Macrophage migration inhibitory factor (MIF) family in arthropods : cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in Mud crabs, Scylla paramamosain

5. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (Nos. 31172438 and U1205123), the Natural Science Foundation of Fujian Province (No. 2012J06008 and 201311180002) and the projects-sponsored by SRF. TW received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions.

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

与黑素生成相关的生化指标测定为主的美白评价体系的初建

近年来，随着黑素细胞生物学的发展和对美白剂功效研究的不断深入，美白剂对皮肤正常生理潜在的负面影响正逐渐为人们所重视 ，仅仅通过临床皮肤敏感试验已远不能给以科学的解释，因而建立一个科学量化的美白剂评价体系就显得尤为迫切和必要。本文工 作旨在将体外黑素细胞原代培养与黑素生成相关的生化指标（包括：黑度，细胞量，铬氨酸酶TYR，多巴色素异构酶DT酶活的）， 检测相结合，并联系细胞学观察，从而形成一整套相对完善，定性定量的研究美白剂功效的评价体系。通过测定外加a-MSH,内皮素 ET-1及其拮抗剂GD2168后黑素细胞内上述生化指标的变化，不仅验证了a-MSH，内皮素ET-1的促黑作用，内皮素拮抗剂GD2168的美 白功效，而且还体现出本评价体系不同于国内现有的其它美白评价体系的独特优势。首先，本评价体系在国内为首家将原代培养的 黑素细胞用于美白评价，由于原代培养环境与体内皮肤生理环境的相似性，在体外对10-8mol/La-MSH产生了正常的应答，所以在评 价美白剂的功效时该培养体较之将黑素细胞孤立生长的纯化培养体系更为科学也更具有说服力。尤其值得一提的是，国内现有的生 化水平的美白评价多局限于对TYR活力的测定，而本体系的另一特点就是除了TYR外还增加了对TRP-2即DT酶活的测定，由于DT在维 持正常黑素细胞及皮肤生理上的重要意义，对其变化的研究往往可揭示出美白剂对皮肤的潜在毒性，本文通过测定并分析内皮素拮 抗剂GD2168对体外黑素细胞DT活力的下调作用，对其潜在的副作用进行了科学的预测，这项工作国内尚无人开展。Over the past few years, melanin cell research has experienced unprecedent impetus, which also contribute to the study on lightener's function especially it's potent skin damage. As a result, it's the high time to build a more accurate and complete evaluation system to investigate lightener's effects and by-effects as well. After normal human melanocytes were cultured primarily in vitro, the effects of a-MSH, endothelin-1(ET-1)and ET -1's antagonist GD2168 on melanogenesis were studied biochemically by measurement of melanin content, cell-number, tyrosinaseTYR activity and dopachrome tautomeraseDT activity. Compared with untreated cells, the treated cells responsed to 10-8mol/L a-MSH with the increase in all items. ET-1 induced both an increae in DT activity and melanin concent; however, the melanosynthesis increase was inhibited significantly in the present of GD2168. Trough above work, a new evaluation system of lightener has been established and confirmed to be feasible. Different from other evaluation systems present in country, this system used the primary culture, which was more consistent to the physiological circumstance. Moreover, the system added DT activity assay that help reveal the GD2168's potent side-effects, which would have been clouded or ignored if only TYR activity was assayed.

Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development

Wnt1 signaling has been implicated as one factor involved in neural crest-derived melanocyte (NC-M) development. Mice deficient for both Wnt1 and Wnt3a have a marked deficiency in trunk neural crest derivatives including NC-Ms. We have used cell lineage-directed gene targeting of Wnt signaling genes to examine the effects of Wnt signaling in mouse neural crest development. Gene expression was directed to cell lineages by infection with subgroup A avian leukosis virus vectors in lines of transgenic mice that express the retrovirus receptor tv-a. Transgenic mice with tva in either nestin-expressing neural precursor cells (line Ntva) or dopachrome tautomerase (DCT)-expressing melanoblasts (line DCTtva) were analyzed. We overstimulated Wnt signaling in two ways: directed gene transfer of Wnt1 to Ntva+ cells and transfer of β-catenin to DCTtva+ NC-M precursor cells. In both methods, NC-M expansion and differentiation were effected. Significant increases were observed in the number of NC-Ms [melanin+ and tyrosinase-related protein 1 (TYRP1)+ cells], the differentiation of melanin− TYRP1+ cells to melanin+ TYRP1+ NC-Ms, and the intensity of pigmentation per NC-M. These data are consistent with Wnt1 signaling being involved in both expansion and differentiation of migrating NC-Ms in the developing mouse embryo. The use of lineage-directed gene targeting will allow the dissection of signaling molecules involved in NC development and is adaptable to other mammalian developmental systems.

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. ^

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.