Stabilization of ß-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

The canonical Wnt/ß-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, ß-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of ß-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of ß-catenin in cells expressing tyrosinase. Constitutive activation of ß-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating ß-catenin at various stages of development (E8.5-E11.5), we showed that the activation of ß-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/ß-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, ß-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

Biological and mathematical modeling of melanocyte development.

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function ß-catenin mutants in the melanocyte lineage. We found that any alteration of ß-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through ß-catenin.

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4.

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.

Effects of n-3 polyunsaturated fatty acid supplementation on cognitive functions, electrocortical activity and neurogenesis in a non-human primate, the grey mouse lemur (Microcebus murinus).

Among environmental factors that may affect on brain function, some nutrients and particularly n-3 polyunsaturated fatty acids (n-3 PUFA) are required for optimal brain development. Their effects on cognitive functions, however, are still unclear, and studies in humans and rodents have yielded contradictory results. We used a non-human primate model, the grey mouse lemur, phylogenetically close to human. The aim of this study was to demonstrate the impact of n-3 PUFA supplementation on cognitive functions, neuronal activity and neurogenesis. Two groups of animals whose diet was supplemented with either fish oil (rich in n-3 PUFA) or olive oil as a control. These two groups were subjected to a visual discrimination task and to a test of anxiety in the open-field. In parallel, cortical activity was measured with telemetric ECoG recordings. Finally, adult neurogenesis was investigated ex vivo by means of immunohistochemistry. Animals supplemented with fish oil exhibited better visual discrimination performance and tended to have lower anxiety levels. Furthermore, supplementation increased the power of alpha, beta and gamma frequency bands in the EEG, which are related to various aspects of memory and decision-making. This study also provides the first evidence of the existence of adult neurogenesis process in a prosimian primate. Notably, lemurs supplemented with n-3 PUFAs for 21 months exhibited a higher number of newly born neurons in brain areas related to memory and emotions, compared to control animals. Altogether, these results point to long-term positive effects of dietary n-3 PUFAs on various functions of the primate brain. Further studies will be needed to determine a formal causal link between behavioral improvement and creation of new neurons.

Brn-2 expression controls melanoma proliferation and is directly regulated by beta-catenin.

Constitutive activation of the Wnt/beta-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/beta-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing beta-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.

Impaired fasting blood glucose is associated to cognitive impairment and cerebral atrophy in middle-aged non-human primates.

Age-associated cognitive impairment is a major health and social issue because of increasing aged population. Cognitive decline is not homogeneous in humans and the determinants leading to differences between subjects are not fully understood. In middle-aged healthy humans, fasting blood glucose levels in the upper normal range are associated with memory impairment and cerebral atrophy. Due to a close evolutional similarity to Man, non-human primates may be useful to investigate the relationships between glucose homeostasis, cognitive deficits and structural brain alterations. In the grey mouse lemur, Microcebus murinus, spatial memory deficits have been associated with age and cerebral atrophy but the origin of these alterations have not been clearly identified. Herein, we showed that, on 28 female grey mouse lemurs (age range 2.4-6.1 years-old), age correlated with impaired fasting blood glucose (rs=0.37) but not with impaired glucose tolerance or insulin resistance. In middle-aged animals (4.1-6.1 years-old), fasting blood glucose was inversely and closely linked with spatial memory performance (rs=0.56) and hippocampus (rs=-0.62) or septum (rs=-0.55) volumes. These findings corroborate observations in humans and further support the grey mouse lemur as a natural model to unravel mechanisms which link impaired glucose homeostasis, brain atrophy and cognitive processes.

Involvement of cadherins 7 and 20 in mouse embryogenesis and melanocyte transformation.

We have determined the expression profiles of cdh7, and the related cdh20 during development. Both transcripts are found in the adult brain, but only cadherin-20 mRNA was detected during embryogenesis. In mouse embryos, cadherin-20 is synthesized by the forebrain, anterior neural ridge, developing visual system, primitive external granular layer of the cerebellum and a subset of neural crest cells likely to develop into melanoblasts. We found that the other embryonic tissues in which cadherin-20 was synthesized depended on genetic background. Melanoma cell lines contained transcripts for cadherin-7 but not for cadherin-20. The majority of the malignant melanoma cell lines produced N-cadherin (N-Cad) and/or cadherin-7 whereas melanocyte cell lines did not. The converse was observed for E-cadherin (E-Cad). Our data suggest that during development cadherin-20 is a key player in compartmentalization of the neural tube and establishment of neural circuitry. Finally, during oncogenesis, cadherin-7, N-cad and E-cad could be used as an efficient marker set for melanoma.

A subpopulation of smooth muscle cells, derived from melanocyte-competent precursors, prevents patent ductus arteriosus.

BACKGROUND: Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/ß-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.

Transcriptomic analysis of mouse embryonic skin cells reveals previously unreported genes expressed in melanoblasts.

Studying the development of melanoblasts, precursors of melanocytes, is challenging owing to their scarcity and dispersion in the skin embryo. However, this is an important subject because diverse diseases are associated with defective melanoblast development. Consequently, characterizing patterns of expression in melanoblasts during normal development is an important issue. This requires isolating enough melanoblasts during embryonic development to obtain sufficient RNA to study their transcriptome. ZEG reporter mouse line crossed with Tyr::Cre mouse line was used to label melanoblasts by enhanced green fluorescent protein (EGFP) autofluorescence. We isolated melanoblasts by FACS from the skin of E14.5-E16.5 embryos, and obtained sufficient cells for large-scale transcriptomic analysis after RNA isolation and amplification. We confirmed our array-based data for various genes of interest by standard quantitative real-time RT-PCR. We demonstrated that phosphatase and tensin homolog was expressed in melanoblasts but BRN2 was not, although both are involved in melanomagenesis. We also revealed the potential contribution of genes not previously implicated in any function in melanocytes or even in neural crest derivatives. Finally, the Schwann cell markers, PLP1 and FABP7, were significantly expressed in melanoblasts, melanocytes, and melanoma. This study demonstrates the feasibility of the transcriptomic analysis of purified melanoblasts at different embryonic stages and reveals the involvement of previously unreported genes in melanoblast development.

Phosphorylation of BRN2 modulates its interaction with the Pax3 promoter to control melanocyte migration and proliferation.

MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues were replaced with alanines (BRN2TS→BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat color phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by nonphosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level.

Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction.

Intestinal ganglioneuromatosis is a benign proliferation of nerve ganglion cells, nerve fibers, and supporting cells of the enteric nervous system (ENS) that can result in abnormally large enteric neuronal cells (ENCs) in the myenteric plexus and chronic intestinal pseudoobstruction (CIPO). As phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a phosphatase that is critical for controlling cell growth, proliferation, and death, we investigated the role of PTEN in the ENS by generating mice with an embryonic, ENC-selective deletion within the Pten locus. Mutant mice died 2 to 3 weeks after birth, with clinical signs of CIPO and hyperplasia and hypertrophy of ENCs resulting from increased activity of the PI3K/PTEN-AKT-S6K signaling pathway. Further analysis revealed that PTEN was only expressed in developing mouse embryonic ENCs from E15.5 and that the rate of ENC proliferation decreased once PTEN was expressed. Specific deletion of the Pten gene in ENCs therefore induced hyperplasia and hypertrophy in the later stages of embryogenesis. This phenotype was reversed by administration of a pharmacological inhibitor of AKT. In some human ganglioneuromatosis forms of CIPO, PTEN expression was found to be abnormally low and S6 phosphorylation increased. Our study thus reveals that loss of PTEN disrupts development of the ENS and identifies the PI3K/PTEN-AKT-S6K signaling pathway as a potential therapeutic target for ganglioneuromatosis forms of CIPO.