Endothelin 3 (Edn3) encodes a ligand important to developing neural crest cells and is allelic to the spontaneous mouse mutation occurring at the lethal spotting (ls) locus. Edn3(ls/ls) mutants exhibit a spotted phenotype due to reduced numbers of neural crest-derived melanocyte precursors in the skin. In this study, we show that when Edn3 is driven by the keratin 5 promoter and thereby placed proximal to melanocyte lineage cells, adult mice manifest pigmented skin harboring dermal melanocytes. Using a tetracycline inducible system, we show that the postnatal expression of Edn3 is required to maintain these dermal melanocytes, and that early expression of the Edn3 transgene is important to the onset of the hyperpigmentation phenotype. Crosses into Edn3(ls/ls) mutants demonstrate that the Edn3 transgene expression does not fully compensate for the endogenous expression pattern. Crosses into tyrosine kinase receptor Kit(Wv) mutants indicate that Edn3 can partially compensate for Kit's role in early development. Crosses into A(y) mutant mice considerably darkened their yellow coat color suggesting a previously unreported role for endothelin signaling in pigment switching. These results demonstrate that exogenous Edn3 affects both precursors and differentiated melanocytes, leading to a phenotype with characteristics similar to the human skin condition dermal melanocytosis.

1. Authentic human acetylcholinesterase (AChE) was expressed in Escherichia coli under regulation of the constitutive deo promoter or the thermo-inducible lambda PL promoter. 2. To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT. These modifications did not alter the amino acid sequence but resulted in ample production of the protein. 3. rhAChE accumulated in the cells and reached a level of 10% of total bacterial proteins. A partially purified inactive recombinant protein was recovered from inclusion bodies. 4. Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low. A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine. 5. The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity.

a mild subtype of HPS and results from mutations in HPS3, encoding a 1004 amino acid protein of unknown function. HPS3 protein contains a clathrin-binding motif (LLDFE) at residues 172-176 and a potential di-lysine ER retention signal at residues 1000-1003. HPS-3 fibroblasts show a clustered perinuclear distribution of early endosomes (EEA1) and lysosomes (LAMPs), when compared with normal fibroblasts. This abnormal distribution is corrected by expression of GFP-HPS3 in HPS-3 cells. The cellular localization of GFP-HPS3, as well as constructs lacking the putative clathrin binding domain (GFP-HPS3delCBD) or its ER retention motif (GFP-HPS3delER), was studied. In fibroblasts, GFP-HPS3 and GFP-HPS3delER partially colocalize with LAMP1 and clathrin, but GFP-HPS3delCBD lost this co-localization. In melanocytes, GFP-HPS3 and GFP-HPS3delER were localized in perinuclear aggregates and small perinuclear vesicles that partially co-localized with LAMP1 and clathrin. GFP-HPS3delCBD, however, displayed a markedly more diffuse localization and did not co-localize with either LAMP1 or clathrin. In addition, clathrin was co-immunoprecipitated by HPS3 antibodies in normal but not HPS-3 null melanocytes. Additionally, immuno-EM of melanocytes expressing GFP-HPS3 demonstrated co-localization of GFP-HPS3 and clathrin on perinuclear vesicles and vesicles near the cell membrane. These data suggest that HPS3 has a functional clathrinbinding domain that is necessary for proper trafficking of endosomal and lysosomal vesicles in both fibroblasts and melanocytes. CL-6 EARLY OVER AND MIS-EXPRESSION OF ENDOTHE-LIN RECEPTOR B CAUSES COAT COLOR SPOTTING.A. Ittah and L. Kos. Biological Sciences, Florida International University, Miami, FL, USA The endothelin signaling system plays a crucial role in the development of various neural crest derivatives. Endothelin 3 signaling (edn3) mediated through endothelin receptor B (ednrB) is required for normal melanocyte and enteric ganglia development. EdnrB expression is mostly restricted to neural crest precursors and its derivatives. To further elucidate the role and timing of action of ednrB signaling, we generated transgenic mice that express ednrB under the control of the human nestin gene second intron enhancer sequence (Nes-ednrB). This enhancer drives expression throughout the entire neural tube, in a transient manner between E7.5 and E12.5. Expression is observed in neural crest precursors but is downregulated in migrating neural crest derivatives. The Nes-ednrB mice showed a white belly spot phenotype. These mice did not exhibit overt deficits in neuronal or glial lineages as ascertained by immunohistochemisty with specific markers. The hypopigmentation phenotype was augmented when the transgene was placed in a piebald lethal (ednrB null mutant) or lethal spotting (edn3 null mutant) background. Interestingly, the aganglionic megacolon phenotype characteristic of piebald lethal mice was rescued by the transgene. These results indicate that early over and mis-expression of ednrB can disrupt norma...