Melanocytes in the developing and adult atrioventricular valves of the murine heart

The Neural Crest (NC) is a multipotential group of cells that arises from the dorsal aspect of the neural tube early in development. It is well established that a group of NC cells named Cardiac Neural Crest (CNC) migrates to the heart and plays a critical role in the remodeling of the aortic arch arteries and septation of the outflow tract. In this study, using the mouse mutant Pax3sp/sp that has CNC deficits I have identified a putative novel role for the CNC in regulating apoptosis in the atrioventricular (AV) endocardial cushion. The AV endocardial cushion undergoes remodeling to give rise to the cardiac AV valves. Using a transgenic mouse that carries the LacZ reporter gene under the control of the Dopachrome tautomerase promoter (Dct-LacZ), I found that another NC derived population, melanocyte precursors, also contribute to the AV endocardial cushion and developing AV valves. The analysis of Dct-LacZ embryos at different stages showed that NC cells already committed to the melanocytic fate migrate to the heart along the same initial pathway taken by those that will populate the skin. Hypopigmented mice carrying mutations in the Kit and Endothelin receptor b genes, that are critical for the proper development of skin melanocytes, do not have cardiac melanocytes indicating that cardiac and skin melanocyte precursors share the same initial signaling requirements. The analysis of murine adult hearts showed that melanocytes are mostly found in the atrial sides of the tricuspid and mitral valve leaflets. The distribution of melanocytes in the AV valves corresponds exactly to areas of high Versican B expression, a proteoglycan essential for the process of AV valve remodeling. To evaluate a potential role for melanocytes in the AV valves, a nanoindentation analysis of the tricuspid valves of wild type, hypopigmented and hyperpigmented mice was performed. The storage modulus, a measure of stiffness, for the leaflets obtained from hyperpigmented mice was considerably higher (10.5GPa) than that for the leaflets from wild type (7.5GPa) and hypopigmented animals (between 3.5 and 5.5 GPa) suggesting that melanocytes may contribute to the mechanical properties of the AV valves.

Endothelin receptor B and neural crest derived melanocyte development

Neural Crest cells (NCC) constitute a unique embryonic cell population that arises between the prospective epidermis and the dorsal aspect of the neural tube of vertebrates. NCC migrate ventromedially and dorsolaterally throughout the developing embryo giving rise to the peripheral nervous system constituents and melanocytes that ultimately reside in the skin and hair follicles respectively. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene manifest strikingly similar phenotypes characterized by hypopigmentation, hearing loss and megacolon these are due to absence of melanocytes in the skin and inner ear and lack of enteric ganglia in the distal part of the gut, respectively. Piebald lethal mice and humans with Hirschsprung's disease or Waardenburg syndrome carry different mutations in the Ednrb gene. The major goals of this project were to determine whether the action of Ednrb in NCC is required prior to commitment of these cells to the melanocytic lineage and to investigate its potential participation in the actual process of commitment. In order to achieve these goals transgenic mice that express Ednrb under two different regulatory elements were created. The first, Dct-Ednrb, expresses Ednrb under the control of the DOPAchrome tautomerase (Dct) promoter to direct expression to already committed melanocyte precursors. The second, Nes-Ednrb, expresses Ednrb under the regulation of the human nestin gene second enhancer to direct expression to pre-migratory NCC. Crosses of the Dct-Ednrb mouse with piebald lethal showed that the transgene was capable of rescuing the hypopigmentation phenotype of the later. This result indicates that the action of Ednrb after NCC commit to the melanocytic lineage is sufficient for normal melanocyte development. The Dct-Ednrb was further crossed with two other hypopigmentation mutants that carry mutations in the transcription factors Sox10 and Pax3. The transgene rescued the phenotype of the Sox10 mutant only. This suggests that Ednrb interacts with Sox10 but not with Pax3 during melanocyte development. The Nes-Ednrb mice developed a hypopigmentation phenotype that was augmented when crossed with piebald lethal or lethal spotting (mutation in Edn3, the ligand for Ednrb) mice but was rescued by over expression of Edn3. These results suggest that alterations in Ednrb expression early in development affect melanocyte development. This study provides novel information necessary to better understand the early embryonic development of NCC, clarifies specific interactions between different melanogenic genes and, could eventually help in the implementation of therapies for human pigmentary genetic disorders. ^

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

Mus Musculus Neural Crest Development: Is Pax3 Regulating the Expression of Endothelin Receptor B?

The vertebrate Neural Crest (NC) is formed during early embryonic development at the neurulation stage. This group of multi potent cells gives rise to a variety of derivatives such as the skin's pigmented cells (Melanocytes), the peripheral nervous system with its associated components, and the endocrine cells of the adrenal medulla amongst others. There are several molecular mechanisms that underlie the development and migration of NC derived cells. For example, during melanocyte differentiation and migration the Endothelin Receptor B and its ligand Endothelin 3 (EdnrB/Edn3), the kit/ Steel factor and the FGF receptor I FGF pathways amongst others play important roles. Additionally, several transcription factors such as Pax3, SoxlO and Mitfalso intervene during the NC cells differentiation processes. In this work, the possible regulatory interaction of Pax3 and EdnrB was assessed by in situ hybridization methods with EdnrB, SoxlO and Dct riboprobes in Pax3 homozygous embryos. To further characterize this interaction, genetic crosses between Pax3 heterozygous mutants and EdnrB heterozygous animals were established. Coat pigmentation was used as an indicator of genetic interaction on the progeny. Experimental results indicated that Pax3 does not directly regulate the expression of EdnrB during neural crest development but interact to produce normal coat color. I propose two possible models to explain the epistatic relationship of Pax3 and EdnrB during normal melanocyte development.

Effects of Endothelin 3 on the Tumor-Angiogenic Response in a Novel Melanoma Mouse Model

Skin cancer is the most common form of cancer in the United States. Melanoma is a particular type of skin cancer, which arises from the malignant transformation of melanocytes and generally exhibits a high propensity to metastasize. Melanoma progression is dependent on angiogenesis to deliver the oxygen and nutrients required to maintain the altered metabolism of rapidly proliferating tumorigenic cells. Recent studies have implicated the growth factor Endothelin 3 (Edn3) in melanoma progression and metastasis. The aim of this study was to examine the role that Edn3 plays in the angiogenesis of melanocytic lesions. For this purpose, Dct-Grm1 transgenic mice, which spontaneously acquire melanocytic lesions through the aberrant expression of the metabotropic glutamate receptor 1 (mGluR1), were crossed with K5-Edn3 transgenic mice that overexpress Edn3. Tumors in the Dct-Grm1/K5-Edn3 experimental population were examined and compared to the control Dct-Grm1 population using immuno-fluorescent staining targeted against the vascular endothelial cell marker CD31. Proteomic arrays were also used and identified changes in the expression of specific angiogenic factors. CD31 antibody staining results revealed an increased vascular density in Dct-Grm1/K5-Edn3 tumors compared with tumors from the Dct-Grm1 controls. Analysis of the relative expression of angiogenic proteins showed an upregulation of various vascular factors in tumors from the Dct-Grm1/K5-Edn3 population, including VEGF-B, MMP-8, MMP-9, and Angiogenin. These results suggest that endothelin signaling promotes angiogenesis in melanocytic lesions. Targeting the factors upregulated by Edn3 signaling may prove effective in hindering melanoma progression.

Inducible Overexpression of Endothelin-3 in a Transgenic Mouse Leads to Hyperpigmentation That Decreases Over Time

Endothelin-3 (Edn3) has been shown to be an essential environmental cue in melanocyte development. Edn3 and its receptor, EdnrB, are allelic to mouse mutations occurring at the lethal spotting and piebald loci, respectively; these mutations result in hypopigmentation phenotypes. Mutations in the genes for both Edn3 and EdnrB are implicated in human pigmentation disorders such as Waardenburg-Shah syndrome, which is characterized by pigmentation defects, deafness, and megacolon. In this study, a tetracycline-inducible transgenic mouse model that overexpresses Edn3 under the control of the Keratin 5 promoter was shown to produce a hyperpigmentation phenotype that decreases over time. The expression pattern of transgenic Edn3 and its effects on the melanocyte population were examined in transgenic embryos, postnatal skin, and the skin of adult mice that exhibit faded hyperpigmentation. These studies suggest that overexpression of Edn3 in this model is restricted primarily to the roof plate of the neural tube and surface ectoderm in the developing embryo and to keratinocytes in the epidermis of postnatal mice. A decline in transgenic expression and a reduction in the dermal melanocytes and free melanin that characterize the phenotype in juvenile mice were shown to correlate with the fading of the hyperpigmentation phenotype. Transgenic mice in which transgenic expression was repressed (resulting in the disappearance of the hyperpigmentation phenotype) also exhibited a decrease in the dermal melanocyte population. The Edn3-overexpressing mice used in this study might be helpful m understanding human skin conditions characterized by dermal melanocytosis.

Transgenic Expression of Endothelin Receptor-B Rescues the Aganglionosis Phenotype of Piebald Lethal Mice

Neural crest cells (NCC) are a unique population of cells in vertebrates that arise between the presumptive epidermis and the dorsal most region of the neural tube. During neurulation, NCC migrate to many regions of the body to give rise to a wide variety of cell types. NCC that originate from the neural tube at the levels of somite 1-7 colonize the gut and give rise to the enteric ganglia. The endothelin signaling pathway has been shown to be crucial for proper development of some neural crest derivatives. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene exhibit similar phenotypes characterized by hypopigmentation, hearing loss, and megacolon. Thesephenotypes are due to lack of melanocytes in the skin, inner ear and enteric ganglia in the distal portion of the colon, respectively. It is well established that Ednrb is required early during the embryonic development for normal innervation of the gut. However, it is not clear if Ednrb acts on enteric neuron precursor cells or in pre-committed NC precursors. Additionally, it is controversial whether the action of Ednrb is cell autonomous or non- autonomous. We generated transgenic mice that express Ednrb under the control of the Nestin second intron enhancer (Nes) which drives expression to pre-migrating NCC. These mice were crosses to the spontaneous mouse mutant piebald lethal, which carriers a null mutation in Ednrb and exhibits enteric aganglionosis. The Nes-Ednrb was capable of rescuing the aganglianosis phenotype of piebald lethal mutants demonstrating that expression of Ednrb in pre-committed precursors is sufficient for normal enteric ganglia development. This study provides insight in early embryonic development of NCC and could eventually have potential use in cellular therapies for Hirschsprung's disease.

The role of the transcription factor Ets1 in melanocyte development

Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. ^ Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. ^ At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.^

UV-induced melanoma mouse model dependent on endothelin 3 over-expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.^

Interactions between Endothelin Receptor B and Transcription Factors Sox10 and Pax3 in the Melanocyte Lineage

Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

UV-Induced Melanoma Mouse Model Dependent on Endothelin 3 Over-Expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.

The Role of the Transcription Factor Ets1 in Melanocyte Development

Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.