Geneviève Aubin-Houzelstein

Melanoblasts' proper location and timed differentiation depend on Notch/RBP-J signaling in postnatal hair follicles.

The Notch/RBP-J pathway is involved in a variety of developmental processes and in tissue homeostasis. In the melanocyte lineage, it has been shown that Notch signaling acts through Hes1 to maintain the melanocyte stem cell population in the hair follicle. This study was designed to determine whether Notch signaling is implicated in other steps of melanocyte-lineage postnatal development. For this purpose, we developed mice in which the RBP-J gene was conditionally ablated in the melanocyte lineage and used the Dct-lacZ reporter transgene to track melanocytes and their precursors in individual hair follicles. We determine that Notch/RBP-J-deficient melanoblasts are in reduced number within the hair follicle and gather within its lower permanent part. Moreover, our results show that Notch signaling is necessary to prevent differentiation of melanocyte stem cells and of melanoblasts before they reach the hair bulb. Finally, our data show that Notch signaling is involved in proper location of melanoblasts in the outer root sheath and of melanocytes in the hair matrix. These findings reveal previously unrecognized roles for Notch signaling in the melanocyte lineage.

Notch Signaling and the Developing Hair Follicle

Notch function in the hair follicle has been mainly studied by use of transgenic mice carrying either loss or gain of function mutations in various members of the pathway. These studies revealed that whereas embryonic development of the hair follicle can be achieved without Notch, its postnatal development requires an intact Notch signaling in the hair bulb and the outer root sheath. Among the many roles played by Notch in the hair follicle, two can be highlighted: in the bulge, Notch controls a cell fate switch in hair follicle stem cells or their progenitors, preventing them from adopting an epidermal fate. In the hair bulb, Notch controls cell differentiation, ensuring the proper development of every layer of the hair shaft and inner root sheath. Notch function in the hair follicle is both cell autonomous and cell non autonomous and involves intercellular communication between adjacent layers.

Centronuclear Myopathy in Labrador Retrievers: A Recent Founder Mutation in the PTPLA Gene Has Rapidly Disseminated Worldwide

Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ∼70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLAcnm mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLAcnm carriers in 13 countries. Haplotype analysis demonstrated that the PTPLAcnm allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM.

RACK1, a clue to the diagnosis of cutaneous melanomas in horses

BackgroundMelanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward. To improve diagnosis and prognosis, markers of malignancy are needed. Receptor for activated C kinase 1 (RACK1) protein may be such a marker. RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients. Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses.ResultsUsing immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples. Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes. Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas. Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features.ConclusionsThis study confirms that horses provide an interesting model for melanoma genesis studies. It establishes MITF and PAX3 as markers of horse melanocytic cells. RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.

Pax3 GFP , a new reporter for the melanocyte lineage, highlights novel aspects of PAX3 expression in the skin

The paired box gene 3 (Pax3) is expressed during pigment cell development. We tested whether the targeted allele Pax3GFP can be used as a reporter gene for pigment cells in the mouse. We found that enhanced green fluorescent protein (GFP) can be seen readily in every melanoblast and melanocyte in the epidermis and hair follicles of Pax3GFP/+ heterozygotes. The GFP was detected at all differentiation stages, including melanocyte stem cells. In the dermis, Schwann cells and nestin‐positive cells of the piloneural collars resembling the nestin‐positive hair follicle multipotent stem cells exhibited a weaker GFP signal. Pigment cells could be purified by fluorescent activated cell sorting and grown in vitro without feeder cells, giving pure cultures of melanocytes. The Schwann cells and nestin‐positive cells of the piloneural collars were FACS‐isolated based on their weak expression of GFP. Thus Pax3GFP can discriminate distinct populations of cells in the skin.

Canine Melanoma Diagnosis RACK1 as a Potential Biological Marker

Melanoma diagnosis in dogs can be challenging due to the variety of histological appearances of canine melanocytic neoplasms. Markers of malignancy are needed. Receptor for activated C-kinase 1 (RACK1) was found to characterize melanomas in other mammals. We investigated the value of RACK1 detection in the classification of 19 cutaneous and 5 mucosal melanocytic neoplasms in dogs. These tumors were categorized as melanocytomas or benign and melanomas or malignant after evaluation of their morphology, mitotic index, and Ki-67 growth fraction. Using immunofluorescence, we confirmed microphthalmia-associated transcription factor (MITF) as a marker of normal and transformed melanocytic cells in dog tissues. All control (n = 10) and tumoral (n = 24) samples stained positively for MITF (34/34, 100%). Whereas RACK1 was not detected in healthy skin melanocytes, melanocytic lesions were all positive for RACK1 signal (24/24, 100%). RACK1 cytoplasmic staining appeared with 2 distinct distribution patterns: strong, diffuse, and homogeneous or granular and heterogeneous. All melanoma samples (13/13, 100%) stained homogeneously for RACK1. All melanocytomas (11/11, 100%) stained heterogeneously for RACK1. Immunohistochemistry was less consistent than immunofluorescence for all labelings in melanocytic lesions, which were often very pigmented. Thus, the fluorescent RACK1-MITF labeling pattern helped to distinguish melanomas from melanocytomas. Furthermore, RACK1 labeling correlated with 2 of 11 morphological features linked to malignancy: cell and nuclear size. These results suggest that RACK1 may be used as a marker in dog melanomas.

Histopathological atlas and proposed classification for melanocytic lesions in Tyr::NRas(Q61K) ; Cdkn2a(-/-) transgenic mice

Reference EPFL-ARTICLE-188431doi:10.1111/pcmr.12115View record in Web of Science Record created on 2013-09-09, modified on 2017-05-12

Haplosufficiency of PAX3 for melanoma development in Tyr: NRASQ61K; Cdkn2a-/- mice allows identification and sorting of melanoma cells using a Pax3GFP reporter allele

The role of the Pax3 gene in embryonic development of pigment cells is well characterized. By contrast, the function of Pax3 in melanoma development is controversial. Indeed, data obtained from cultured cells suggest that PAX3 may contribute to melanomagenesis. PAX3 is found to be overexpressed in melanomas and also in nevi compared with normal skin samples. Pax3 homozygous loss of function is embryonic lethal. To assess the role of Pax3 in melanoma development in vivo, we analyzed Pax3 haploinsufficiency in a mouse model of melanoma predisposition. The Pax3GFP/+ knock-in reporter system was combined with the Tyr::NRASQ61K; Cdkn2a−/− mouse melanoma model. Melanoma development was followed over 18 months. Histopathological, immunohistochemical, and molecular analyses of lesions at different stages of melanoma progression were carried out. Fluorescence-activated cell sorting on GFP of cells from primary or metastatic melanoma was followed by ex-vivo transformation tests and in-vivo passaging. We report here that Tyr::NRASQ61K; Cdkn2a−/−; Pax3GFP/+ mice developed metastasizing melanoma as their Tyr::NRASQ61K; Cdkn2a−/− littermates. Histopathology showed no differences between the two genotypes, although Pax3 mRNA and PAX3 protein levels in Pax3GFP/+ lesions were reduced by half. The Pax3GFP allele proved to be a convenient marker to identify and directly sort heterogeneous populations of melanoma cells within the tumor bulk at each stage of melanoma progression. This new mouse model represents an accurate and reproducible means for identifying melanoma cells in vivo to study the mechanisms of melanoma development.

Les cellules souches des mélanocytes de l'adulte

Melanocyte stem cells have been recently localized in mice, in the outer root sheath of the lower permanent portion of the hair follicle. Specific depletion of melanocyte stem cell population is responsible for natural hair greying in aging mice and humans. Melanocyte stem cells also seem to drive the growth of malignant melanomas. A few mutations, either spontaneous or genetically engineered, accelerate the natural process of hair greying with age. These mutations allowed the identification of genes and signalling pathways controlling emergence, maintenance and/or differentiation of melanocyte stem cells. This review summarizes recent studies on the melanocyte stem cells and defines a few major unanswered questions in the field.