Distinct classes of motor neurons and ventral interneurons are generated by the graded signaling activity of Sonic hedgehog (Shh). Shh controls neuronal fate by establishing different progenitor cell populations in the ventral neural tube that are defined by the expression of Pax6 and Nkx2.2. Pax6 establishes distinct ventral progenitor cell populations and controls the identity of motor neurons and ventral interneurons, mediating graded Shh signaling in the ventral spinal cord and hindbrain.

Loss of Crumbs homologue 1 (CRB1) function causes either the eye disease Leber congenital amaurosis or progressive retinitis pigmentosa, depending on the amount of residual CRB1 activity and the genetic background. Crb1 localizes specifically to the sub-apical region adjacent to the adherens junction complex at the outer limiting membrane in the retina. We show that it is associated here with multiple PDZ protein 1 (Mupp1), protein associated with Lin-7 (Pals1 or Mpp5) and Mpp4. We have produced Crb1-/- mice completely lacking any functional Crb1. Although the retinas are initially normal, by 3-9 months the Crb1-/- retinas develop localized lesions where the integrity of the outer limiting membrane is lost and giant half rosettes are formed. After delamination of the photoreceptor layer, neuronal cell death occurs in the inner and outer nuclear layers of the retina. On moderate exposure to light for 3 days at 3 months of age, the number of severe focal retinal lesions significantly increases in the Crb1-/- retina. Crb2, Crb3 and Crb1 interacting proteins remain localized to the sub-apical region and therefore are not sufficient to maintain cell adhesion during light exposure in Crb1-/- retinas. Thus we propose that during light exposure Crb1 is essential to maintain, but not assemble, adherens junctions between photoreceptors and Müller glia cells and prevents retinal disorganization and dystrophy. Hence, light may be an influential factor in the development of the corresponding human diseases.

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.

Developmental genetics has shown that the Brachyury (T) gene has a key role in mesoderm formation during gastrulation in the mouse. Homozygous embryos have a defective allantois, degenerate or absent notochord and disrupted primitive streak and node. The neural tube is kinked and somite formation interrupted. The T gene has been cloned and is expressed during the early stages of gastrulation, being restricted to the primitive streak region, nascent mesoderm and notochord. Neither the sequence of the gene nor its expression pattern define its developmental function. To study the cell autonomy of the T mutation we have isolated and genetically characterized embryonic stem cell lines and studied their behaviour in chimaeras. T/+ embryonic stem cells form normal chimaeras, whereas T/T in equilibrium with +/+ chimaeras mimic the T/T mutant phenotype. The results indicate that the T gene acts cell autonomously in the primitive streak and notochord but may activate a signalling pathway involved in the specification of other mesodermal tissues.

In the proliferative zone of the developing cerebral cortex, multipotential progenitors predominate early in development and divide to increase the progenitor pool. As corticogenesis progresses, proportionately fewer progenitors are produced and, instead, cell divisions yield higher numbers of postmitotic neurones or glial cells. As the switch from the generation of progenitors to that of differentiated cells occurs, the orientation of cell division alters from predominantly symmetrical to predominantly asymmetrical. It has been hypothesised that symmetrical divisions expand the progenitor pool, whereas asymmetrical divisions generate postmitotic cells, although this remains to be proved. The molecular mechanisms regulating these processes are poorly understood. The transcription factor Pax6 is highly expressed in the cortical proliferative zone and there are morphological defects in the Pax6Sey/Sey (Pax6 null) cortex, but little is known about the principal cellular functions of Pax6 in this region. We have analysed the cell-cycle kinetics, the progenitor cleavage orientation and the onset of expression of differentiation markers in Pax6Sey/Sey cortical cells in vivo and in vitro. We showed that, early in corticogenesis at embryonic day (E) 12.5, the absence of Pax6 accelerated cortical development in vivo, shortening the cell cycle and the time taken for the onset of expression of neural-specific markers. This also occurred in dissociated culture of isolated cortical cells, indicating that the changes were intrinsic to the cortical cells. From E12.5 to E15.5, proportions of asymmetrical divisions increased more rapidly in mutant than in wild-type embryos. By E15.5, interkinetic nuclear migration during the cell cycle was disrupted and the length of the cell cycle was significantly longer than normal in the Pax6Sey/Sey cortex, with a lengthening of S phase. Together, these results show that Pax6 is required in developing cortical progenitors to control the cell-cycle duration, the rate of progression from symmetrical to asymmetrical division and the onset of expression of neural-specific markers.

The mouse nude mutation inactivates the gene encoding the Foxn1 transcription factor, causing defective hair morphogenesis. Here, we show for the first time that Foxn1 is required for proper assembly of the hair medulla, and we identify Foxn1-regulated genes by transcript profiling. One such gene encodes the desmosomal cadherin, Dsc2. Significantly, Foxn1-dependent Dsc2 expression is restricted to the hair medulla, and within these cells, Dsc2 protein is predominantly localized to specialized adhesion junctions between the cortex and the medulla. Our results reveal Foxn1 as an essential regulator of tissue assembly in the growing hair shaft and implicate Dsc2 as a downstream effector of this activity. Developmental

Pax6 is a member of an evolutionarily conserved family of transcription factors. It is developmentally regulated and is required for the normal embryonic development of the central nervous system, eye and pancreas. Pax6 mutations in the mouse result in the Small eye (Sey) phenotype. Heterozygous mice have eye defects and homozygotes die immediately after birth lacking eyes, nasal cavities and with severe brain abnormalities, including a malformed cerebral cortex. Recent work has established that there are changes in expression of cell adhesion molecules and these may underlie at least a part of the Pax6(Sey/Sey) phenotype. Here we used cell transplants and explant cultures to investigate the role of Pax6 in cell adhesion. Pax6(Sey/Sey) embryonic cortical cells transplanted into wild-type embryonic cortex were observed to segregate from wild-type cells and form dense clusters. Cells migrating from explants of Pax6(Sey/Sey) embryonic cortex clustered to a greater extent than cells migrating from wild-type controls. These new data support the hypothesis that Pax6 exerts a cell-autonomous effect on the adhesiveness of cortical cells.