Cell context reveals a dual role for Maf in oncogenesis.

Maf b-Zip transcription factors are involved in both terminal differentiation and oncogenesis. To investigate this apparent contradiction, we used two different primary cell types and performed an extensive analysis of transformation parameters induced by Maf proteins. We show that MafA and c-Maf are potent oncogenes in chicken embryo fibroblasts, while MafB appears weaker. We also provide the first evidence that MafA can confer growth factor independence and promote cell division at low density. Moreover, using MafA as a model, we identified several parameters that are critical for Maf transforming activities. Indeed, MafA ability to induce anchorage-independent cell growth was sensitive to culture conditions. In addition, the transforming activity of MafA was dependent on its phosphorylation state, since mutation on Ser65 impaired its ability to induce growth at low density and anchorage-independent growth. We next examined transforming activity of large Maf proteins in embryonic neuroretina cells, where they are known to induce differentiation. Unlike v-Jun, MafA, MafB and c-Maf did not show oncogenic activity in these cells. Moreover, they counteracted transformation induced by constitutive activation of the Ras/Raf/MEK pathway. Taken together, our results show that Maf proteins could display antagonistic functions in oncogenesis depending on the cellular context, and support a dual role for Maf as both oncogenes and tumor suppressor-like proteins.

Expression pattern of the drm/gremlin gene during chicken embryonic development.

The drm gene encodes a cystine knot-containing secreted and cell membrane-associated glycoprotein shown to be an antagonist of BMPs. Drm was recently reported to play a crucial role in limb bud development, by its capacity to bind BMPs. Here, we have studied the expression pattern of drm transcripts during chicken development, by using whole-mount in situ hybridization. We show that, from stage 22HH to stage 26HH, in addition to limb buds, drm is expressed in cephalic neural crest-derived branchial arches I, II and III, in the medio-dorsal lip of the myotome and in the superficial dermatome

Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro.

Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3 in vitro on the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the present in vitro experiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCC in vivo when they migrate to the skin under the ectoderm that expresses ET3.

Cloning and characterization of a novel endothelin receptor subtype in the avian class.

Endothelin 3 (EDN 3) and the endothelin receptor B (EDNRB) are involved in the development of neural crest and particularly of the melanocytes and the enteric nervous system. We reported previously that the avian EDNRB gene is expressed in the neural fold before crest cell migration and later on in all the neural crest derivatives except, at any developmental stage, in the melanocytic lineage. However, quail melanoblasts proliferate in response to EDN 3 stimulation in vitro. These observations prompted us to search for another type of endothelin receptor (EDNR). We report here the cloning by reverse transcriptase-PCR of an avian cDNA encoding a subtype of EDNR, which we have called EDNRB2, because its deduced amino acid sequence is more closely related to that of EDNRB than to either the mammalian EDNRA or to the Xenopus EDNRC. Its expression pattern differs from that of the "classical" avian EDNRB because it is strongly expressed in melanoblasts and melanocytes. EDNRB2 transcripts are also abundant in the liver and kidney. Our pharmacological studies showed that EDNRB2 binds with similar affinity to EDN 1, EDN 2, and EDN 3, further confirming that this receptor belongs to the B type, although it displays a low affinity for sarafotoxin-c, a known EDNRB-selective agonist.

Endothelin-B receptor is expressed by neural crest cells in the avian embryo.

Disruptions of the genes encoding endothelin 3 (EDN3) and its receptor endothelin-B receptor (EDNRB) in the mouse result in defects of two neural crest (NC)-derived lineages, the melanocytes, and the enteric nervous system. To assess the mechanisms through which the EDN3/EDNRB signaling pathway can selectively act on these NC derivatives, we have studied the spatiotemporal expression pattern of the EDNRB gene in the avian embryo, a model in which NC development has been extensively studied. For this purpose, we have cloned the quail homologue of the mammalian EDNRB cDNA. EDNRB transcripts are present in NC cells before and during their emigration from the neural tube at all levels of the neuraxis. At later developmental stages, the receptor remains abundantly expressed in the peripheral nervous system including the enteric nervous system. In a previous study, we have shown that EDN3 enhances dramatically the proliferation of NC cells when they are at the pluripotent stage. We propose that the selective effect of EDN3 or EDNRB gene inactivation is due to the fact that both melanocytes and enteric nervous system precursors have to colonize large embryonic areas (skin and bowel) from a relatively small population of precursors that have to expand considerably in number. It is therefore understandable that a deficit in one of the growth-promoting pathways of NC cells has more deleterious effects on long-range migrating cells than on the NC derivatives which develop close to the neural primordium like the sensory and sympathetic ganglia.

Origin of the c-kit-positive interstitial cells in the avian bowel.

Interstitial cells of Cajal (ICC) aroused much interest among neuroanatomists at the beginning of the century. These small cells, organized into networks, are intercalated between nerve fibers and muscle cells, and are now considered by many authors to be responsible for the pacemaker activity of the gut. Renewed interest in these cells arose recently when the receptor tyrosine kinase, c-kit, was shown to be associated with their functional activity. The embryonic origin of interstitial cells has remained a controversial issue ever since their discovery. Some authors consider them to be of neural or glial nature and thus of neural crest origin. Others consider them to be of fibroblastic or muscular nature. We have applied the quail-chick marker system to solve this problem. ICC were identified by means of a chicken-c-kit nucleic probe which cross-reacts with the quail c-kit gene product. We constructed chimeric bowels by grafting isotopically quail vagal neural crest into chick embryos at embryonic day 2 (E2). The enteric innervation of the chimeras was then of quail origin. In situ hybridization of the chimeric bowels showed that all the c-kit-positive cells were of the chick type, and therefore belonged to the gut mesenchyme and were not neural crest-derived cells. This observation was confirmed by culturing aneural chick guts on the chorio-allantoic membrane. Typical ICC, as defined at the EM level and by their expression of the c-kit receptor, developed in the gut wall in the complete absence of enteric innervation. One can conclude the ICC are of mesodermal origin and develop independently from enteric neurons with which they later establish anatomical and functional relations.

Steel and c-kit in the development of avian melanocytes: a study of normally pigmented birds and of the hyperpigmented mutant silky fowl.

We describe here the expression of c-kit and Steel (Sl) genes during the development of melanocytes in normally pigmented strains of chick and quail compared to unpigmented (White Leghorn) and hyperpigmented (Silky Fowl) strains of chickens. By using the quail/chick chimera system, we found that the neural crest cells, which migrate dorso-laterally in the subectodermal mesenchyme to give rise to the melanocytes, express c-kit as early as E4, that is about 2 days after they have left the neural primordium. The Sl gene is expressed from E4 onward in the epidermis but not at all in the dermis at any developmental stage. As feather buds develop, Sl mRNA becomes restricted to the apical region of the feather filaments. During formation of the barbs and barbules of the down feather, production of the Steel factor is restricted to the external epidermal cells of the barbules. The cell bodies of the c-kit-positive melanocytes are then located in the internal border of the epidermal ridges and extend their processes toward the source of the Steel factor. We propose that the spatial restriction of Sl gene activity at that stage accounts for the morphology of the melanocytes and their vectorial secretion of melanin to the external barbule cells. As a whole, these results show that during skin development c-kit positive cells are present in the Steel factor-producing areas at the time when melanoblasts proliferate and differentiate. Interestingly, in the mouse, previous studies showed that the Sl gene is activated in the dermis where melanoblasts undergo most of their expansion (Nishikawa et al. [1991] EMBO J. 10:2111-2118). In the unpigmented and hyperpigmented mutants that we studied, expression of the Sl message, as judged quantitatively in Northern blots (for the SF embryos) or spatially by in situ hybridization, is similar to that observed in normal birds. In SF embryos the c-kit expressing melanoblasts migrate initially in the dorso-lateral migration pathway as in normal birds. However their number increases considerably in the dermis from E5 onward. From E7, they invade mesodermally derived organs that do not express the Sl gene. This suggests that another, still unknown, factor(s) is responsible for the survival, the proliferation, and the extensive spreading of melanocytic cells within the mesoderm of this mutant.

Effect of the Steel gene product on melanogenesis in avian neural crest cell cultures.

Mutations at the Steel (Sl) and dominant white spotting (W) loci affect three embryonic lineages: primordial germ cells, hemopoietic stem cells and neural-crest-derived melanocytes. The gene products of these loci are a peptide growth factor, called here stem cell factor (SCF), and its tyrosine kinase receptor, the proto-oncogene c-kit. We have studied how chicken recombinant SCF affects the development of melanocytes from quail neural crest cells in secondary culture under defined conditions. We observed that the total number of neural crest cells, of melanocytes and of their precursors was higher in the presence than in the absence of SCF. Labelling with bromodeoxyuridine showed that SCF had a modest and transient mitogenic effect on the neural crest population. SCF also enhanced the differentiation rate of melanocyte precursors, recognized by the "melanocyte early marker" monoclonal antibody (MelEM MAb), and of melanocytes, since the proportion of both subpopulations significantly increased in the presence of SCF. Finally, SCF increased the survival of the neural crest population since in its presence the total number of cells remained stable while it gradually declined in control cultures. Our results support the notion that SCF sustains the survival of the neural crest population and stimulates the rate of the melanogenic differentiation process.

Growth of neural crest cells in vitro is enhanced by extracts from Silky Fowl embryonic tissues.

In the Silky Fowl (SF) breed of chicken, most of the internal organs are infiltrated with melanocytes. Previous studies have shown that this generalized mesodermal pigmentation is not due to a cell autonomous abnormality of the melanocytes but to environmental factors able to promote both the homing of pigment cell precursors in abnormal embryonic sites and their proliferation and differentiation. To analyse the mode of these environmental cues, we tested the effect of SF embryo extract (SFEE) on cultured quail neural crest cells as compared with that of EE from normal chickens of the JA57 strain (JA57EE). We found that SFEE enhances crest cell proliferation as judged by 3H-TdR incorporation and cell counting. In contrast, no effect of SFEE was observed either on the proportion of cultured cells that are engaged into the melanocytic differentiation pathway or on the amount of melanin produced by each differentiated pigment cell. The simple observation, however, reveals that SFEE has a significant effect on pigmentation of the cultured quail neural crest cells. This effect has therefore to be accounted for by the general increase in cell number induced by SFEE. The question is raised as to whether the in vivo SF phenotype is generated exclusively by this mechanism.