Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.

Ca 2؉ -dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca 2؉ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca 2؉ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca 2؉ -dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca 2؉ -induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca 2؉ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca 2؉ -induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.Ca 2ϩ -induced exocytosis is a widely conserved mechanism of importance for a broad range of secretory systems, such as the release of neurotransmitters and neuropeptides in the central and peripheral nervous system. To attain fusion competence, secretory vesicles must first dock at the target membrane and undergo a process called priming. The release of fusion-ready vesicles is coupled to the opening of Ca 2ϩ channels in the plasma membrane, producing a transient high Ca 2ϩ concentration (10 -100 M for ϳ1 ms) in close proximity to the channels. The inward Ca 2ϩ current then diffuses into the cytoplasm, resulting in a residual concentration in the range of 1 M during tens of seconds. It is widely accepted that this residual Ca 2ϩ concentration induces short term changes in exocytotic strength (e.g. see Refs. 1 and 2), although the mechanisms that contribute to this phenomenon remain largely unclear. As a result, the secretory strength of neuronal and endocrine release sites is modulated in an activity-dependent way. This phenomenon is important to avoid vesicle depletion during repetitive stimulation and is furthermore considered in neurons to contribute to memory and learning.Although Ca 2ϩ is the most important messenger after high frequency activation of the release site, exocytotic potentiation can also be induced by diacylglycerol (synthesized by members of the phospholipase family) or by its phorbolester analogues. Diacylglycerol has multiple intracellular targets (e.g. protein kinase C isoforms), but the potentiating effect in regulated exocytosis is mediated by the priming factor Munc13-1. Munc13-1-deficient mice die shortly after birth and show a defect in synapt...

The formation of a functionally integrated nervous system is dependent on a highly organized sequence of events that includes timely division and differentiation of progenitors. Several apical polarity proteins have been shown to play crucial roles during neurogenesis, however, the role of Crumbs 2 (CRB2) in cortical development has not previously been reported. Here, we show that conditional ablation of Crb2 in the murine dorsal telencephalon leads to defects in the maintenance of the apical complex. Furthermore, within the mutant dorsal telencephalon there is premature expression of differentiation proteins. We examined the physiological function of Crb2 on wild type genetic background as well as on background lacking Crb1. Telencephalon lacking CRB2 resulted in reduced levels of PALS1 and CRB3 from the apical complex, an increased number of mitotic cells and expanded neuronal domain. These defects are transient and therefore only result in rather mild cortical abnormalities. We show that CRB2 is required for maintenance of the apical polarity complex during development of the cortex and regulation of cell division, and that loss of CRB2 results in cortical abnormalities.

The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. The majority of mutant mice died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival.