A direct link between MITF, innate immunity, and hair graying.

Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitfmi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.

Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence.

Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed, but how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glial­ILC3­epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably, ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit, revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals.

Rescue of an in vitro neuron phenotype identified in Niemann-Pick disease, type C1 induced pluripotent stem cell-derived neurons by modulating the WNT pathway and calcium signaling.

Niemann-Pick disease, type C1 (NPC1) is a familial disorder that has devastating consequences on postnatal development with multisystem effects, including neurodegeneration. There is no Food and Drug Administration-approved treatment option for NPC1; however, several potentially therapeutic compounds have been identified in assays using yeast, rodent models, and NPC1 human fibroblasts. Although these discoveries were made in fibroblasts from NPC1 subjects and were in some instances validated in animal models of the disease, testing these drugs on a cell type more relevant for NPC1 neurological disease would greatly facilitate both study of the disease and identification of more relevant therapeutic compounds. Toward this goal, we have generated an induced pluripotent stem cell line from a subject homozygous for the most frequent NPC1 mutation (p.I1061T) and subsequently created a stable line of neural stem cells (NSCs). These NSCs were then used to create neurons as an appropriate disease model. NPC1 neurons display a premature cell death phenotype, and gene expression analysis of these cells suggests dysfunction of important signaling pathways, including calcium and WNT. The clear readout from these cells makes them ideal candidates for high-throughput screening and will be a valuable tool to better understand the development of NPC1 in neural cells, as well as to develop better therapeutic options for NPC1.

A somatic cell defect is associated with the onset of neurological symptoms in a lysosomal storage disease.

Mutations in individuals with the lysosomal storage disorder Niemann-Pick disease, type C1 (NPC1) are heterogeneous, not localized to specific protein domains, and not correlated to time of onset or disease severity. We demonstrate direct correlation of the time of neurological symptom onset with the severity of lysosomal defects in NPC1 patient-derived fibroblasts. This is a novel assay for NPC1 individuals that may be predictive of NPC1 disease progression and broadly applicable to other lysosomal disorders.

The exon junction complex component Magoh controls brain size by regulating neural stem cell division.

Brain structure and size require precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, and mechanistic explanations of how aberrant NSC division causes the reduced brain size seen in microcephaly are lacking. Here we show that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly because of INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes, as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number and genomic stability. In utero rescue experiments showed that a key function of Magoh is to control levels of the microcephaly-associated protein Lis1 during neurogenesis. Our results uncover requirements for the EJC in brain development, NSC maintenance and mitosis, thereby implicating this complex in the pathogenesis of microcephaly.

Comparison of melanoblast expression patterns identifies distinct classes of genes.

A full understanding of transcriptional regulation requires integration of information obtained from multiple experimental datasets. These include datasets annotating gene expression within the context of an entire organism under normal and genetically perturbed conditions. Here we describe an expression dataset annotating pigment cell-expressed genes of the developing melanocyte and retinal pigmented epithelium lineages. Expression images are annotated and available at http://research.nhgri.nih.gov/manuscripts/Loftus/March2009/. Data are also summarized in a standardized manner using a universal melanoblast scoring scale that accounts for the embryonic location of cells and regional cell density. This approach allowed us to classify 14 pigment genes into four groupings classified by cell lineage expression, temporal-spatial context, and differential alteration in response to altered MITF and SOX10 status. Significant differences in regional populations were also observed across inbred strain backgrounds, highlighting the value of this approach to identify modifier allele influences on melanoblast number and distributions. This analysis revealed novel features of in vivo expression patterns that are not measurable by in vitro-based assays, providing data that in combination with genomic analyses will allow modeling of pigment cell gene expression in development and disease.

A Sox10 expression screen identifies an amino acid essential for Erbb3 function.

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10(LacZ/+). Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1-4 (msp1-4), which show altered NC patterning. In homozygous msp1 embryos, Sox10(LacZ) expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3(msp1), that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting.

Analysis of ocular hypopigmentation in Rab38cht/cht mice.

PURPOSE: To characterize the ocular phenotype resulting from mutation of Rab38, a candidate gene for Hermansky-Pudlak syndrome. METHODS: Chocolate mice (cht, Rab38(cht/cht)) and control heterozygous (Rab38(cht/)(+)) and wild-type mice were examined clinically, histologically, ultrastructurally, and electrophysiologically. Mice homozygous for both the Rab38(cht) and the Tyrp1(b) alleles were similarly examined. RESULTS: Rab38(cht/cht) mice showed variable peripheral iris transillumination defects at 2 months of age. Patches of RPE hypopigmentation were noted clinically in 57% of Rab38(cht/cht) eyes and 6% of Rab38(cht/)(+) eyes. Rab38(cht/cht) mice exhibited thinning of the iris and RPE and larger b-wave amplitudes in the scotopic range when compared with the control animals. Compared with wild-type mice, Rab38(cht/cht) melanosomes were smaller and there were fewer in neuroectodermally derived retinal pigment epithelium; in neural crest-derived choroid melanocytes, they were smaller in size only. Mutation of both Rab38 and Tyrp1 produced mice with ocular and coat color pigment dilution greater than that seen with either mutation alone. Comprehensive clinical and pathologic analyses showed no other organ system or blood defects in Rab38(cht/cht) mice. CONCLUSIONS: Rab38(cht/cht) mice show ocular characteristics reminiscent of human oculocutaneous albinism, as well as iris and RPE thinning. The synergistic effects of the Rab38(cht) and Tyrp1(b) alleles suggest that TYRP1 is not the only target of RAB38 trafficking. This mouse line provides a useful model for studying melanosome biology and its role in human ocular diseases.

Informatic and genomic analysis of melanocyte cDNA libraries as a resource for the study of melanocyte development and function.

As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5' end and 96-97% complete coding sequence at the 3' end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease.

Mutation of melanosome protein RAB38 in chocolate mice.

Mutations of genes needed for melanocyte function can result in oculocutaneous albinism. Examination of similarities in human gene expression patterns by using microarray analysis reveals that RAB38, a small GTP binding protein, demonstrates a similar expression profile to melanocytic genes. Comparative genomic analysis localizes human RAB38 to the mouse chocolate (cht) locus. A G146T mutation occurs in the conserved GTP binding domain of RAB38 in cht mice. Rab38(cht)/Rab38(cht) mice exhibit a brown coat similar in color to mice with a mutation in tyrosinase-related protein 1 (Tyrp1), a mouse model for oculocutaneous albinism. The targeting of TYRP1 protein to the melanosome is impaired in Rab38(cht)/Rab38(cht) melanocytes. These observations, and the fact that green fluorescent protein-tagged RAB38 colocalizes with end-stage melanosomes in wild-type melanocytes, suggest that RAB38 plays a role in the sorting of TYRP1. This study demonstrates the utility of expression profile analysis to identify mammalian disease genes.