Zic Proteins Research Articles

Zic3z defines the dorsal and vegetal neuroectoderm in the zebrafish embryonic development

The Zic family is a group of genes encoding zinc finger proteins that are highly expressed in the mammalian cerebellum. Zic genes are the vertebrate homologue of Drosophila pair‐rule gene, odd‐paired (opa), which plays important roles in the parasegmental subdivision as well as in the visceral mesoderm development of Drosophila embryos. Recent studies on human, mouse, frog, fish and ascidian Zic homologues support that Zic genes are involved in a variety of developmental processes, including neurogenesis, myogenesis, skeletal patterning, and left‐right axis establishment. In an effort to explore possible functions of Zic proteins during vertebrate embryogenesis, we initially examined more detailed expression pattern of zebrafish homologue of zic3 (zic3z). zic3z transcripts are detected in the neuroectoderm, neural plate, dorsal neural tube, and brain regions including eye field during early embryonic development. Marker DNA studies found that zic3z transcription is modulated by BMP, Wnt, and Nodal signals particularly in the dorsal and vegetal neuroectoderm at gastrula. Interfering with zic3z translation with zic3z‐specific morpholino causes abnormal brain formation and expansion of the optic stalk cells. Retinal ganglion cells (RGCs) undergo abnormal neuronal differentiation. These findings suggest that zic3z defines the dorsal and vegetal neuroectoderm to specify brain formation and retinal neurogenesis during early embryonic development.

The Zic family member,odd-paired, regulates theDrosophilaBMP,decapentaplegic, during adult head development

The eye/antennal discs of Drosophila form most of the adult head capsule. We are analyzing the role of the BMP family member decapentaplegic (dpp) in the process of head formation, as we have identified a class of cis-regulatory dpp mutations (dpp(s-hc)) that specifically disrupts expression in the lateral peripodial epithelium of eye/antennal discs and is required for ventral head formation. Here we describe the recovery of mutations in odd-paired (opa), a zinc finger transcription factor related to the vertebrate Zic family, as dominant enhancers of this dpp head mutation. A single loss-of-function opa allele in combination with a single copy of a dpp(s-hc) produces defects in the ventral adult head. Furthermore, postembryonic loss of opa expression alone causes head defects identical to loss of dpp(s-hc)/dpp(s-hc), and dpp(hc)/+;opa/+ mutant combinations. opa is required for dpp expression in the lateral peripodial epithelium, but not other areas of the eye/antennal disc. Thus a pathway that includes opa and dpp expression in the peripodial epithelium is crucial to the formation of the ventral adult head. Zic proteins and members of the BMP pathway are crucial for vertebrate head development, as mutations in them are associated with midline defects of the head. The interaction of these genes in the morphogenesis of the fruitfly head suggests that the regulation of head formation may be conserved across metazoans.

3P036 C2H2型ジンクフィンガータンパク質、Zicタンパク質の構造とGli及びGlisタンパク質との系統樹的関係の解析(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

3P036 C2H2型ジンクフィンガータンパク質、Zicタンパク質の構造とGli及びGlisタンパク質との系統樹的関係の解析(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

3P035 Zicタンパク質の構造と物性(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

3P035 Zicタンパク質の構造と物性(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

A wide-range phylogenetic analysis of Zic proteins: Implications for correlations between protein structure conservation and body plan complexity

We compared Zic homologues from a wide range of animals. Striking conservation was found in the zinc finger domains, in which an exon–intron boundary has been kept in all bilateralians but not cnidarians, suggesting that all of the bilateralian Zic genes are derived from a single gene in a bilateralian ancestor. There were additional conserved amino acid sequences, ZOC and ZF-NC. Combined analysis of the zinc finger, ZOC, and ZF-NC revealed the presence of two classes of Zic, based on the degree of protein structure conservation. The “conserved” class includes Zic proteins from the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates and cephalochordates), whereas the “diverged” class contains those from the Platyhelminthes, Cnidaria, Nematoda, and Chordata (urochordates). The result indicates that the ancestral bilateralian Zic protein had already acquired an entire set of conserved domains, but that this was lost and diverged in the platyhelminthes, nematodes, and urochordates.

Myogenic repressor I-mfa interferes with the function of Zic family proteins

Zinc finger proteins belonging to the Zic family control several developmental processes such as patterning of the axial skeleton. Here we mapped the transcriptional regulatory domains in Zic2 protein and identified a protein which specifically binds to one of them. In the mapping experiments, an amino-terminal region was identified as transcriptional regulatory domains. A search for proteins binding to the amino terminal domain of Zic2 revealed that inhibitor of MyoD family (I-mfa) protein, which has been identified as a repressor of myogenic helix–loop–helix class transcription factors, can physically interact with the amino terminal domain. When Zic1-3 and I-mfa proteins were co-expressed in cultured cells, nuclear import of the Zic proteins was inhibited. Consequently, I-mfa inhibited transcriptional activation by the Zic proteins in cultured cells. These results suggest that the physical and functional interaction between Zic and I-mfa proteins can play a role in the vertebrate development.

Zic1 is a Transcriptional Repressor Through the Lamin A/C Promoter and has an Intrinsic Repressive Domain

Zic family proteins are expressed in the cerebellum and play important roles in vertebrate development. However, very few observations have been made regarding the function of Zic proteins in transcription. We previously showed, using P19 cells as a model system, that the lamin A/C promoter has a retinoic acid responsive element (L-RARE), and that Sp1 and Sp3 bind the CACCC box of the L-RARE. Here, Zic1 is identified as a binding protein of the L-RARE by yeast one-hybrid screening. We show that Zic1 can bind to and repress transcription through the CACCC box of the L-RARE. Moreover, reporter assays using various fusion proteins of Zic1 with the GAL4 DNA binding domain reveal that Zic1 has an intrinsic transcriptional repressive and activation domain.

Physical and Functional Interactions between Zic and Gli Proteins

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.

Molecular Properties of Zic Proteins as Transcriptional Regulators and Their Relationship to GLI Proteins

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 cultured cells, Zic proteins enhanced or suppressed sequence-dependent, 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.

Transcription Factors Zic1 and Zic2 Bind and Transactivate the Apolipoprotein E Gene Promoter

We have used the yeast one-hybrid system to identify transcription factors that bind to specific sequences in proximal regions of the apolipoprotein E gene promoter. The sequence between -163 and -124, that has been previously defined as a functional promoter element, was used as a bait to screen a human brain cDNA library. Ten cDNA clones that encoded portions of the human Zic1 (five clones) and Zic2 (five clones) transcription factors were isolated. Electrophoretic mobility shift assays confirmed the presence of a binding site for Zic1 and Zic2 in the -136/-125 region. Displacement of binding with oligonucleotides derived from adjacent sequences within the APOE promoter revealed the existence of two additional Zic-binding sequences in this promoter. These sequences were identified by electrophoretic mobility shift assays and mutational analysis in regions -65/-54 and -185/-174. Cotransfection of Zic1 and Zic2 expression vector and different APOE promoter-luciferase reporter constructs in U87 glioblastoma cell line showed that the three binding sites partially contributed to the trans-stimulation of the luciferase reporter. Ectopic expression of Zic1 and Zic2 in U87 cells also trans-stimulated the expression of the endogenous gene, increasing the amount of apolipoprotein E produced by glial cells. These data indicate that Zic proteins might contribute to the transcriptional activity of the apolipoprotein E gene and suggest that apolipoprotein E could mediate some of the developmental processes in which Zic proteins are involved.

Genetic Aspects of Host-Parasite Relationships

The formation of the highly organized cortical structure depends on the production and correct placement of the appropriate number and types of neurons. The <i>Zic</i> family of zinc-finger transcription factors plays essential roles in regulating the proliferation and differentiation of neuronal progenitors in the medial forebrain and the cerebellum. Examination of the expression of <i>Zic</i> genes demonstrated that <i>Zic1</i>, <i>Zic2</i>, and <i>Zic3</i> were expressed by the progenitor cells in the septum and cortical hem, the sites of generation of the Cajal-Retzius (CR) cells. Immunohistochemical studies have revealed that Zic proteins were abundantly expressed in the meningeal cells and that the majority of the CR cells distributed in the medial and dorsal cortex also expressed Zic proteins in the mid-late embryonic and postnatal cortical marginal zones. During embryonic cortical development, <i>Zic1</i>/<i>Zic3</i> double-mutant and hypomorphic <i>Zic2</i> mutant mice showed a reduction in the number of CR cells in the rostral cortex, whereas the cell number remained unaffected in the caudal cortex. These mutants also showed mislocalization of the CR cells and cortical lamination defects, resembling the changes noted in type II (cobblestone) lissencephaly, throughout the brain. In the <i>Zic1/3</i> mutant, reduced proliferation of the meningeal cells was observed before the thinner and disrupted organization of the pial basement membrane (BM) with reduced expression of the BM components and the meningeal cell-derived secretory factor. These defects correlated with the changes in the end feet morphology of the radial glial cells. These findings indicate that the <i>Zic</i> genes play critical roles in cortical development through regulating the proliferation of meningeal cells and the pial BM assembly.