Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.
Xenopus Zic3 is a Xenopus homologue of mouse Zic and Drosophila pair-rule gene, odd-paired. We show here that Zic3 has significant roles both in neural and neural crest development in Xenopus embryo. Expression of Zic3 is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed chordin expression. The expression was induced by blockade of BMP4 signal. Overexpression of Zic3 resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induced expression of all the proneural genes and neural crest marker genes. These findings suggest that Zic3 can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.
Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, we investigated the functional properties of Zic proteins and their relationship to the GLI proteins. We first established an optimal binding sequence for Zic1, Zic2, and Zic3 proteins by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence was almost identical to the GLI binding sequence. However, the binding affinity was lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins was less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activated a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins were cotransfected into culturedcells,Zicproteinsenhancedorsuppressedsequencedependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors.
Zic and Gli family proteins are transcription factors that share similar zinc finger domains. Recent studies indicate that Zic and Gli collaborate in neural and skeletal development. We provide evidence that the Zic and Gli proteins physically and functionally interact through their zinc finger domains. Moreover, Gli proteins were translocated to cell nuclei by coexpressed Zic proteins, and both proteins regulated each other's transcriptional activity. Our result suggests that the physical interaction between Zic and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway.Zic and Gli transcription factors share a highly conserved zinc finger domain and have critical roles in multiple developmental processes. In human, mutations in ZIC2, ZIC3, and GLI3 genes result in various developmental abnormalities. ZIC2 results in malformation of the forebrain (holoprosencephaly), ZIC3 in a disturbance of the left to right body axis (heterotaxy), and GLI3 in complex anomalies of the brain and digits (cephalopolysyndactyly syndrome) (1-3). Studies in other vertebrates indicated that Zic1, Zic2, Zic3, Gli1, Gli2, and Gli3 are involved in multiple aspects of the neural and skeletal development (4 -14). Zic and Gli families are also critical in invertebrate development as shown by the studies on their Drosophila homologues, Odd-paired (15) and Cubitus interruptus (Ci) (16).Although a number of studies suggest the importance of the two zinc finger protein families, the relationship between them has not been fully understood. However, recent studies have shown significant Zic-Gli genetic interaction in neural and skeletal patterning. Xenopus Zic2 and Gli2 are counter-active in the patterning of neural tube along the dorsoventral axis (13). On the other hand, the double mutation of Zic1 and Gli3 showed a synergistic disturbance in the segmentation of the vertebral lamina (17).Gli proteins bind a consensus nonamer target DNA sequence (GLI-BS) (18) to which Zic proteins can also bind (19). However, we recently found that the DNA-binding affinity of the Zic proteins was lower than that of Gli (20) and that Zic proteins significantly enhance gene expression but less efficiently in the absence of GLI-BS. When Zic and Gli are expressed together in cultured cells, they synergistically enhance, or mutually suppress, GLI-BS-mediated transcription depending on the cell type (20). Here we show that Zic and Gli proteins physically interact through their zinc finger domains and regulate each other's subcellular localization and transcriptional activity. EXPERIMENTAL PROCEDURESPlasmids-To express Flag-tagged and hemagglutinin (HA) 1 -tagged proteins, the relevant sequence was amplified by PCR, verified by DNA sequencing, and subcloned into pCMVtag2 (Stratagene) and pcDNA3HA (a gift from Dr. T. Nakajima). Sequences cloned included the full-length mouse Zic1-(1-447) and mouse Zic2 and Zic3 (19,21). The deletion seri...
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