During development, cells undergo dynamic morphological changes by rearrangements of the cytoskeleton including microtubules. However, molecular mechanisms underlying the microtubule remodeling between orientated and disoriented formations are almost unknown. Here we found that novel subtypes of collapsin response mediator proteins (CRMP-As) and the originals (CRMP-Bs), which occur from the alternative usage of different first coding exons, are involved in this conversion of microtubule patterns. Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization.

In the developing retina, a retinoic acid (RA) gradient along the dorso-ventral axis is believed to be a prerequisite for the establishment of dorso-ventral asymmetry. This RA gradient is thought to result from the asymmetrical distribution of RA-generating aldehyde dehydrogenases along the dorso-ventral axis. Here, we identified a novel aldehyde dehydrogenase specifically expressed in the chick ventral retina, using restriction landmark cDNA scanning (RLCS). Since this molecule showed enzymatic activity to produce RA from retinaldehyde, we designated it retinaldehyde dehydrogenase 3 (RALDH-3). Structural similarity suggested that RALDH-3 is the orthologue of human aldehyde dehydrogenase 6. We also isolated RALDH-1 which is expressed in the chick dorsal retina and implicated in RA formation. Raldh-3 was preferentially expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region and then in the ventral retina, earlier than Raldh-1 in chick and mouse embryos. High level expression of Raldh-3 was also observed in the nasal region. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. These results suggested that Raldh-3 is the key enzyme in the formation of an RA gradient along the dorso-ventral axis during the early eye development, and also in the development of the olfactory system.

Delta is a major transmembrane ligand for Notch receptor that mediates numerous cell fate decisions. The Notch signaling pathway has long been thought to be mono-directional, because ligands for Notch were generally believed to be unable to transmit signals into the cells expressing them. However, we showed here that Notch also supplies signals to neighboring mouse neural stem cells (NSCs). To investigate the Notch–Delta signaling pathway in a bi-directional manner, we analyzed functional roles of the intracellular domain of mouse Delta like protein 1 (Dll1IC). In developing mouse NSCs, Dll1IC, which is released from cell membrane by proteolysis, is present in the nucleus. Furthermore, we screened for transcription factors that bind to Dll1IC and demonstrated that Dll1IC binds specifically to transcription factors involved in TGF-β/Activin signaling—Smad2, Smad3 and Smad4—and enhances Smad-dependent transcription. In addition, the results of the present study indicated that over-expression of Dll1IC in embryonic carcinoma P19 cells induced neurons, and this induction was blocked by SB431542, which is a specific inhibitor of TGF-β/Activin signaling. These observations strongly suggested that Dll1IC mediates TGF-β/Activin signaling through binding to Smads and plays an important role for bi-directional Notch–Delta signaling pathway.

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In the canonical Notch signaling pathway, intramembrane cleavage by gamma-secretase serves to release an intracellular domain of Notch that has activity in the nucleus through binding to transcription factors. In addition, we showed that Notch also supplies signals to Delta, a major Notch ligand, to release the intracellular domain of Delta by gamma-secretase from the cell membrane, which then translocates to the nucleus, where it mediates the transcription of specific genes. Therefore, the Notch-Delta signaling pathway is bi-directional and similar mechanisms regulated by gamma-secretase are involved in both directions. Recently, it was demonstrated that many type 1 transmembrane proteins including Notch, Delta and amyloid precursor protein (APP) are substrates for gamma-secretase and release intracellular domains of these proteins from cell membranes. These observations that the common enzyme, gamma-secretase, modulates proteolysis and the turnover of possible signaling molecules have led to the attractive hypothesis that mechanisms similar to the Notch-Delta signaling pathway may widely contribute to gamma-secretase-regulated signaling pathways, including APP signaling which leads to Alzheimer's disease. Here, we review the molecular mechanisms of the Notch-Delta signaling pathway in a bi-directional manner, and discuss the recent progress in understanding the biology of gamma-secretase-regulated signaling with respect to neurodegeneration.

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Although -secretase was first identified as a protease that cleaves amyloid precursor protein (APP) within the transmembrane domain and produces A peptides, which are thought to be pathogenic in Alzheimer's disease (AD), the physiological functions of -secretase have not been fully elucidated. In the canonical Notch signaling pathway, intramembrane cleavage by -secretase serves to release an intracellular domain of Notch that has activity in the nucleus through binding to transcription factors.Recently, it was demonstrated that many type 1 transmembrane proteins, including Notch, Delta, and APP, are substrates for -secretase, and the intracellular domains of these substrates are released from the cell membrane by -secretase. The common enzyme -secretase modulates proteolysis and the turnover of possible signaling molecules has led to the attractive hypothesis that mechanisms similar to Notch signaling contribute widely to proteolysis-regulated signaling pathways. It is likely that APP also has a signaling mechanism, although the physiological functions of APP have not been elucidated.Indeed, we have shown that the intracellular domain of APP (AICD) alters gene expression and induces neuron-specific apoptosis. These results suggest that APP signaling responds to the onset of AD. Here, we review the possibility of -secretase-regulated signaling, including APP signaling, which leads to AD.