concentrations of fetal bovine serum (FBS; 2 to 20%). For each explant, the total number of cells that migrated and the percent of differentiated melanophores were recorded. At concentrations of FBS above 2% melanophore differentiation was essentially equivalent (32 to 59%) for both the white and dark neural crest cultures, but subtle differences in cell behavior and differentiation were found between the two phenotypes. By contrast there was a significant difference in the percent melanization of cells in serum-free control cultures, wherein melanophore differentiation in dark neural crest cultures was, on average, 18% compared to 5% in white cultures. Thus, contrary to all previously published work, white and dark neural crest cells are not intrinsically equivalent. Our culture results are discussed with regard to the probable in vivo conditions that cause the white phenotype.

Follow-up of prospective randomized trial of ampicillin or chloramphenicol versus moxalactam treatment of Haemophilus influenzae type b meningitis.

In a prospective randomized trial, moxalactam administered to 66 children was compared with ampicillin or chloramphenicol given to 68 children for the treatment of Haemophilus influenzae type b meningitis. Acute morbidity and mortality rates were equivalent between the two treatment groups. At 2 years after discharge, the results of psychologic tests (Bayley Scales of Infant Development or McCarthy Scales of Children's Abilities) were also equivalent between the two treatment groups for patients remaining in the study.

Prospective comparative trial of moxalactam versus ampicillin or chloramphenicol for treatment of Haemophilus influenzae type b meningitis in children.

In a prospective, randomized study, moxalactam in 44 children was compared with ampicillin or chloramphenicol in 47 children for the treatment of Haemophilus influenzae type b meningitis. Both groups were comparable in terms of clinical and laboratory findings at admission. The hospital course, neurologic sequelae including deafness, and number of deaths were the same for both groups. The incidence of adverse reactions also was the same except that diarrhea and thrombocytosis occurred significantly (P less than or equal to 0.04) more frequently in children given moxalactam. Moxalactam was equivalent to ampicillin or chloramphenicol in the treatment of H. influenzae type b meningitis in children.