Lethal Spot Research Articles

Endothelin 3 Induces Skin Pigmentation in a Keratin-Driven Inducible Mouse Model

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.

What do knockout models teach us about the enteric nervous system?

Since the first histological studies, enormous strides have been made in understanding the genetics and cell biology of enteric nervous system (ENS) formation. Several mitogenic and trophic factors have been implicated in the process of neural cell proliferation and differentiation. A number of natural (piebald-lethal mice [s l], lethal spotting mice [ls], spotting lethal rats [sl]) or target (Gfralpha1-deficient mice, ret.k - mice, and NT-4 knockout mice) mutations have been reported to produce developmental defects in neural crest cell migration, differentiation or survival. Study of these mutations continues to provide new insights into this complex system. In the present investigation, we showed that a lack of basic fibroblast growth factor (FGF) or growth hormone (GH) leads to morphological abnormalities of the enteric nervous system. Because knockouts, neither of FGF nor of GH, produce enteric nervous system defects substantial enough to compromise the ability of the gut to support life, we postulate that FGF and GH affect only a relatively small subset of neurons and/or that compensatory effects of other growth factors might occur.

SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Inhibitory neurotransmission in lethal spotted mutant mice: A model for Hirschsprung's disease

BACKGROUND & AIMS: The pathogenesis of Hirschsprung's disease is not well understood. The suitability of the animal model for the unknown pathogenesis of inhibitory neurotransmission in Hirschsprung's disease was investigated. METHODS: Circular smooth muscle strips from the internal anal sphincter (IAS) and distal colon (2, 6, 8, 16, and 24 mm from the anal verge) from normal and Ls/Ls mice (mice homozygous for the lethal spotting mutation that develop fetal megacolon after aganglionosis of the terminal colon) were prepared to record changes in isometric tensions in response to different agents and nonadrenergic, noncholinergic nerve stimulation by electrical field stimulation. RESULTS: Bethanechol was used to produce contraction of the smooth muscle strips of distal colon to record a decrease in the tension. Conversely, the IAS smooth muscle strips developed spontaneous tone. In the normal homozygous mice, electrical field stimulation caused a biphasic response, an initial decrease followed by an after- contraction, whereas in Ls/Ls mice, the predominant response was contraction. All smooth muscle strips from normal and Ls/Ls mice produced relaxation in response to sodium nitroprusside and vasoactive intestinal polypeptide. CONCLUSIONS: Ls/Ls mice may serve as an appropriate animal model to investigate the pathogenesis of the inhibitory neurotransmission in Hirschsprung's disease in the distal colon and IAS. (Gastroenterology 1997 May;112(5):1575-85)

Neural and skin cell‐specific expression pattern conferred by steel factor regulatory sequence in transgenic mice

We have produced transgenic mice expressing a lacZ reporter gene under the control of a fragment of a Steel factor (SLF). The function of this gene is essential for the development of hematopoietic cells, germ cells, melanocytes and pacemaker cells of the intestine. The expression of the transgene, containing 2 kb DNA 5′ regulatory sequence, was restricted to neural and skin tissues in appropriate spatial and temporal pattern compared with endogenous SLF mRNA expression. This indicates that the regulatory elements necessary for the neural and skin specific expression are present in this 2 kb DNA sequence, although strong position-dependence of transgene expression was observed. As we could not detect transgene expression in hematopoietic tissues and germ cells after extending 10 kb upstream, elements important for these organs must reside in other regions. Our results indicate that neural crest derived enteric ganglion cells provide SLF to the neighboring pace maker cells expressing c-kit, the receptor for SLF. Cells expressing the transgene in the intestine are ganglion cells derived from neural crest since homozygosity for the lethal spotting (ls) mutation results in loss of such ganglion cells in transgenic mice. We have also shown that the dermal papillae of the hair follicle expresses the transgene, suggesting its roles to support the c-kit dependent growth and development of melanocytes in the hair follicle. © 1996 Wiley-Liss, Inc.

Neural and skin cell-specific expression pattern conferred by steel factor regulatory sequence in transgenic mice.

We have produced transgenic mice expressing a lacZ reporter gene under the control of a fragment of a Steel factor (SLF). The function of this gene is essential for the development of hematopoietic cells, germ cells, melanocytes and pacemaker cells of the intestine. The expression of the transgene, containing 2 kb DNA 5' regulatory sequence, was restricted to neural and skin tissues in appropriate spatial and temporal pattern compared with endogenous SLF mRNA expression. This indicates that the regulatory elements necessary for the neural and skin specific expression are present in this 2 kb DNA sequence, although strong position-dependence of transgene expression was observed. As we could not detect transgene expression in hematopoietic tissues and germ cells after extending 10 kb upstream, elements important for these organs must reside in other regions. Our results indicate that neural crest derived enteric ganglion cells provide SLF to the neighboring pace maker cells expressing c-kit, the receptor for SLF. Cells expressing the transgene in the intestine are ganglion cells derived from neural crest since homozygosity for the lethal spotting (Is) mutation results in loss of such ganglion cells in transgenic mice. We have also shown that the dermal papillae of the hair follicle expresses the transgene, suggesting its roles to support the c-kit dependent growth and development of melanocytes in the hair follicle.

A high-resolution linkage map of the lethal spotting locus: a mouse model for Hirschsprung disease.

Mice homozygous for the lethal spotting (ls) mutation exhibit aganglionic megacolon and a white spotted coat owing to a lack of neural crest-derived enteric ganglia and melanocytes. The ls mutation disrupts the migration, differentiation, or survival of these neural crest lineages during mammalian development. A human congenital disorder, Hirschsprung disease (HSCR), is also characterized by aganglionic megacolon of the distal bowel and can be accompanied by hypopigmentation of the skin. HSCR has been attributed to multiple loci acting independently or in combination. The ls mouse serves as one animal model for HSCR, and the ls gene may represent one of the loci responsible for some cases of HSCR in humans. This study uses 753 N2 progeny from a combination of three intersubspecific backcrosses to define the molecular genetic linkage map of the ls region and to provide resources necessary for positional cloning. Similar to some cases of HSCR, the ls mutation acts semidominantly, its phenotypic effects dependent upon the presence of modifier genes segregating in the crosses. We have now localized the ls mutation to a 0.8-cM region between the D2Mit113 and D2Mit73/D2Mit174 loci. Three genes, endothelin-3 (Edn3), guanine nucleotide-binding protein alpha-stimulating polypeptide 1 (Gnas), and phosphoenolpyruvate carboxykinase (Pck1) were assessed as candidates for the ls mutation. Only Edn3 and Gnas did not recombine with the ls mutation. Mutational analysis of the Edn3 and Gnas genes will determine whether either gene is responsible for the neural crest deficiencies observed in ls/ls mice.

Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons

Defects in the gene encoding the endothelin-B receptor produce aganglionic megacolon and pigmentary disorders in mice and humans. We report that a targeted disruption of the mouse endothelin-3 ligand ( EDN3) gene produces a similar recessive phenotype of megacolon and coat color spotting. A natural recessive mutation that results in the same developmental defects in mice, lethal spotting ( ls), failed to complement the targeted EDN3 allele. The ls mice carry a point mutation of the EDN3 gene, which replaces the Arg residue at the C-terminus of the inactive intermediate big EDN3 with a Trp residue. This mutation prevents the proteolytic activation of big EDN3 by ECE-1. These findings indicate that interaction of EDN3 with the endothelin-B receptor is essential in the development of neural crest-derived cell lineages. We postulate that defects in the human EDN3 gene may cause Hirschsprung's disease.

Mapping studies of the distal imprinting region of mouse chromosome 2.

The known limits of the distal imprinting region of mouse Chromosome (Chr) 2 are defined by the breakpoints of the translocations T(2;8)2Wa, (T2Wa), and T(2;16)28H, (T28H), in distal H3, and proximal H4 respectively. We have shown that T2Wa and T(2;4)1Go, (T1Go), which has a breakpoint in central H3 map close to a, non-agouti. Ada, adenosine deaminase, lies very near the proximal boundary and Ra, ragged, maps very close to the distal boundary, and is less than 0.2 cM from wasted, wst. From the current data Ada can be taken as the proximal, and Ra as the distal gene marker of the imprinting region on the linkage map. From consensus maps twenty three other markers, including fourteen genes, lie between Ada and Ra, some of which may be useful in investigations of imprinting. Of the markers included in the study reported here, four, Ada, ls, lethal spotting, Ra and wst lie or probably lie within the region but none display any evidence of imprinting. We suggest that recombination frequency is elevated in distal Chr 2, because in none of the crosses could the most closely linked marker be ordered in relation to the translocation breakpoint due to the high frequency of double crossovers.

Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.

Mice with hereditary retinal degeneration have provided excellent models for human disease of the biochemical and physiological events occurring in retinal degeneration. Since a number of mouse models are available for other human conditions, more mouse retinal degenerations would be expected to be known; however, finding new models has proved difficult since the search has usually involved laborious histologic screening. We applied the clinical technique of indirect ophthalmoscopy to screen mice for retinal degeneration and then used electroretinography and histology to determine whether true retinal degeneration was present. A Dawson-Trick-Litzkow microfiber corneal electrode was used to record the electroretinogram since the fiber does not occlude the pupil in these small eyes. Normal control values were developed. As an example of the success of the technique, one strain, lethal spot (ls) on indirect ophthalmoscopy appeared to have a retinal degeneration, but these mice had a normal electroretinogram indicating a primary optic atrophy. Likewise, one ls heterozygote that was tested as a control animal and was not suspected of having a retinal degeneration had an abnormal electroretinogram and peripheral retinal degeneration.

An experimental investigation of pattern development in lethal spotting and belted mouse embryos

This investigation is a study of the site of gene action in producing coat spotting in two mutant mice, belted and lethal spotting. An analysis of the pigment-forming potency of skin from the two mutants was made through the use of the technique of intracoelomic grafting. Transplants from potentially pigmented regions of 12 1 2 - and 13 1 2 - day bt bt embryos produced grafts which showed extensive pigment development in the hair and dermis of the graft and in the coelomic lining of the host. Skin isolates taken from the potentially spotted area yielded grafts possessing pigment-free hair. The dermis and coelomic lining were populated by melanocytes in essentially the same number as found in the control group. Embyros used in the lethal spotting series resulted from matings of ls + mice, since ls ls mice usually die between 2 and 3 weeks of age with megacolon. Ten embryos from a group of 45 used in this series produced grafts in which at least some pigment-free hair was found. Those grafts which produced only white hair also failed to develop pigment in the dermis and in the coelomic lining. These embryos were considered to be of the ls ls genotype, and their number corresponds well to the predicted percentage. Transplants of skin from 35 embryos of this series produced grafts which contained pigmented hair only. These embryos are considered to represent ls + and +/+ genotypes. The results of the grafting series clearly indicate that melanoblasts migrate freely throughout all areas of the skin of belted mice and that the genetic block is at the level of the hair follicle. The ls gene appears to act at a different site. An examination of the pigment content of the Harderian gland, the retina and choroid of the eye, the leg musculature, ankle skin, and the membranous labyrinth of the ear was undertaken in normal and spotting mice in an attempt to clarify further the site of gene action. These results substantiate the view that bt acts specifically on the hair follicle. Pigmentation in other tissues of bt bt mice was at the normal level. In contrast to the action of bt, ls reduces the level of pigmentation in the mouse as a whole. The melanocytes in all the tissues studied in ls ls mice, with the exception of those of the retina, were greatly reduced in number or entirely absent. The occurrence of megacolon, the variable nature of the spotting, and the evidence from the descriptive and experimental studies support the hypothesis that the neural crest is abnormal in ls ls mice.