Multipotent cells from mammalian iris pigment epithelium

The regeneration of lens tissue from the iris of newts has become a classical model of developmental plasticity, although little is known about the corresponding plasticity of the mammalian iris. We here demonstrate and characterize multipotent cells within the iris pigment epithelium (IPE) of postnatal and adult rodents. Acutely-isolated IPE cells were morphologically homogeneous and highly pigmented, but some produced neurospheres which expressed markers characteristic of neural stem/progenitor cells. Stem/progenitor cell markers were also expressed in the IPE in vivo both neonatally and into adulthood. Inner and outer IPE layers differentially expressed Nestin (Nes) in a manner suggesting that they respectively shared origins with neural retina (NR) and pigmented epithelial (RPE) layers. Transgenic marking enabled the enrichment of Nes-expressing IPE cells ex vivo, revealing a pronounced capacity to form neurospheres and differentiate into photoreceptor cells. IPE cells that did not express Nes were less able to form neurospheres, but a subset initiated the expression of pan-neural markers in primary adherent culture. These data collectively suggest that discrete populations of highly-pigmented cells with heterogeneous developmental potencies exist postnatally within the IPE, and that some of them are able to differentiate into multiple neuronal cell types.     

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.     

c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

Cooperation between the PDGF receptors in cardiac neural crest cell migration

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) alpha in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRbeta, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRalpha and PDGFRbeta in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRalpha plays a predominant role in NCC development, the PDGFRbeta is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.     

Insulin is imprinted in the placenta of the marsupial, Macropus eugenii

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.     

Smad signaling in the neural crest regulates cardiac outflow tract remodeling through cell autonomous and non-cell autonomous effects

Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.