(Received for publication, July 25, 1995; and in revised form, October 31, 1995)
From the
The mouse Zic gene, which encodes a zinc finger protein, is expressed in the developing or matured central nervous system in a highly restricted manner. We identified two novel Zic-related genes (Zic2, Zic3) through genomic and cDNA cloning. Both genes are highly similar to Zic(1), especially in their zinc finger motif. A comparison of genomic organization among the three Zic genes showed that they share common exon-intron boundaries and belong to the same gene family. Zic1, Zic2, and Zic3 were determined to mouse chromosome 9, 14, and X using an interspecific backcross panel. Northern blotting and ribonuclease protection showed that Zic2 and Zic3 are expressed in a restricted manner in the cerebellum at the adult stage. However, the temporal profile of the mRNA expression in the developing cerebella differ in the three Zic genes. Furthermore, we found that the Drosophila pair-rule gene, odd-paired is highly homologous to the Zic gene family. The similarity was not only the zinc finger motif, but also the exon-intron boundary was the same as those of mouse Zic gene family. These findings suggest that the Zic gene family and Drosophila odd-paired are derived from a common ancestral gene.
A molecular analysis of the early development of Drosophila elucidated the basic molecular mechanisms that establish the
regional specification of the developing organism (Ingham, 1988). A
parallel attempt has been made to understand developmental mechanisms
of the mammalian central nervous system (CNS). ()The Drosophila genes involved in the generation of
anterior-posterior differences have vertebrate homologues that appear
to have developmental roles in the CNS. Several gene families that
control the regionalization of the mammalian CNS have been isolated
(Rubenstein et al., 1994). Often, structurally related genes
that belong to the same gene family control the coordinated process of
CNS development.
The Zic protein (Aruga et al., 1994) is a zinc finger protein with a molecular mass of 48 kilodaltons. The expression of Zic is restricted to the dorsal region of the neural tube at a specific embryonic stage, whereas in the adult, it is restricted to the cerebellar granule cells and neurons of a few other nuclei (Zic, zinc finger protein of the cerebellum). We speculate that the Zic gene is involved in regional specification and cell lineage determination based upon its expression profile.
In this study, we searched structurally Zic-related genes and identified novel genes (Zic2 and Zic3) that were highly homologous to the Zic gene. Their predicted amino acid sequences and genomic organization suggest that they belong to the same class of gene family. The chromosomal locations of Zic1, Zic2, and Zic3 were determined by means of an interspecific backcross panel. The expression was largely restricted to the cerebellum in adults.
Furthermore, we found homology between the Zic gene family and odd-paired, a Drosophila pair-rule segmentation gene (Benedyk et al., 1994). odd-paired is a gene required for the timely activation of engrailed and wingless in Drosophila embryos.
The cDNA clones were isolated from cDNA libraries generated from
mice cerebella (Furuichi et al., 1989) or mice embryos (12
days postcoitum). ()Screening and sequencing analyses
proceeded as described (Aruga et al., 1994). The nucleotide
sequence data reported in this paper appears in the DDBJ, EMBL, and
GenBank nucleotide sequence data bases with the following accession
numbers, D70848 and D70849.
Figure 1: A, schematic drawing of the genomic organization of mouse Zic1, Zic2, and Zic3. The cross-hatched boxes indicate the exons, and the closed bar indicates the flanking and intron regions, respectively. The crossed region in the exons shows the location of the zinc finger domain. Arrows indicate the positions of the primers of the polymerase chain reaction for the backcross panel analysis. B, BamHI; E, EcoRI. B and C, the predicted amino acid sequence of the Zic2 (B) and Zic3 (C). In both panels, the underlines indicate the location of the zinc finger motif. In C, the first and second methionine are both candidates for translation initiation.
Zic2 and Zic3 proteins had a calculated molecular mass of 55.3 and 50.6 kilodaltons. A comparison of the predicted amino acid sequences of the Zic1, Zic2, and Zic3 ( Fig. 1and Fig. 2) revealed that there are several similar regions in the three genes. The conserved regions are located in a linear fashion. The most extensive homology was found in the zinc finger domain (97% between Zic1 and Zic2, 91% between Zic1 and Zic3 and 91% Zic2 and Zic3 over 158 amino acids). We found that Zic1 bound to DNA probes containing the 5`-GACAACAAAC-3` sequence recognized by GLI protein (Aruga et al., 1994). The high similarity in the zinc finger domain suggests that Zic2 and Zic3 bind similar sequences.
Figure 2: A and B, the amino acid sequence alignment of Zic1 (Aruga et al., 1994), Zic2, Zic3, and Opa (Benedyk et al., 1994) proteins in their zinc finger domain (A) and a conserved region other than the zinc finger domain (B). The bold letters indicate the conserved residues among all four proteins. Similar residues are also indicated by bold letters. The asterisks above the Zic1 line indicate the cysteine and histidine residues of the typical C2H2 motif. C, homology plot showing the similarity between the putative amino acid sequences of mouse Zic1 (vertical, Aruga et al.(1994)) and Opa (horizontal, Benedyk et al.(1994)). D, the exon-intron boundaries in the three Zic genes and opa. The location of first intron is based on Benedyk et al.(1994), whereas that of the second intron was determined in this study.
There was also similarity in the three regions including the amino and carboxyl termini ( Fig. 1and Fig. 2). The overall homologies between the proteins are 64% (between Zic1 and Zic2), 64% (between Zic1 and Zic3), and 59% (between Zic2 and Zic3). Except for the zinc finger domain, the amino acid compositions were similar to that of the Zic1 protein. There were some polyalanine and polyhistidine sequences. The carboxyl-terminal flanking the zinc finger motif is enriched with serine/threonine residues. Zic2 is relatively large because of the insertion of extra sequences in the serine-rich region (Aruga et al., 1994).
In addition to the Zic2 and Zic3, we isolated Zic1 genomic clones. All three genes were composed of three exons, containing the zinc finger domain. The third exon contains large 3`-untranslated regions. The nucleotide sequences of the two introns adjoining the splice junctions are consistent with the recognized consensus sequence GT/AG (Fig. 2D). The exon-intron boundaries are conserved in homologous sites in the zinc finger motif. Thus, it seems likely that these three genes are derived from a common ancestral gene.
To determine the chromosomal locations of the mouse Zic genes, we analyzed the segregation of the gene by means of a BSS-Ros interspecific backcross. Differences in the length of the PCR products or in single strand conformation polymorphism analysis were used to segregate the alleles from C57BL/6Ros or M. spretus. The segregation of these differences was determined in progeny of this cross (Fig. 3, A and B). A comparison of the segregation of Zic1, -2, and -3 with characterized loci (Hayashizaki et al., 1994) revealed a close linkage between Zic1 and D9Ncvs13 (3/56 recombinants), D9Ncvs15 (2/56 recombinants), Zic2 and D14Ncvs12, D14Ncvs59 (both 0/55 recombinants), Zic3 and DXNcvs1, Hprt (both 1/55 recombinants) (Fig. 3, A and B). The gene orders shown in Fig. 3C were indicated from the minimization of recombinations. Thus, the Zic1, -2, and -3 genes are located in mouse chromosomes 9, 14, and X, respectively.
Figure 3:
Interspecific backcross analyses of
assignment of the chromosomal locus of mouse Zic genes. A, DNA isolated from backcross progeny of (C57BL/6 M. spretus) female and M. spretus male (Hayashizaki et al., 1994) were analyzed by electrophoresis of the PCR
products on nondenaturing (Zic1) or denaturing gels (Zic2 and Zic3). While mapping the Zic3 gene, the
C57BL/6 hemizygous type (B/Y) appeared (data not shown). B,
distribution of the haplotypes in the backcross progeny. B represents heterozygous, S homozygous. The markers are
described by Hayashizaki et al. (1994). C, a
schematic drawing of the position of the Zic1, Zic2,
and Zic3 in each chromosome. The numbers in the left of the bars indicate the distance from the
centromere of each chromosome.
In the regions where the Zic1 and Zic3 genes were mapped, there are some mutations that result in abnormal behavior. ducky (Snell, 1955) which causes abnormal behavior and histologically severe dysgenesis of hindbrain and spinal cord, as well as myelin deficiency, is located near the position of Zic1 and trembly which causes tremors and seizures in hemizygous males (Taylor et al., 1978) is 1 centimorgan proximal to the Hprt gene, at the position of Zic3 gene. We do not, however, have any concrete data linking these genes.
We found by means of
Southern blotting that Zic1 has at least one homologous gene
in various species including fish, amphibians, reptiles, and avians
(Aruga et al., 1994). Correspondingly, we isolated Zic-related cDNAs from various species. ()It is
likely that the Zic-opa gene family is phylogenically
conserved.
We compared the genomic organization of the three Zic genes and that of opa. The positions of introns in the three Zic genes were conserved in the opa gene (Fig. 2D). The genomic organization of opa, which is composed of three exons with a relatively long first intron (Benedyk et al., 1994), was also similar to that of Zic gene family. These similarities in the genomic organization suggest that the mouse Zic genes and Drosophila opa are derived from a common ancestral gene.
The homology between the Zic-opa and GLI-ci gene family is significantly high in their zinc finger domain.
However, we consider that the Zic-opa gene family belongs to
another subclass of zinc finger proteins for the following reasons. The
exon-intron organization of the Zic-opa gene family is
different from that of GLI(-ci
-tra1) gene family
(Ruppert et al., 1988; 1990; Hui et al., 1994). The
similarity in the non-zinc finger region in GLI, GLI2, GLI3, and
Ci
is not conserved in Zic1, Zic2, Zic3, and Opa, and vice
versa.
Drosophila opa is a pair-rule gene, which is essential for the parasegmental subdivision of the Drosophila embryo; opa mRNA and protein are found throughout all segment primordia. It appeared that Opa does not act in a spatially restricted manner. However, the expression of Zic1 is a highly restricted in the adult mouse CNS, although the mRNA was found in a broad domain, namely, a large area of the alar plate of entire neural tube and the surrounding mesenchyme at specific embryonic stages (Aruga et al., 1994). There is apparent similarity in their expression during development.
Figure 4:
The spatiotemporal distribution of Zic2 and Zic3 mRNA. In each panel, Zic2 and Zic3 mRNA were analyzed by Northern blotting and by the
ribonuclease protection assay, respectively, except for B. A, tissue distribution in adult mice. B, Northern
blots of the three Zic genes. In each panel, 20 µg of the
total RNA from the cerebella was separated by electrophoresis. The
hybridized membranes were exposed to the films for 12 h (Zic1), 2 days (Zic2), and 14 days (Zic3)
with an intensifying screen. C, expression in the developing
brain. RNAs were extracted from the forebrain, midbrain, and hindbrain
of developing mice. The upper band in the Zic2 panel
seemed to be an artifact arising from the presence of the ribosomal RNA
because the band disappeared when the poly(A) RNA were
used (data not shown). D, temporal change in postnatal
developing cerebellum. E, densitometric scanning of D and Zic1 (Aruga et al., 1994) are summarized in
a graph. Closed circles, open circles, and squares represent the relative amounts of Zic1, Zic2, and Zic3 mRNA. The relative amounts were
calculated as percentages of the maximal value in each
mRNA.
We examined the mRNA levels in younger mice because Zic1 is expressed in the developing neural tissue at days postcoitus. The developmental profile in the whole brain (fore-, mid-, hindbrain) showed that throughout the embryonic days 12 to 18, the mRNA levels seemed to be relatively constant. The high level of mRNA in embryonic day 12 observed using the Zic1 probe (Aruga et al., 1994) was not found with Zic2 or Zic3. In the postnatal developing cerebella (Fig. 4D), the profile differed among the three genes. The Zic3 gene peaked soonest at 5 days after birth, whereas Zic2 peaked at 11 days. Zic1 mRNA peaked in 5 to 7 days. These periods correspond to the stage when the precursors of granule cells are differentiating into the mature neurons. However, whether or not Zic2 and Zic3 are expressed in the granule cells as seen in Zic1 must be determined before their role in cerebellar development can be investigated.
Our preliminary
data indicated that another gene (Zic4) is also expressed in
the cerebellum. Combining with the highly restricted
expression of the Zic1 gene in the cerebellar granule cells
(Aruga et al., 1994), the Zic gene family is unique
in that all of the members of the family are expressed in the adult
cerebellum in a highly restricted manner.