M. Lynn Lamoreux

Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences

Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4.

Oculocutaneous albinism (OCA) type 4 is a newly identified human autosomal recessive hypopigmentary disorder that disrupts pigmentation in the skin, hair and eyes. Three other forms of OCA have been previously characterized, each resulting from the aberrant processing and/or sorting of tyrosinase, the enzyme critical to pigment production in mammals. The disruption of tyrosinase trafficking occurs at the level of the endoplasmic reticulum (ER) in OCA1 and OCA3, but at the post-Golgi level in OCA2. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the membrane-associated transporter protein (MATP). To characterize OCA4, we investigated the processing and sorting of melanogenic proteins in primary melanocytes derived from uw/uw mice and from wild-type mice. OCA4 melanocytes were found to be constantly secreted into the medium dark vesicles that contain tyrosinase and two other melanogenic enzymes, Tyrp1 (tyrosinase-related protein 1) and Dct (DOPAchrome tautomerase); this secretory process is not seen in wild-type melanocytes. Although tyrosinase was synthesized at comparable rates in wild-type and in uw-mutant melanocytes, tyrosinase activity in uw-mutant melanocytes was only about 20% of that found in wild-type melanocytes, and was enriched only about threefold in melanosomes compared with the ninefold enrichment in wild-type melanocytes. OCA4 melanocytes showed a marked difference from wild-type melanocytes in that tyrosinase was abnormally secreted from the cells, a process similar to that seen in OCA2 melanocytes, which results from a mutation of the pink-eyed dilution (P) gene. The P protein and MATP have 12 transmembrane regions and are predicted to function as transporters. Ultrastructural analysis shows that the vesicles secreted from OCA4 melanocytes are mostly early stage melanosomes. Taken together, our results show that in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the melanosome is disrupted and the enzyme is abnormally secreted from the cells in immature melanosomes, which disrupts the normal maturation process of those organelles. This mechanism explains the hypopigmentary phenotype of these cells and provides new insights into the involvement of transporters in the normal physiology of melanocytes.

The Tyr (albino) locus of the laboratory mouse

The albino mouse was already known in ancient times and was apparently selectively bred in Egypt, China, and Japan. Thus, it is not surprising that the c or albino locus (now the Tyr locus) was among the first used to demonstrate Mendelian inheritance in mammals at the dawn of the past century. This locus is now known to encode tyrosinase, the rate-limiting enzyme in the production of melanin pigment, and the molecular basis of the albino (Tyrc) mutation is known. Here we describe the congenic series of Tyr-locus alleles, from wild type to null (albino). We compare eye and skin pigmentation phenotypes and the genetic lesions that cause each. We suggest that this panel of congenic mutants contains rich, untapped resources for the study of many questions of basic cell biological interest.

The colors of mice : a model genetic network

Preface. Acknowledgments. Statement regarding the use of pictures. Statement regarding nomenclature. Part I: Introduction to the Pigmentary System. 1. Introduction to the Pigmentary System. 1.1. Introduction. 1.2. Colors of vertebrate animals. 1.3. Other pigment cells. 1.4. The epidermal melanin unit. 1.5. Mammalian hair. 1.6. Melanosome biogenesis and translocation. 1.7. Melanin. 1.8. Hair growth. 1.9. Hair growth cycles. 1.10. Embryonic development of the pigment cell lineage. 1.11. Pigment cells in culture. 1.12. Conclusion. Appendix: color loci of the mouse. Part II: The Pigmentary Loci. 2. Introduction to Mutant Pigmentary Genes. 2.1. Defects of normal melanocyte development: white spotting and graying with age. 2.2. Defects in normal melanosome development: albinism. 2.3. Transport of melanosomes to other cells: the dilute phenotype. 2.4. Pigment-type switching: from eumelanogenesis to pheomelanogenesis. 3. White Spotting and Progressive Graying. 3.1. Definitions and general background . 3.2. Pigment cell development: developmental biology. 3.3. Cellular signaling pathways for melanocytes. 3.4. Pigment phenotypes and the classical white-spotting genes. 3.5. The head, heart, ears, and eyes. 4. Albinism and the Failure of Normal Melanosome Development. 4.1. Background. 4.2. The melanosomal matrix. 4.3. The enzymes that catalyze melanogenesis. 4.4. Membrane proteins that regulate the internal milieu of the melanosome. 4.5. Protein processing and routing to the maturing melanosome. 4.6. Melanosome transport. 5. Pigment-Type Switching. 5.1. Introduction. 5.2. Yellow phenotypes. 5.3. Melanin pigment . 5.4. Melanogenesis and the eumelanin/pheomelanin switch mechanism. 5.5. Signaling the switch mechanism at the cellular level. 5.6. Yellow genes. Part III: Technology and Resources. 6. Novel Mouse Pigmentary Mutants Generated by Genetic Manipulation. 6.1. Introduction. 6.2. Mouse transgenesis: generation of genetically engineered mice. 6.3. Coat-color transgenic mice. 6.4. The coat-color mutants generated by gene targeting. 6.5. Influence of the genetic background. 6.6. Conclusions. 7. Other Species and Other Resources. 7.1. Introduction. 7.2. Resources. 7.3. Other species. References. Index.

Mutations in dopachrome tautomerase (Dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein.

Dopachrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194-->Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486-->Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.

Agouti protein, mahogunin, and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes: a cAMP-independent pathway

Melanocortin‐1 receptor (MC1R) and its ligands, α‐melanocyte stimulating hormone (αMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan‐a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200‐fold increases in the pheomelanin to eumelanin ratio, and a tan‐yellow color in pelletted cells. Moreover, ASIP‐treated cells showed reduced proliferation and a melanoblast‐like appearance, seen also in melanocyte lines from yellow (Ay/a and Mc1re/ Mc1re) mice. However ASIP‐YY, a C‐terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP‐YY inhibited the cAMP rise induced by αMSH analog NDP‐MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrnmg‐3J/mg‐3J or Mgrn1md‐nc/md‐nc) also responded to both ASIP and ASIP‐YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP–MC1R signaling includes a cAMP‐independent pathway through attractin and mahogunin, while the known cAMP‐dependent component requires neither attractin nor mahogunin.

New regulatory factors for melanogenesis: Developmental changes in neonatal mice of various genotypes☆

The biosynthesis of melanin occurs through sequential steps known as the Mason-Raper pathway. The initial steps are the conversion of tyrosine to dihydroxyphenylalanine (dopa) and of dopa to dopaquinone by the enzyme tyrosinase (EC 1.10.3.1). Until recently it was assumed that once these first two conversions were completed, the subsequent reactions occurred spontaneously. However, studies with mouse melanoma cells in culture revealed that subsequent steps in the pathway are also regulated. In this report, we demonstrate that these steps are also regulated in skins of fetal and newborn mice. The specific activities of the regulatory factors change during the first week after birth and differ in mice of different genotypes. The findings provide new insights into genetic and developmental regulation of the pigmentary system in mammals.

Number of mast cells in the peritoneal cavity of mice: Influence of microphthalmia transcription factor through transcription of newly found mast cell adhesion molecule, spermatogenic immunoglobulin superfamily

The mi (microphthalmia) locus of mice encodes a transcription factor, MITF. B6-tg/tg mice that do not express any MITF have white coats and small eyes. Moreover, the number of mast cells decreased to one-third that of normal control (+/+) mice in the skin of B6-tg/tg mice. No mast cells were detectable in the stomach, mesentery, and peritoneal cavity of B6-tg/tg mice. Cultured mast cells derived from B6-tg/tg mice do not express a mast cell adhesion molecule, spermatogenic immunoglobulin superfamily (SgIGSF). To obtain in vivo evidence for the correlation of nonexpression of SgIGSF with decrease in mast cell number, we used another MITF mutant, B6-mi(vit)/mi(vit) mice that have a mild phenotype, ie, black coat with white patches and eyes of normal size. B6-mi(vit)/mi(vit) mice had a normal number of mast cells in the skin, stomach, and mesentery, but the number of peritoneal mast cells decreased to one-sixth that of +/+ mice. Cultured mast cells and peritoneal mast cells of B6-mi(vit)/mi(vit) mice showed a reduced but apparently detectable level of SgIGSF expression, demonstrating the parallelism between mast cell number and expression level of SgIGSF. The number of peritoneal mast cells appeared to be influenced by MITF through transcription of SgIGSF.