odd-paired
Cnidaria are the first class of organisms in animal evolution with a nervous system. The cnidarian Hydra has two types of neuronal cells: nerve cells and nematocytes. Both differentiate from the same pool of pluripotent stem cells. Yet the molecular regulation of neural differentiation in Hydra is largely unknown. Hyzic, a homolog of the Zn-finger transcription factor gene zic/odd-paired, is expressed in the early nematocyte differentiation pathway, starting at the level of interstitial stem cells. Expression of Hyzic is restricted to the proliferative stages of nematoblasts. Hyzic acts before and possibly directly upstream of Cnash, a homolog of the proneural bHLH transcription factor gene achaete-scute, and of Nowa, an early nematocyte differentiation marker gene. Hyzic may determine stem cells to differentiate into nematocytes. The data are consistent with a role of Hyzic in inhibiting nematocyte differentiation, by keeping committed nematoblast cells in the cell cycle. A similar role has been demonstrated for Zic genes in vertebrates. These results suggest, that genetic cascades of neural development may be conserved from Hydra to vertebrates, indicating that the molecular regulation of neural development evolved only once (Lindgens, 2004).
During larval development in C. elegans, some of the cells of the ventral epidermis, the Pn.p cells, fuse with the growing epidermal syncytium hyp7. The pattern of these cell fusions is regulated in a complex, sexually dimorphic manner. It is essential that some Pn.p cells remain unfused in order for some sex-specific mating structures to be generated. The pattern of Pn.p cell fusion is regulated combinatorially by two genes of the C. elegans Hox gene cluster: lin-39 and mab-5. Some of the complexity in the Pn.p cell fusion pattern arises because these two Hox proteins can regulate each otherís activities. A zinc-finger transcription factor, REF-2, is described that is required for the Pn.p cells to be generated and to remain unfused. ref-2 encodes a zinc-finger transcription factor of the odd-paired (opa)/Zic family. REF-2 functions with the Hox proteins to prevent Pn.p cell fusion. ref-2 may also be a transcriptional target of the Hox proteins (Alber, 2002).
Multiple functions of a Zic-like zinc finger transcription factor gene (Cs-ZicL) were identified in Ciona savignyi embryos. cDNA clones for Cs-ZicL, a ß-catenin downstream gene, were isolated: the gene is transiently expressed in the A-line notochord/nerve cord lineage and in B-line muscle lineage from the 32-cell stage and later in a-line CNS lineage from the 110-cell stage. Suppression of Cs-ZicL function with specific morpholino oligonucleotide indicates that Cs-ZicL is essential for the formation of A-line notochord cells but not of B-line notochord cells, essential for the CNS formation, and essential for the maintenance of muscle differentiation. The expression of Cs-ZicL in the A-line cells is downstream of ß-catenin and a ß-catenin-target gene, Cs-FoxD, which is expressed in the endoderm cells from the 16-cell stage and is essential for the differentiation of notochord. In spite of its pivotal role in muscle specification, the expression of Cs-ZicL in the muscle precursors is independent of Cs-macho1, which is another Zic-like gene encoding a Ciona maternal muscle determinant, suggesting another genetic cascade for muscle specification independent of Cs-macho1. Cs-ZicL may provide a future experimental system to explore how the gene expression in multiple embryonic regions is controlled and how the single gene can perform different functions in multiple types of embryonic cells (Imai, 2002).
In ascidian embryos, Brachyury is expressed exclusively in
blastomeres of the notochord lineage and plays an essential role in the
notochord cell differentiation. The genetic cascade leading to the
transcriptional activation of Brachyury in A-line notochord cells of
Ciona embryos begins with maternally provided ß-catenin, which
is essential for endodermal cell specification. ß-catenin directly
activates zygotic expression of a forkhead transcription factor gene,
FoxD, at the 16-cell stage, which in turn somehow activates a zinc
finger transcription factor gene, ZicL (Drosophila homolog: Odd-paired), at the 32-cell stage, and
then Brachyury at the 64-cell stage. One of the key questions to be
answered is whether ZicL functions as a direct activator of Brachyury transcription, and this was addressed in the present study. A fusion protein
was constructed in which a zinc finger domain of Ciona ZicL was
connected to the C-terminus of GST. Extensive series of PCR-assisted binding site selection assays and electrophoretic mobility shift assays demonstrated that the most plausible recognition sequence of Ciona ZicL was CCCGCTGTG. The elements CACAGCTGG
(complementary sequence: CCAGCTGTG) at -123 and
CCAGCTGTG at -168 bp upstream of the putative transcription start site of
Ci-Bra are found in a previously identified basal enhancer of this gene. In vitro binding assays indicate that the ZicL fusion protein binds to these elements efficiently. A fusion gene construct in which lacZ was fused with the upstream sequence of Ci-Bra showed the reporter gene expression exclusively in notochord cells when the construct was introduced into fertilized eggs. In contrast, fusion constructs with mutated ZicL-binding-elements failed to show the reporter expression. In addition,
suppression of Ci-ZicL abolished the reporter gene expression, while
ectopic and/or overexpression of Ci-ZicL resulted in ectopic reporter expression in non-notochord cells. These results provide evidence that ZicL directly activates Brachyury, leading to specification and subsequent differentiation of notochord cells (Yagi, 2004).
Two axial structures, a neural tube and a notochord, are key structures in
the chordate body plan, and a closer look at these structures furthers understanding of the origin of chordates.
The neural tube of ascidian larvae is composed of about 340 cells, and is divided into three regions along the
anteroposterior axis, which are, from anterior to posterior, the sensory vesicle, the visceral ganglion and the caudal
neural tube. The sensory vesicle is composed solely of the a-line (anterior-animal) cells. The
visceral ganglion present at the junction between the trunk and tail consists of the A-line (anterior-vegetal) cells. The caudal neural tube running along
the length of the tail consists of four (dorsal, ventral and two lateral) rows of ependymal cells: the lateral and ventral cells are of A-line origin and the
dorsal cells are of b-line (posterior-animal) origin. Beneath the neural tube, a stack of exactly 40 notochord cells runs along the tail. The anterior 32
cells (primary notochord) and the posterior 8 cells (secondary notochord) are derived from A-line and b-line cells, respectively. To expand
knowledge on mechanisms of development of the neural tube in lower
chordates, isolation and characterization of HrzicN, a new member of the Zic family gene of the ascidian, Halocynthia roretzi, was undertaken. HrzicN expression is detected by whole-mount in
situ hybridization in all neural tube precursors, all notochord precursors,
anterior mesenchyme precursors and a part of the primary muscle precursors.
Expression of HrzicN in a- and b-line neural tube precursors was
detected from early gastrula stage to the neural plate stage, while expression
in other lineages is observed between the 32-cell and the 110-cell stages.
HrzicN function was investigated by disturbing translation using a
morpholino antisense oligonucleotide. Embryos injected with HrzicN
morpholino ('HrzicN knockdown embryos') exhibit failure of
neurulation and tail elongation, and develop into larvae without a neural
tube and notochord. Analysis of neural marker gene expression in
HrzicN knockdown embryos has revealed that HrzicN plays critical
roles in distinct steps of neural tube formation in the a-line- and A-line-precursors. In particular HrzicN is required for early specification
of the neural tube fate in A-line precursors. Involvement of HrzicN
in the neural tube development was also suggested by an overexpression
experiment. However, analysis of mesodermal marker gene expression in
HrzicN knockdown embryos revealed unexpected roles for this gene in
the development of mesodermal tissues. HrzicN knockdown leads to loss
of HrBra (Halocynthia roretzi Brachyury) expression in all
of the notochord precursors: this may be the cause for notochord deficiency.
Hrsna (Halocynthia roretzi snail) expression is also lost
from all the notochord and anterior mesenchyme precursors. By contrast,
expression of Hrsna and the actin gene is unchanged in the primary
muscle precursors. These results suggest that HrzicN is responsible
for specification of the notochord and anterior mesenchyme. Finally,
regulation of HrzicN expression by FGF-like signaling was
investigated. This signaling has been shown to be involved in induction of the a- and b-line neural tube, the notochord and the mesenchyme cells in
Halocynthia embryos. Using an inhibitor of FGF-like signaling, it has been shown that HrzicN expression in the a- and b-line neural tube, but
not in the A-line lineage and mesodermal lineage, depends on FGF-like
signaling. Based on these data, roles of HrzicN as a key
gene in the development of the neural tube and the notochord are discussed (Wada, 2002).
Patterning of the embryonic ectoderm is dependent upon the action of negative (antineural) and positive (neurogenic) transcriptional regulators. Msx1 and Dlx3 are
two antineural genes for which the anterior epidermal-neural boundaries of expression differ, probably due to differential sensitivity to BMP signaling in the ectoderm.
In the extreme anterior neural plate, Dlx3 is strongly expressed while Msx1 is silent. While both of these factors prevent the activation of genes specific to the nascent
central nervous system, Msx1 inhibits anterior markers, including Otx2 and cement gland-specific genes. Dlx3 has little, if any, effect on these anterior neural plate
genes, instead providing a permissive environment for their expression while repressing more panneural markers, including prepattern genes belonging to the Zic
family and BF-1. Zic3 is activated by chordin and suppressed by BMP4; overexpression of this factor results in conversion of ectoderm to anterior neural tissue. The finding that Dlx3 is able to suppress the activation of Zic3 suggests that a Dlx3-mediated regulatory step might exist between the initial disruption of BMP signaling and activation of this gene. To test this hypothesis, truncated BMP-4 receptor, Dlx3 and Zic3 RNAs were injected in combinations, followed by animal cap excision, culture and RNA isolation for Northern blot analysis. Dlx3 blocks the activation of the panneural marker Nrp1 by truncated BMP-4 receptor. Addition of Zic3 RNA to the injection mixture restores Nrp1 expression to levels comparable to those of truncated BMP-4 induced caps. Based on these results, it is concluded that the inductive effects of Zic3 function downstream of the antineurogenic stem mediated by Dlx3. These properties define a molecular mechanism for translating the organizer-dependent morphogenic gradient of BMP activity into spatially restricted gene expression in the prospective anterior neural plate (Feledy, 1999).
Reported here is the expression of the zebrafish zic1 gene, also known as opl, a homolog to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To determine whether BMP2 signaling affects the extent of zic1 expression, swirl and chordino mutant embryos were examined. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, it was found that zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. However, the most rostral expression is not affected by Shh, suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme (Rohr, 1999).
Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric
nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo alters the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 is expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 is effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, both of which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3 (Kitaguchi, 2000).
The mouse Zic gene, which encodes a zinc finger protein, is expressed in a highly restricted manner in the
developing or matured central nervous system. Two novel Zic-related genes (Zic2, Zic3) are structurally very similar to Zic(1), the zinc finger motif in particular. A
comparison of genomic organization among the three Zic genes shows that they
share common exon-intron boundaries and belong to the same gene family . Zic1,
Zic2, and Zic3 are found, respectively, on mouse chromosomes 9, 14, and X. 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
cerebellum differs among in the three Zic genes. Drosophila
Odd-paired is highly homologous to the Zic gene family. Not only is the zinc finger motif similar in both genes, but the exon-intron boundary in Odd-paired is the same as
that found in the mouse Zic gene family. This suggests that the Zic gene family and
Drosophila odd-paired are derived from a common ancestral gene (Aruga, 1996a).
Zic4 is highly similar to Zic1, Zic2 and Zic3, especially in its Zf motif. An analysis of the genomic organization of Zic4 shows that the gene shares a common exon-intron boundary with Zic1, Zic2, Zic3 and opa. Zic4 is located on mouse chromosome 9, in the vicinity of Zic1. Zic4 is expressed only in the cerebellum during the adult stage, as are the other Zic genes. The temporal profile of mRNA expression in the developing cerebellum is similar to that of Zic3, which has
a peak on postnatal day 5. This suggests that Zic4 is a gene which works
cooperatively with other Zic genes during cerebellar development (Aruba, 1996b).
Zic1, Zic2 and Zic3 are vertebrate homologs of odd-paired, and are expressed in a restricted manner in the adult mouse cerebellum. The expression of the three Zic genes is first detected at gastrulation in a spatially restricted pattern. Zic2 is detected only in the presumptive headfold region, whereas Zic3 is expressed mainly in the primitive streak. The expression of both genes continues during neural tube formation. At the late primitive streak stage, Zic1 expression in the neuroectoderm is detected in the presumptive dorsal region. All three Zic genes are expressed only in dorsal axial structures. During organogenesis, the three genes are expressed in specific regions of several developing organs, including dorsal areas of the brain, spinal cord, paraxial mesenchyme, and epidermis, and the marginal zone of the neural retinal and distal regions of the developing limb. In all cases significant differences are observed in the spatial patterns of the three genes. Expression is not regulated by Pax3, Wnt-1 or Wnt-3a, but the pattern of expression is altered in Wnt-3a mutants and in open brain, a mutant with severe neural tube defects. The changed expression pattern in Wnt-3a mutants suggests that Zic genes in the neural tube are regulated by factors from notochord. While opa is involved in visceral mesoderm, Zic may be involved in dorsal mesoderm. Both insect and vertebrate genes are expressed in neural regions (Nagai, 1997).
Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, mice deficient in Zic1 were generated. Homozygous mice show remarkable ataxia during postnatal development. Nearly all of the mice die within 1 month. Their cerebella are hypoplastic and missing a lobule in the anterior lobe. There is a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL (Aruga, 1998).
Holoprosencephaly (HPE) is the most common structural anomaly of the human brain and is one of the anomalies seen in patients with deletions and duplications of chromosome 13. On the basis of molecular
analysis of a series of patients with hemizygous deletions of the long arm of chromosome 13, a discrete region in band 13q32 has been defined where deletion leads to major developmental anomalies (the
13q32 deletion syndrome). This approximately 1-Mb region lies between markers D135136 and D13S147. Patients in which this region is deleted usually have major congenital malformations, including brain anomalies such as HPE or exencephaly, and digital anomalies such as absent thumbs. Human ZIC2 maps to this critical deletion region and heterozygous mutations in ZIC2 are associated with HPE. Haploinsufficiency for ZIC2 is likely to cause the brain malformations seen in 13q deletion patients (Brown, 1998).
Skeletal abnormalities are described that appeared in Zic1-deficient mice. These mice show multiple abnormalities in the axial skeleton.
The deformities are severe in the dorsal parts of the vertebrae (the vertebral arches), but less so in the vertebral bodies (spina bifida occulta). The
proximal ribs are deformed by ectopic processes. The abnormalities found in the vertebral arches can be traced back to disturbed
segmental patterns of dorsal sclerotome. The Zic1/Gli3 double mutants show severe abnormalities of vertebral arches that are not found in single
mutants. The abnormalities in the vertebral arches are less severe in Zic1/Pax1 mutants (Pax1 is a homolog of Drosophila Pax-meso) than in Zic1/Gli3 mutants, but significantly more
pronounced than in Zic1 single mutants. The three genes may act synergistically in the development of the vertebral arches (Aruga, 1999).
The vertebral arch phenotypes in the Zic1/Gli3 and Zic1/Pax1 combined mutants indicate that these two sets of the
genes cooperate in the AP patterning of the dorsal sclerotome. Based on these results, the role of the three genes in the development and compartmentalization of the sclerotome can be considered. In terms of the expression in the axial structures, Zic1/Gli3 co-expression is observed in the dorsolateral sclerotome, the dorsal dermomyotome and
the dorsal neural tube whereas Zic1/Pax1 co-expression is detected in the intermediate part of the sclerotome. Zic1/Gli3 synergism may be related to the Zic1/Gli3 coexpression in the dorsolateral sclerotomes and/or in the dorsal spinal cord. Although it is not yet known whether the neural tube is involved in the segmental organization of the axial skeleton, it is possible that the loss of the most dorsal vertebral arches secondarily affects the segmental organization of the vertebral laminae. A detailed examination of the spinal cord of these mutants should clarify the roles of Zic and Gli genes in the interactions between spinal cord and sclerotome (Aruga, 1999).
From a molecular point of view, Zic1 and Gli3 proteins
might be functionally redundant during vertebral arch
development, since Gli3 expression is not affected in Zic1
mutants and Zic1 expression is not affected in Gli3 mutants. The two proteins may interact with the same target sequences in vivo and cooperate in the transcriptional regulation of the same target genes. It is also
possible that the same target molecules other than DNA
are recognized by the conserved zinc finger domain.
Although this study suggests synergism between the Zic
and the Gli proteins, Xenopus Zic2 and Gli genes counteract one another during neurogenesis. Different set of downstream genes or associating
factors for Zic and Gli proteins might function in neurogenesis
versus vertebral arch development (Aruga, 1999).
The sclerotome cells expressing both Zic1 and Pax1 may
contribute to the formation of vertebral arches. In support of
this possibility, the vertebral arches are known to be derived
in part from lateral sclerotome. Interestingly, the mitosis of sclerotome cells is most active in the
Zic1/Pax1 co-expressing region.
In the Pax1-/- single mutant, the impaired proliferation
in this sclerotome subregion leads to insufficient expansion
of sclerotome cells. Therefore, Zic1 protein could be
involved in the proliferation of the sclerotome cells in this
region in collaboration with Pax1 protein. As an alternative
explanation, Zic1 and Pax1 proteins might synergistically
affect the responsiveness of sclerotome cells to vertebral
arch-inductive signals, such as BMP4 and PDGF (Aruga, 1999).
Recent studies showed that Shh signal is essential for the
development of the vertebral column by establishing the dorsoventral axis of the somite and subsequently that of the sclerotome. Gli genes are considered to act as a downstream factor of the Shh-mediated signaling cascade. Although the role of Gli3 has not been clarified, involvement in the Shh-mediated signaling
cascade is possible, judging from the phenotypes of the
Gli2/Gli3 mutant. Pax1 is also a mediator
of Shh signals. Genetic interactions of
Zic1 mutation with Gli3 and Pax1 suggest the involvement
of Zic1 gene product in the Shh-mediated signaling cascade (Aruga, 1999).
Mutation in human ZIC2, a zinc finger protein homologous to Drosophila odd-paired, causes holoprosencephaly (HPE), which is a
common, severe malformation of the brain in humans. However, the pathogenesis is largely unknown. Reduced
expression (knockdown) of mouse Zic2 causes neurulation delay, resulting in HPE and spina bifida. Differentiation of the most dorsal
neural plate, which gives rise to both roof plate and neural crest cells, is also delayed as indicated by the expression lag of a roof plate
marker, Wnt3a. In addition the development of neural crest derivatives, such as dorsal root ganglion, were impaired. These results suggest
that the Zic2 expression level is crucial for the timing of neurulation. Because the Zic2 knockdown mouse is the first mutant with HPE and spina bifida to survive to
the perinatal period, the mouse will promote analyses of not only the neurulation but also the pathogenesis of human HPE (Nagai, 2000).
An extracellular signaling molecule acts on several types of cells, evoking characteristic and different responses depending on intrinsic factors in the signal-receiving cells. In ascidian embryos, notochord and mesenchyme are induced in the anterior and posterior margins, respectively, of the vegetal hemisphere by the same FGF signal emanating from endoderm precursors. The difference in the responsiveness depends on the inheritance of the posterior-vegetal egg cytoplasm. odd-paired related zinc finger transcription factor macho-1, first identified as a localized muscle determinant, is also required for mesenchyme induction, and it plays a role in making the cell response differ between notochord and mesenchyme induction. A zygotic event involving snail expression downstream of maternal macho-1 mediates the suppression of notochord induction in mesenchyme precursors (Kobayashi, 2003)
Xenopus Zic5, which belongs to a novel class of the Zic family, has been characterized. Zic5 is more specifically expressed in the prospective
neural crest than other Zic genes. At the late gastrula stage (stages 11.5-13), Zic5 expression is restricted to the prospective neural fold
region and anterior neural plate border region. Expression becomes enhanced in the prospective midbrain and hindbrain region at the late gastrula stage.
The expression in the neural plate border region continues in the neurula stage. However, anterior neural expression is more enhanced. Zic5-specific staining
is seen in the form of four longitudinal lines, which represent the neural plate edges and part of the neural fold, similar to the staining of Zic1, Zic2, Zic3. In the tailbud stages (stages 21-22) Zic5 is expressed in the eyes, dorsal brain, and the posterior part of the dorsal spinal cord in a very
restricted manner. In the brain, the expression was still
higher in the mesencephalon and rhombencephalon (stage 30). Overexpression of Zic5 in embryos leads to ectopic expression of the early neural crest markers, Xsna and
Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there is marked induction of neural crest
markers, without mesodermal and anterior neural markers. This is in contrast to other Xenopus Zic genes, which induce both anterior and
the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These
results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest
tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene (Nakata, 2000).
A number of regulatory genes have been implicated in neural crest
development. However, the molecular mechanism of how neural crest
determination is initiated in the exact ectodermal location still remains
elusive. The cooperative function of Pax3 and
Zic1 determines the neural crest fate in the amphibian ectoderm.
Pax3 and Zic1 are expressed in an overlapping manner in the
presumptive neural crest area of the Xenopus gastrula, even prior to
the onset of the expression of the early bona fide neural crest marker genes
Foxd3 and Slug. Misexpression of both Pax3 and
Zic1 together efficiently induces ectopic neural crest
differentiation in the ventral ectoderm, whereas overexpression of either one
of them only expands the expression of neural crest markers within the
dorsolateral ectoderm. The induction of neural crest differentiation by
Pax3 and Zic1 requires Wnt signaling. Loss-of-function
studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1
is essential for the initiation of neural crest differentiation. Thus,
co-activation of Pax3 and Zic1, in concert with Wnt, plays a
decisive role for early neural crest determination in the correct place of the
Xenopus ectoderm (Sato, 2005).
Teneurin-2, a vertebrate homolog of the Drosophila pair-rule gene ten-m/odz, is revealed to be a membrane-bound transcription regulator. In the nucleus, the intracellular domain of teneurin-2 colocalizes with promyelocytic leukemia (PML) protein in nuclear bodies implicated in transcription control. Since Drosophila ten-m acts epistatically to another pair-rule gene opa, whether gene regulation by the mammalian opa homolog zic-1 was influenced by the intracellular domain of teneurin-2 was investigated. zic-mediated transcription from the apolipoprotein E promoter was inhibited. Release of the intracellular domain of teneurin-2 can be stimulated by homophilic interaction of the extracellular domain, and the intracellular domain is stabilized by proteasome inhibitors. Teneurin-2 is expressed by neurons belonging to the same functional circuit. Therefore, it is hypothesized that homophilic interaction enables neurons to identify their targets and that the release of the intracellular domain of teneurin-2 provides them with a signal to switch their gene expression program from growth towards differentiation once the proper contact has been made (Bagutti, 2003).
In Drosophila, Ten-m was postulated to modulate the activity of Opa protein. It was therefore of interest to investigate whether the zinc finger transcription factor zic, a vertebrate homolog of Opa, would influence or would be influenced by the intracellular domain of teneurin-2.
When both proteins are expressed in COS-7 cells by transient transfections, a marked downregulation of the intracellular domain I of teneurin-2 was observed compared with its usual expression level. The zic-induced downregulation of the intracellular domain I of teneurin-2 is counteracted by the addition of the proteasome inhibitor lactacystin. Thus the nuclear intracellular domain of teneurin-2 seems to be subject to degradation by the proteasome pathway. By immunofluorescence staining of the transfected cells it was observed that zic-transfected cells reveal a relatively diffuse nuclear staining and in nuclei containing high amounts of zic protein, the punctate staining of teneurin-2 I disappears and becomes diffuse. Thus, the presence of zic prevents the association of the teneurin-2 intracellular domain with PML bodies and makes it amenable to proteasome-mediated degradation (Bagutti, 2003).
The intracellular domain of teneurin-2 appears to have an inhibiting effect on the transcriptional activity of zic, and this effect is more pronounced in the presence of the proteasome inhibitor ALLN, which stabilizes teneurin-2 I.
To be a functional regulator of transcription, wild-type transmembane teneurin-2 would have to be specifically cleaved in or at the plasma membrane, possibly upon a signal by ligand binding. In turn its intracellular part must be released and translocated to the nucleus in a manner similar to that established for proteins regulated by RIP. To test this hypothesis a sensitive method was developed to detect the released intracellular domain of teneurin-2 in the nucleus. Fusion proteins of full-length teneurin-2 (or of smaller transmembrane versions truncated in their extracellular domain) were fused to a Gal4 DNA-binding domain (BD) and a NFkappaB activation domain (AD). If cleavage and translocation to the nucleus occurred, BDAD-I could be detected by binding to specific Gal4 recognition sequences in the promotor of the cotransfected luciferase reporter plasmid, and subsequent initiation of luciferase gene expression activated by AD could be monitored (Bagutti, 2003).
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, the functional properties of Zic proteins and their relationship to the GLI proteins has been investigated. An optimal binding sequence for Zic1, Zic2, and Zic3 proteins was establised by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence is almost identical to the GLI binding sequence. However, the binding affinity is lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins is less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activate a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins are cotransfected into cultured cells, Zic proteins enhance or suppress 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 (Mizugishi, 2001).
In the present study, a consensus binding sequence for Zics was established by EMSA-based target selection and mutational analysis. The Zic binding sequence is essentially identical to the GLI-BS, 5'-TGGGTGGTC -3', and has a minimum consensus sequence of 5'-GGGTGGTC-3'. The binding affinities for this sequence are very similar among the three Zic proteins examined. However, the Zics-ZF bind the GLI-BS much more weakly than GLI3-ZF, as shown by competition experiments and the calculated binding constant. The Kd values of Zics are much higher than those of other transcription factors that function in a sequence-specific manner. Therefore, it is unlikely that Zic proteins compete with GLI for the GLI-BS (Mizugishi, 2001).
The binding properties are consistent with the results of the reporter assay, in which the dependence of Zic proteins on the GLI-BS for transcriptional activation is much less than that of GLI1. Instead, Zics activated transcription even in the absence of the GLI-BS via various promoters (TK promoter, adenovirus major late promoter, SV40 early promoter, and Zic1 promoter). On the basis of these facts, rather than being the transcription factors that regulate transcription by direct binding to DNA, Zic family proteins may function as transcriptional coactivators, which potentiate the activity of other transcription regulatory factors. It is possible that Zics interact with the transcription machinery or other factors that regulate transcriptional efficiency (Mizugishi, 2001).
The relationship between Zic and GLI proteins was examined. In C3H10T1/2 cells, Zic-GLI1 or Zic-GLI3 coactivates reporter gene expression, whereas in 293T cells, coexistence of the Zic and GLI proteins has a reverse effect. These results suggest a significant regulatory relationship between Zic and GLI proteins; however, the nature of this interaction remains unclear (Mizugishi, 2001).
The interaction between Zic and GLI proteins may be entirely independent of DNA binding. This direct or indirect interaction between Zic and GLI proteins may be modulated by cell type-specific cofactors. One well characterized cell type-specific cofactor is Oct-binding factor 1 (OBF-1); this is expressed in B-lymphocyte lineages and interacts with the POU-homeodomain proteins Oct-1 and Oct-2 to enhance transcriptional activation in the B-cell lineage. Similar cell type-specific cofactors might modify the GLI-Zic interactions. It is also possible that GLI proteins may be differentially modified post-translationally depending on the presence of Zics in different cell types. Recently, it was shown that Gli3 was processed depending on cAMP-activated protein kinase to generate a phosphorylated repressor form. Zic proteins might be involved in this pathway (Mizugishi, 2001).
Alternatively, the differential binding affinities of the Zic and GLI proteins for the target sequence may underlie the regulatory relationship between these two protein families. Although Zics-ZF have much lower binding affinities to the GLI-BS, there is a DNA binding transcription factor that has a Kd value similar to those of Zics. Moreover, the different human homeodomain proteins, despite having similar homeodomains, bind their target sequence with different affinities and thereby generate a complex regulatory network in the developmental process. In that case, less conserved domains other than the zinc finger may modulate binding in vivo to determine final binding specificity, because the recombinant proteins used in these experiments only included zinc finger domains. It is necessary to examine the downstream target genes in the developmental context to understand the Zic-DNA interaction in detail (Mizugishi, 2001).
Zic1/Gli3 double mutant mice showed severe abnormalities of vertebral arches not found in single mutants, strongly suggesting that these two proteins act synergistically in the development of the vertebral arches. However, in Xenopus laevis it was shown that Zic2 antagonizes the Gli proteins in the patterning of the neural plate. These findings suggest that Zic and GLI proteins may interact to variously repress or activate gene expression in vivo (Mizugishi, 2001).
In conclusion, Zic1, Zic2, and Zic3-ZF specifically recognize and bind the GLI-BS but with a much lower binding affinity than that of the GLI3-ZF. Zic proteins activated a wide range of promoters. These results suggest that Zic proteins may function as transcriptional coactivators or as factors generally involved in the gene expression process. How can such general factors regulate specific developmental processes, including the patterning of forebrain, cerebellum, axial skeleton, vasculature, and visceral organs? A clue to solving this problem may be the relationship with Gli family proteins as shown in this study. To clarify the regulatory networks under a broad range of developmental processes, the relationships between Zic proteins and other molecules in the hedgehog signaling pathway and transforming growth factor beta superfamily, which are closely related to each other, should also be examined in both in vitro and in vivo studies (Mizugishi, 2001).
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. Evidence suggests 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. These 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 (Koyabu, 2001).
Xenopus Zic3 is a Xenopus homolog of the mouse gene Zic and the Drosophila pair-rule gene odd-paired. Zic3 has significant roles both in neural and neural crest development in the Xenopus
embryo. Expression of Zic3 is first detected in the prospective neural plate region at gastrulation. Onset of
the expression is earlier than most proneural genes and follows chordin expression. The expression
is induced by blockade of BMP4 signal. Overexpression of Zic3 results in hyperplastic neural and
neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induces 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 (Nakata, 1997).
In a differential screen for downstream genes of the neural inducers, two
extremely early neural genes induced by Chordin and suppressed by BMP-4 have been identified: Zic-related-1 (Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a
Sry-related HMG factor. Expression of the two genes is first detected widely in the prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal
caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2
makes the ectoderm responsive to extracellular signals, demonstrating that the early phase
of neural induction involves simultaneous activation of multiple functions (Mizuseki, 1998).
In order to study the mechanism of neural patterning in Xenopus, subtractive cloning was used to isolate genes activated early during this process. One gene isolated was opl, (odd-paired-like), which resembles the Drosophila pair-rule gene odd-paired and encodes a zinc finger protein that is a member of the Zic gene family. At the onset of gastrulation, opl is expressed throughout the presumptive neural plate, indicating that neural determination has begun at this stage; in contrast, by the neurula stage, opl expression is restricted to the dorsal neural tube and neural crest. opl encodes a transcriptional activator, with a carboxy terminal regulatory domain, which when removed, increases opl activity. opl both sensitizes animal cap ectoderm to the neural inducer noggin and alters the spectrum of genes induced by noggin, allowing activation of the midbrain marker engrailed. Consistent with the later dorsal neural expression of opl, the activated form of opl is able to induce neural crest and dorsal neural tube markers both in animal caps and whole embryos. In ventral ectoderm, opl induces formation of loose cell aggregates that may indicate neural crest precursor cells. Aggregates do not express an epidermal marker, indicating that opl suppresses ventral fates. Together, these data suggest that opl may mediate neural competence and may be involved in activation of midbrain, dorsal neural and neural crest fates (Kuo, 1998).
In order to study forebrain determination and patterning in the zebrafish Danio rerio, zebrafish homologs of two neural markers were isolated: odd-paired-like (opl), which encodes a zinc finger protein, and fkh5,
which encodes a forkhead domain protein. At mid-gastrula, expression of these genes defines a very early pattern in the
presumptive neurectoderm, with opl later expressed in the telencephalon, and fkh5 in the diencephalon and more posterior
neurectoderm. Using in vitro explant assays, it was shown that forebrain induction had occurred even earlier, by the onset of
gastrulation (shield stage). Signaling from the early gastrula shield, previously shown to be an organizing center, is sufficient
for activation of opl expression in vitro. In order to determine whether the organizer is required for opl regulation, either the presumptive prechordal plate, marked by goosecoid (gsc) expression, or the entire
organizer, marked by chordin (chd) expression was removed
from late blastula stage embryos. opl is correctly expressed after removal of the presumptive prechordal plate; consistently, opl is correctly expressed in one-eyed pinhead (oep) mutant embryos, where the prechordal plate fails to
form. However, after removal of the entire organizer, no opl expression is observed, indicating that this region is crucial for
forebrain induction. Continued organizer function is required for forebrain induction, since beads of
BMP4, which promote ventral fates, also prevent opl expression when implanted during gastrulation. These data show that
forebrain specification begins early during gastrulation, and that a wide area of dorsal mesendoderm is required for its
patterning (Grinblat, 1998).
The posteriorizing agent
retinoic acid can accelerate anterior neuronal
differentiation in Xenopus laevis embryos. To
elucidate the role of retinoic acid in the primary
neurogenesis cascade, an investigation was carried out to see whether retinoic acid
treatment of whole embryos can change the spatial
expression of a set of genes known to be involved in
neurogenesis. Retinoic acid expands the N-tubulin,
X-ngnr-1, X-MyT1, X-Delta-1 and Gli3 domains
and inhibits the expression of Zic2 and sonic hedgehog
in the neural ectoderm, whereas a retinoid antagonist
produces the opposite changes. In contrast, sonic and banded
hedgehog overexpression reduce the N-tubulin stripes,
enlarge the neural plate at the expense of the neural crest,
downregulate Gli3 and upregulate Zic2. Thus, retinoic
acid and hedgehog signaling have opposite effects on the
prepattern genes Gli3 and Zic2 and on other genes acting
downstream in the neurogenesis cascade. In addition,
retinoic acid cannot rescue the inhibitory effect of
NotchICD, Zic2 or sonic hedgehog on primary neurogenesis.
These results suggest that retinoic acid acts very early,
upstream of sonic hedgehog, and a model is proposed for
regulation of differentiation and proliferation in the neural
plate, showing that retinoic acid might be activating
primary neurogenesis by repressing sonic hedgehog
expression (Franco, 1999).
RA treatment can accelerate
neuronal differentiation in the anterior neural plate of whole
embryos. Could RA also alter
neuronal differentiation in the posterior neural plate where
endogenous RA might mainly play its role and where primary
neurogenesis occurs?
It has been shown that RA exposure during gastrulation
greatly expands the normal domains of N-tubulin expression
at the neural plate stage. In contrast, retinoic acid antagonist Ro treatments decrease N-tubulin
expression, in agreement with the loss of primary
neurons produced by the microinjection of dominant negative
forms of retinoic acid receptors. RA treatment increases the
domains of genes previously shown to promote
neuronal differentiation, such as X-ngnr-1, X-MyT1
and Gli3. The deletion of spacing between
the stripes of X-ngnr-1 and X-MyT1 suggests
that RA changes the activity of prepattern
genes, thus directing the neural plate toward a
uniform proneural territory. Indeed, RA produces
a widespread Gli3 expansion in the posterior
neural plate and a dramatic downregulation of
Zic2, a gene proposed to inhibit neuronal
differentiation. The involvement of endogenous
retinoids in this regulatory hierarchy was
confirmed by blocking RA signaling with Ro,
which produced opposite changes in the
expression patterns of these genes (Franco, 1999).
Because RA treatments could not rescue the inhibitory
effect of X-shh on neuronal differentiation, while X-shh
overexpression produces a widespread expansion of Zic2 and
suppresses Gli3, it is suggested that a cascade of interactions occurs,
wherein endogenous retinoids act far upstream, promoting
primary neurogenesis by inhibiting X-shh expression in the
dorsal midline. This in turn changes the balance of
prepattern genes (activation of Gli3 and reduction of Zic2),
thus altering the expression of other intermediary genes,
ultimately leading to N-tubulin activation.
Because in the normal embryo X-shh is expressed along the
dorsal midline, it is evident that endogenous retinoids do not
completely block shh signaling. This fact suggests that a
precise balance between retinoid and hedgehog signaling must
be established, resulting in the normal primary neurogenesis
pattern. While endogenous retinoids constitute an early signal
that promotes primary neuron formation by inclining the entire
neural plate towards a uniform proneural territory, shh
signaling is necessarily required at the same time and at an
accurate level, limited at least by endogenous retinoids, to save
a pool of neuronal precursors from premature differentiation
by retinoid signaling, keeping them in a mitotic,
undifferentiated state for subsequent waves of neurogenesis (Franco, 1999).
The regulatory elements in the 5' flanking region of the Zic1 gene have been analyzed as an initial step to understanding how the Zic1 expression is restricted to the dorsal neural tissue. When a 2.9-kb fragment of the 5' flanking segment of the mouse Zic1 gene was linked to the E. coli beta-galactosidase gene, the enzyme was consistently expressed in the dorsal half of the embryonic spinal cord and in the vestibulocochlear nucleus in all four transgenic mouse lines. The transgene expression mimics the Zic1 expression with respect to the region where it occurs. But this is not so for the neuronal cell types. This suggests that the segment contains a region-specific enhancer. In vivo and in vitro deletion analyses indicate that there are essential regions between -2.0 and -0.9 kb and within the proximal 0.9 kb. The distal element is necessary for the transgene expression in the embryonic dorsal spinal cord whereas the adult vestibulocochlear nucleus expression is regulated by both elements. In these regions, there are sequences similar to the binding sequences for potential regulatory proteins (Aruga, 2000).
The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates -- among them are neural plate formation and neural patterning. The Xenopus Iro (Xiro) function in primary neurogenesis has been studied in detail. Misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, XGadd45-gamma has been identified as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. It is concluded that during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons (de la Calle-Mustienes, 2002).
Mouse Zic genes encode zinc finger proteins and are
expressed in the developing and mature CNS. Reduced expression
of Zic2 in mice results in spina bifida and
holoprosencephaly. However, the disruption of Zic1, a
strong homolog of Zic2 that has an overlapping expression pattern, results in cerebellar malformation with no apparent abnormalities in the forebrain or in posterior neuropore closure. Zic2 and Zic1 cooperatively control cerebellar development by regulating neuronal
differentiation. Both Zic1 and Zic2 are
expressed in the precursor cells of the granule neuron and the
neurons in cerebellar nuclei. Mice carrying one mutated
Zic1 allele together with one mutated Zic2 allele (Zic1+/-Zic2+/kd) show a marked cerebellar folial abnormality similar to, but distinct from that found in mice homozygous for the Zic1 mutation (Zic1-/-). The Zic1+/-Zic2+/kd cerebellum is missing a lobule in the anterior vermis and has a truncation of the most posterior lobule. Expression of transverse zonal
markers is shifted anteriorly in the developing cerebellum, indicating
that the anterior part of the cerebellum is poorly developed. Abnormalities in the developing Zic1+/-Zic2+/kd cerebellum share the following features with those of the Zic1-/- cerebellum: a preceding reduction of cell proliferation in the anterior external germinal layer, a reduction in cyclin D1 expression, and enhanced expression of the mitosis inhibitors p27 and p16, and enhancement of Wnt7a expression. These results indicate that Zic1 and Zic2 may have very similar functions in the regulation of cerebellar development (Aruga, 2002).
The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger
proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal
neural tube. Chick Zic1 is initially expressed evenly along the dorsoventral axis and its expression becomes increasingly
restricted dorsally during the course of neurulation. The dorsal expression of Zic1 is regulated by Sonic hedgehog, BMP4,
and BMP7, as revealed by experimentally induced overexpression of these genes in the spinal cord. When Zic1 is misexpressed on the ventral side of the chick spinal cord, neuronal differentiation is inhibited irrespective of the dorsoventral position. In addition, dorsoventral
properties are not grossly affected as revealed by molecular markers. Concordantly, when Zic1 is overexpressed in the dorsal spinal cord in transgenic mice, hypercellularity is observed in the dorsal spinal cord. The transgene-expressing cells are increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, a significant cell number reduction is observed without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differention is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick (Aruga, 2002).
This may be due to redundancy between different Gadd45 proteins. The spatial and temporal patterns of expression of Gadd45-gamma and the Notch ligand XDl1 largely coincide. Moreover, both XGadd45-gamma and XDl1 are positively regulated by proneural genes and negatively controlled by Notch signaling. According to the lateral inhibition model, activation of the Notch pathway within a cell, by signaling from neighboring cells, maintains the cell's mitotic potential and prevents its differentiation. In contrast, a cell that expresses high levels of Notch ligands and signals strongly, escapes lateral inhibition, exits the cell cycle and differentiates. XGadd45-gamma may provide a link between Notch signaling, cell-cycle arrest and differentiation. Thus, in the neural plate, cells with high levels of proneural genes have also high levels of XDl1 and XGadd45-gamma. The first allows them to escape lateral inhibition, and the second to exit the cell cycle. These cells can then differentiate. Mitotic arrest mediated by XGadd45-gamma probably occurs through interaction with cyclin and inhibitors of cyclin-dependent kinases. In neighboring cells, the Notch pathway is activated, proneural genes and XGadd45-gamma are downregulated, and cell-cycle arrest and differentiation cannot occur. It is of interest that induction of Gadd45 genes in cell culture stops the cell cycle in G1 phase. This phase is compatible with exiting the cell cycle, a requirement for terminal neuronal differentiation. Cells that differentiate outside the neural plate may resort to genes different from the proneural ones to accumulate Notch ligands and XGadd45-gamma (de la Calle-Mustienes, 2002).
This study compares the effects of overexpressing either Xiro1, -2 or -3 in neural development. To make comparisons more meaningful, equivalent constructs were prepared in the pCS2MT plasmid. The overexpression of each Xiro gene causes similar effects, although Xiro3 was approximately five to ten times more potent. Paradoxically, the overexpressions activated Xngnr1 and repressed neuronal differentiation. This may be explained at least in part by the finding that Xiro upregulates the neuronal repressors XHairy2A and XZic2. Indeed, it has been shown that XZic2 antagonizes development of Xngnr1-promoted ectopic neurons. XZic2 antagonizes Xngnr1-promoted XGadd45-gamma and XDl1 expression. Consistently with these findings, in wild type embryos the intermediate stripes of expression of XHairy2A and XZic2 are within the Xiro1 domains. Also in accordance with these results, in the prospective spinal chord, the Xiro1 domain is contained within the broader Xngnr1 domain and neurons arise at the border of the Xiro1 domain. Taken together, these observations suggest that Xiro proteins simultaneously participate in the activation of Xngnr1 and of genes that antagonize primary neuron formation (de la Calle-Mustienes, 2002).
Overexpressions of Xiro genes represses both XGadd45-gamma and XDl1 in territories where primary neurons arise. Consistently, in wild type embryos, XGadd45-gamma and XDl1 are expressed at the borders of Xiro domains. Moreover, XDl1 is activated in embryos expressing a Xiro1 chimera that converts the Xiro1 repressor into an activator (HD-GR-E1A). This activation occurs even in the absence of protein synthesis. Thus, XDl1 is probably directly repressed by Xiro. However, XGadd45-gamma is repressed by HD-GR-E1A, probably because Xngnr1 is also downregulated. Indeed, coinjection of HD-GR-E1A and Xngnr1 mRNAs rescues the expression of XGadd45-gamma. Thus, Xiro-mediated repression of XGadd45-gamma is probably indirect and may take place, at least in part, by Xiro-upregulated neuronal repressors. In this case, interference with Xiro function would suppress neuronal repressors, but would also downregulate Xngnr1, which is needed for XGadd45-gamma expression (de la Calle-Mustienes, 2002).
A model is proposed for the function of Xiro in neural patterning that integrates the above data. Xiro proteins, as well as other factors, participate in the activation of Xngnr1. Within the Xiro domains, Xngnr1 does not activate XDl1 or XGadd45-gamma, and cannot promote differentiation of primary neurons due to the upregulation by Xiro of neuronal repressors, such as XHairy2A and XZic2. In addition, Xiro probably represses XDl1 directly. Outside the Xiro domains, other factors, such as the Gli proteins, activate Xngnr1, which in turn promotes the expression of XDl1 and XGadd45-gamma in those cells that will become primary neurons. XDl1 switches on the lateral inhibition mechanism by which the Notch signaling pathway is activated in neighboring cells. This pathway downregulates proneural genes, XDl1 and XGadd45-gamma. As a consequence, these cells keep dividing and do not differentiate. In contrast, cells with high levels of Xngnr1, XDl1 and XGadd45-gamma escape lateral inhibition, exit the cell cycle (in part due to the presence of XGadd45-gamma) and differentiate as primary neurons. This differentiation is triggered by a genetic program activated by Xngnr1. Thus, Xiro proteins may help coordinate cell cycle and differentiation (de la Calle-Mustienes, 2002).
In intact Xenopus embryos, an increase in intracellular Ca2+ in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this Ca2+ increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between Ca2+ signaling and gene expression during neural induction is complicated by the fact that the dorsal ectoderm receives both planar and vertical signals from the mesoderm. The experimental system may be simplified by using Keller open-face explants where vertical signals are eliminated, thus allowing the interaction between planar signals, Ca2+ transients, and neural induction to be explored. Ca2+ dynamics during neural induction have been imaged in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized Ca2+ transients in groups of cells in the ectoderm. These transients result from the activation of L-type Ca2+ channels. The accumulated Ca2+ pattern correlates with the expression of the early neural precursor gene, Zic3. When the transients are blocked with pharmacological agents, the level of Zic3 expression is dramatically reduced. These data indicate that, in open-face explants, planar signals reproduce Ca2+ -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized Ca2+ transients over time may play a role in controlling the expression pattern of Zic3 (Leclerc, 2003).
The dorsal hindbrain includes distinct classes of neurons for processing various sensory stimuli, but the developmental aspects of these neurons remain largely unknown. Two distinct classes of neurons have been identified in the dorsal hindbrain of developing zebrafish: (1) neurons that express the inhibitory neuronal marker Gad1/2, and (2) neurons that express the zn-5 antigen and Lhx2/9 and require the basic helix-loop-helix transcription factor Atoh1a for development. Neurons were traced to their axon terminals by expressing green fluorescent protein using the Gal4VP16-UAS (UAS, upstream activating sequences) system in combination with the promoter/enhancer regions of gad2 for the Gad1/2(+) neurons and zic1 for the zn-5(+)Lhx2/9(+) neurons. The Gad1/2(+) neurons projected to the contralateral hindbrain, while the zn-5(+)Lhx2/9(+) neurons projected to the contralateral midbrain torus semicircularis, suggesting a role in auditory and lateral line sensory processing. Comparison of these projections with those from the cochlear nuclei to the inferior colliculus in mammals suggests similarities across vertebrate species (Sassa, 2007).
Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic; however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, two new Wnt targets have een identified, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, zic2a and zic5 transcription were shown to be activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair (Nyholm, 2007).
The targeting of retinal ganglion axons toward the optic disc is the first step in axon pathfinding in the visual system. The molecular mechanisms involved in guiding the retinal axons to project towards the optic disc are not well understood. A gene encoding a zinc-finger
transcription factor, Zic3, is expressed in a periphery-high and
center-low gradient in the retina at the stages of active axon extension
inside the retina. The gradient expression of Zic3 recedes towards
the periphery over the course of development, correlating with the progression of retinal cell differentiation and axonogenesis. Disruption of gradient expression of Zic3 by retroviral overexpression resulted in mis-targeting of retinal axons and some axons misrouted to the sub-retinal space at the photoreceptor side of the retina. Misexpression of Zic3 did not affect neurogenesis or differentiation inside the retina, or grossly alter retinal lamination. By stripe assay, it is shown that misexpression of Zic3 may induce the expression of an inhibitory factor to the retinal axons. Zic3 appears to play a role in intra-retinal axon targeting, possibly through regulation of the expression of specific downstream genes involved in axon guidance (Zhang, 2004).
Pathfinding of retinal ganglion cell (RGC) axons at the midline optic chiasm determine whether RGCs project to ipsilateral or contralateral brain visual centers, critical for binocular vision. Using Isl2tau-lacZ knockin mice, it has been shown that the LIM-homeodomain transcription factor Isl2 marks only contralaterally projecting RGCs. The transcription factor Zic2 and guidance receptor EphB1, required by RGCs to project ipsilaterally, colocalize in RGCs distinct from Isl2 RGCs in the ventral-temporal crescent (VTC), the source of ipsilateral projections. Isl2 knockout mice have an increased ipsilateral projection originating from significantly more RGCs limited to the VTC. Isl2 knockouts also have increased Zic2 and EphB1 expression and significantly more Zic2 RGCs in the VTC. It is concluded that Isl2 specifies RGC laterality by repressing an ipsilateral pathfinding program unique to VTC RGCs and involving Zic2 and EphB1. This genetic hierarchy controls binocular vision by regulating the magnitude and source of ipsilateral projections and reveals unique retinal domains (Pak, 2004).
These findings indicate that Isl2 normally represses Zic2 expression in RGCs in the VTC and either directly represses EphB1 expression or indirectly through repression of Zic2 and that the increased ipsilateral projection in Isl2-null mice is due to a loss of this repression and upregulation of Zic2 and EphB1. This model is consistent with several pieces of data. (1) Regarding the timing of expression of Isl2 and Zic2 in VTC RGCs, the onset of Isl2 expression in VTC RGCs is similar to that of Zic2: weak Isl2 expression is detected in the VTC as early as E13.5, and moderate levels of Isl2 expression are evident by E14.5, the age when Zic2 expression in VTC RGCs is first detected. (2) Zic2 and EphB1 colocalize in a subset of RGCs distinct from Isl2 RGCs. (3) Increased expression of Zic2 and EphB1 and a significant increase in Zic2-positive RGCs are found in the VTC of Isl2-null retina. (4) The laterality phenotype of Isl2-null mice complements that of Zic2kd/kd and EphB1 mutants (Pak, 2004).
The yeast one-hybrid system has been used 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 encode 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.
Math1 is a basic helix-loop-helix transcription factor expressed in
progenitor cells that give rise to dorsal commissural interneurons in the
spinal cord, granule cells of the cerebellum, and sensory cells in the inner
ear and skin. Transcriptional regulation of this gene is tightly controlled
both temporally and spatially during nervous system development. The signals
that mediate this regulation are likely integrated at the Math1
enhancer, which is highly conserved among vertebrate species. The zinc-finger transcription factor Zic1 has been identified as a regulator of
Math1 expression. Zic1 binds a novel conserved site within the
Math1 enhancer, and represses both the expression of endogenous Cath1
(chicken homolog of Math1) and the activity of a Math1 enhancer
driven lacZ reporter when expressed in chick neural tubes. Repression
by Zic1 blocks the autoregulatory activity of Math1 itself. Although previous
reports have shown that Zic1 and Math1 are both induced by BMP signaling,
these genes appear to have opposing functions, since Math1 acts to promote
neuronal differentiation in the chick neural tube and excess Zic1 appears to block differentiation. Zic1-mediated repression of Cath1 transcription may modulate the temporal switch between the progenitor state and differentiating dorsal cell types during neural tube development (Ebert, 2003).
The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, whether Xenopus Zic1 acted through the Wnt pathway was examined. Using Wnt signaling inhibitors, it was demonstrated that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated in this study. Together, these data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates (Merzdorf, 2006).
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