odd-paired


EVOLUTIONARY HOMOLOGS

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).

The pair-rule gene odd-paired (opa) is required for the patterning of alternate segment boundaries in the early Drosophila embryo. Mutant phenotypes of opa display a typical pair-rule phenotype in which most of each odd-numbered denticle belt is eliminated. However, among the nine Drosophila pair-rule genes, opa is the only gene that is not expressed in stripes with double segmental periodicity; its transcript and protein are expressed in a broad domain within segmenting embryos. While expression patterns of orthologs of opa have been analyzed in several arthropod species, their regulation and function in segmentation were largely unknown. This study analyzed the expression patterns, regulation, and function of the Tribolium ortholog of opa (Tc-opa). Tc-opa is expressed in segmental stripes in the early stages of segmentation and then is expressed in a broad domain at the growth zone of elongating germbands where new segments form. This broad expression is processed into segmental stripes once the trunk has become segmented. Tc-opa expression is regulated positively and negatively by even-skipped and odd-skipped, respectively. However, knock-down of Tc-opa does not affect embryonic segmentation. These findings suggest that Tc-opa expression is regulated by the pair-rule gene network even though its requirement for segmentation is uncertain in Tribolium (Choe, 2017).

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 other’s 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).

How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. This study establishes a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/β-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. This study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/β-catenin pathway is widely used to control the identity of neuronal lineages (Bertrand, 2009).

Several examples have by now well illustrated that the differentiation of individual neuron types is governed by terminal selector genes that encode transcription factors which directly activate large batteries of terminal differentiation genes. However, how these terminal selector genes are regulated by earlier specification processes, in particular asymmetric divisions, remains poorly understood. This study has uncovered a direct regulatory cascade that links the asymmetric division machinery to the activation of the terminal selector genes ttx-3 and ceh-10 during embryogenesis in C. elegans. These results will first be discussed in the context of the broad concept of progressive regulatory states before analyzing two other general implications of these studies, namely, a common theme of Zic gene function in neural precursors and a potentially broadly conserved role of Wnt signaling in neuronal specification (Bertrand, 2009).

The Zic transcription factor REF-2 is transiently expressed in the SMDD/AIY mother, where it directly activates the expression of the ttx-3 LIM homeobox gene in cooperation with the bHLH transcription factor HLH-2. Following division of the mother cell, TTX-3 is inherited in both SMDD and AIY and activates ceh-10 expression in AIY, but not in SMDD. The difference in ttx-3 activity between AIY and SMDD is due to the Wnt/β-catenin asymmetry pathway. The transcriptional mediators of this pathway, the TCF transcription factor POP-1 and its coactivator the β-catenin SYS-1, are asymmetrically localized after division of the SMDD/AIY mother. In AIY, the POP-1 nuclear concentration is low and SYS-1 concentration is high. This may allow most of the POP-1 proteins to be associated with the coactivator SYS-1 and to activate the transcription of ceh-10 via the predicted POP-1 binding sites present in its promoter. In SMDD, where the POP-1 nuclear concentration is high and SYS-1 concentration is low, most of the POP-1 proteins may not be associated with SYS-1 and therefore repress ceh-10 transcription. Finally, once coexpressed in postmitotic AIY, TTX-3 and CEH-10 directly activate a large battery of terminal differentiation genes responsible for AIY differentiation and specific function. TTX-3 and CEH-10 also maintain their own expression so that the system is locked during larval and adult stages (Bertrand, 2009).

It has been proposed that during development a cell progresses through a succession of 'regulatory states' each characterized by a combination of specific gene regulatory factors. In the case of the AIY terminal division, two regulatory states are observed. The first one (state 1) is characterized by the transient expression of REF-2 and HLH-2 in the SMDD/AIY mother. The second (state 2p) corresponds to the terminal differentiation state defined by the expression of the terminal complex TTX-3/CEH-10 and the battery of terminal differentiation genes. The transition between those two states is driven by a binary decision system based on the Wnt/β-catenin asymmetry pathway (Bertrand, 2009).

These findings provide explicit support for a theoretical model initially proposed by Priess and coworkers (Lin, 1998). In this model a transcription factor 'B' expressed in both daughter cells following the division cooperates with a high POP-1 level in the anterior cell to specify state 2a and cooperates with a low POP-1 level in the posterior cell to specify state 2p. In the case of AIY, this lineage-specific factor 'B' corresponds to the transcription factor TTX-3 (Bertrand, 2009).

Before discussing general principles of Wnt/β-catenin signaling in neuronal specification, ref-2, one specific member of the regulatory network studied here, will be discussed. ref-2 is expressed in several neuronal precursors in the embryo; in contrast, there is no detectable expression of ref-2 in postmitotic neurons at larval and adult stages. Similarly, in Hydra and vertebrates, Zic transcription factors are also expressed in several neural progenitors, while expression in adult postmitotic neurons is only rarely seen. This indicates that Zic transcription factors may have a conserved function in neural precursor development. While in vertebrates Zic transcription factors have been shown to play a role in promoting the proliferation of the progenitors, it is conceivable that they also function as transient initiators of the terminal differentiation program of specific neurons, as observed in the case of AIY. For example, an intriguing parallel can be drawn between the development of the AIY interneurons and the cholinergic projection neurons/interneurons of the vertebrate basal forebrain. These vertebrate cholinergic neurons have an important function in memory formation, as is the case for the cholinergic interneuron AIY. In vertebrates, these postmitotic neurons and their progenitors express the TTX-3-related LIM-homeodomain transcription factor Lhx7/8, which is required for their differentiation. It has been recently reported that the Zic transcription factors Zic1 and Zic3 are also expressed in these progenitors and that inactivation of both genes reduces the number of cholinergic neurons. While these Zic factors seem to regulate primarily the proliferation of the precursors, it would be interesting to test whether, in analogy to ttx-3 initiation by REF-2, they also initiate the expression of Lhx7/8 and endow the progenitors with the ability to generate cholinergic neurons (Bertrand, 2009).

A particular Wnt pathway, the Wnt/β-catenin asymmetry pathway, is involved in many asymmetric blastomere divisions in the early embryo as well as some asymmetric divisions during larval development in C. elegans. Analysis of temperature-sensitive mutants of the upstream kinase gene lit-1(t1512) has shown that this pathway is involved in six successive asymmetric division rounds in the early embryo. However, this pathway has not been shown so far to be implicated in the terminal division of embryonic neuroblasts. This study has observed that the three terminal neuroblast divisions analyzed (giving rise to AIY, AIN, and ASER, respectively) are affected by disrupting this Wnt pathway. Moreover, lit-1(t1512); mom-4(ne1539) embryos shifted at restrictive temperature just before most embryonic neuroblasts undergo their last division give rise to larvae showing strongly uncoordinated movements, suggesting additional defects in motor neuron lineages. These observations predict that the Wnt/β-catenin asymmetry pathway is widely used in terminal neuroblast division in the C. elegans embryo (Bertrand, 2009).

While it was shown that the transcriptional mediators of this pathway, POP-1/TCF and SYS-1/β-catenin, are asymmetrically localized after the terminal division of embryonic neuroblasts, how the asymmetry in this pathway is initially established remains obscure. Both POP-1 and SYS-1 are regulated by this pathway at a posttranslational level (Mizumoto, 2007). In the early embryo POP-1 asymmetry in the AB lineage requires an initial MOM-2/Wnt signal coming from the P1 lineage that may be transmitted among AB blastomeres by a relay mechanism, but POP-1 asymmetry becomes later independent of MOM-2/Wnt. MOM-5/Frizzled is enriched in the posterior pole of early AB derivatives, and in analogy to the planar cell polarity in Drosophila, a Wnt-independent asymmetric Frizzled localization could be responsible for generating asymmetric cell divisions. Additional studies on Wnt requirement and Frizzled localization are required to assess their mode of function in the context of the terminal division of embryonic neuroblasts (Bertrand, 2009).

Neurons are also generated via asymmetric divisions in Drosophila and vertebrates. Recent results suggest a possible role for β-catenin in the asymmetric division of neural progenitors in the mouse brain. For example, it has been proposed that β-catenin may regulate the asymmetric division generating intermediate progenitors from radial glial cells during corticogenesis. A Wnt/β-catenin system, similar to the one shown in this study to operate in terminal neuroblast divisions in C. elegans, may therefore be used in binary cell fate decisions during the development of the nervous system in other organisms (Bertrand, 2009).

Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. This study identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head (the anterior regeneration pole) and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. A model is suggested in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue (Vogg, 2014).

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).

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).

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)

Genomic cis-regulatory networks in the early Ciona intestinalis embryo

Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. This determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, it was found that most of the previously determined interactions are direct. Focus was placed on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. Many interactions were found that had not been predicted by simple gene knockdown experiments, and a significant fraction of TF-DNA interactions were found to make major contributions to the regulatory control of target gene expression (Kubo, 2010).

The developmental fates of blastomeres in the Ciona embryo have been determined by the gastrula stage. A comprehensive study has revealed that 53 TF genes are zygotically expressed and regulate one another in complex networks before gastrulation begins. To dissect the architecture of these networks at the level of protein-DNA interactions, focus was placed on 11 TF genes that play core roles in gene regulatory networks for endomesoderm specification: Brachyury, FoxA-a, FoxD, MyoD, Neurogenin, Otx, Snail, SoxC, Tbx6b, Twist-like1 and ZicL. Because the Ciona genome contains multiple copies of FoxD, Tbx6b and ZicL as gene clusters and their precise copy numbers have not yet been determined, these genes are collectively referred to FoxD, Tbx6b and ZicL in this paper. Likewise, there are two copies of Twist-like1, which are highly similar to each other, and these are collectively referred to as Twist-like1 (Kubo, 2010).

Eleven gene-fusion constructs were prepared that encode GFP-tagged TFs expressed under the control of their own promoters (e.g. a fusion gene that encodes GFP-tagged Brachyury driven by the Brachyury promoter). When these constructs were introduced into eggs, the resultant embryos expressed the fusion genes at the same time and in the same blastomeres as the endogenous genes. Exceptions were the Twist-like1 and the Snail constructs. Twist-like1 is normally expressed in three cell lineages (A7.6, B7.7 and B8.5), but the construct drove Twist-like1-GFP expression only in the B7.7 and B8.5 lines. Snail expression in the notochord lineage is normally very weak. The Snail construct did not recapitulate this expression in the notochord lineage but did drive Snail-GFP expression in the remaining lineages (Kubo, 2010).

Expression of these genes did not affect embryonic morphology at the stage when the embryos were fixed. The fixed embryos were subjected to ChIP using anti-GFP antibodies, and subsequently to microarray analysis. To define significant regions, two programs were used employing totally different algorithms. DNA segments regarded as positive by both programs were defined as significant. To confirm that this approach successfully identified TF binding sites, the sequences of ZicL and Tbx6b binding regions defined with three different false discovery rates (FDRs) were analyzed, as the consensus binding motifs of these two TFs are known. The frequencies of matches to the consensus binding sequences for ZicL and Tbx6b around peaks in 0.1% FDR were generally better than in 0.01% and 1% FDRs. As expected, the frequencies of the consensus binding sequences for ZicL and Tbx6b were markedly higher around peaks in the identified regions, suggesting that the method was able to successfully identify the TF binding regions (Kubo, 2010).

Brachyury and Ci-tropomyosin-like are the only known direct targets of ZicL and Brachyury, respectively. As an independent confirmation, the TF binding sites of these genes was expected. The ZicL ChIP profile showed a sharp peak around two known strong ZicL binding sites. The Brachyury ChIP profile also showed a peak around the known Brachyury binding site in the Ci-tropomyosin-like promoter. These peaks were included in significant regions identified with all the FDRs described above. ChIP-qPCRs were performed for these two known interactions. The ChIP-qPCR results showed excellent agreement with the ChIP-chip results (Kubo, 2010).

Next, the promoters were examined of genes that were identified in previous studies as likely direct targets of one of the 11 TFs on the basis of expression assays and gene knockdown assays. Among 29 interactions that had been found in the gene knockdown assays and for which both the source and target genes are expressed in the same cells, 28, 23 and 19 interactions were indicated to be direct under the FDRs of 1%, 0.1% and 0.01%, respectively. The remainder of the interactions were not regarded as direct. Otx expression in the A-line lineage requires a cis-regulatory module that includes Fox binding sites and is suppressed in FoxA-a morphants. The FoxA-a binding to this cis-regulatory element was counted with FDRs of 1% and 0.1%, but not with the most stringent FDR (0.01%). Similarly, several lines of evidence have suggested that MyoD is directly regulated by ZicL. First, MyoD expression is suppressed in ZicL morphants. Second, MyoD and ZicL are both expressed in presumptive muscle cells and the time windows of their expression overlap. Lastly, there is a putative ZicL binding site near to the peaks found in the MyoD upstream region. This putative binding was observed under the FDRs of 1% and 0.1%, but not under the most stringent FDR of 0.01%. On the basis of the above observations, in the following sections the results obtained at an FDR of 0.1% are generally described (Kubo, 2010).

The frequencies of the consensus sequences for ZicL and Tbx6b binding were markedly higher around peaks in the identified regions. Since the consensus binding motifs of the other nine TFs had not been determined previously, similar analyses was performed with motifs of homologs in other animals. The frequencies of the consensus binding motifs for six of the TFs, but not FoxD, SoxC or Twist-like1, were markedly higher around peaks. Because the position weight matrices (PWMs) for FoxD, SoxC and Twist-like1 gave higher background, no significant changes were seen. However, the number of matches to the motifs was markedly higher around peaks than in flanking regions and the background. These observations suggested that the method was able to successfully identify the TF binding regions (Kubo, 2010).

As has been reported in other animals, it was found that the regions bound by Brachyury, MyoD, Neurogenin, Snail, Tbx6b, Twist-like1 and ZicL, especially around the peaks, showed a marked GC bias. This bias is likely to be related to the consensus sequences, because the consensus sequences for these TFs are generally more GC-rich than those of the remaining TFs. The observed enrichment of recognition sequences was unlikely to be an artifact of GC bias because even if background sequences were picked with a base composition comparable to the averaged GC content of the bound regions (the difference between the average GC content of the bound and background regions was less than 0.8%), matches to the PWMs were enriched around peaks versus each of the GC-adjusted backgrounds (Kubo, 2010).

Next, attempts were made to discover overrepresented motifs in the regions (360 bp) around the peaks identified by each ChIP experiment using the Trawler program. It was found that overrepresented motifs were similar to the PWMs that were determined experimentally (Tbx6b and ZicL) or to those of homologs in other animals (the remaining nine TFs). This further supported the conclusion that the results of the ChIP experiments were of high quality (Kubo, 2010).

It is generally believed that TFs tend to bind near promoters, although many examples are known in which TFs bind to enhancers far from promoters. The distributions of peaks in all experiments, except Snail ChIP, were higher around transcription start sites. The reason why Snail binding sites were not enriched around transcription start sites is unclear, but this does not necessarily indicate that the results of the Snail ChIP were of low quality. Altogether, these observations support the conclusion that all of the ChIP experiments revealed in vivo occupancies of the TFs (Kubo, 2010).

TF genes were significantly enriched among the target genes of the 11 TFs. Among 670 potential TF genes in the Ciona genome, at least 607 encode proteins with known TF motifs or proteins with two or more zinc-finger motifs that potentially bind to DNA. A significantly greater number of TF genes were found among the targets than would be expected from random sampling. This enrichment indicates that the TFs examined bind targets selectively and not randomly (Kubo, 2010).

The ChIP data was compared with the results of the comprehensive gene knockdown experiments of a previously study. Among 76 interactions previously found in the early embryo, the ChIP assays indicated that 58 are direct. In addition, 251 novel interconnections were found. Among 121 (11×11) possible interconnections, 84 were observed in the present study. The data indicate that these genes are tightly interconnected with one another (Kubo, 2010).

Because the gene regulatory network model previously constructed from comprehensive expression profiles and comprehensive knockdowns of regulatory genes is of single-cell resolution, the ChIP data was interpred into this network by assuming that the examined TFs bind to the targets wherever their mRNAs are expressed. The reconstructed networks had a complex architecture (Kubo, 2010).

The reconstructed regulatory networks allow tracing of development at the single-cell level. Figs S8 and S9 in the supplementary material show the interconnections among the core 11 TFs in A-line and B-line blastomeres, which give rise to endomesodermal tissues, from the 8-cell to the early gastrula stage. Two of the three mesenchymal lineages (B-line mesenchymal cells) and 28 out of 36 muscle cells (B-line muscle cells) in the tadpole larvae are derived from B4.1 blastomeres at the 8-cell stage. Thirty-two and eight notochord cells are derived from A4.1 and B4.1 blastomeres, respectively. Previous studies demonstrated that Twist-like1, MyoD and Brachyury are essential for specification of the mesenchyme, muscle and notochord, respectively (Kubo, 2010).

Twist-like1 is expressed exclusively in the mesenchymal lineage and is regulated by FoxA-a, Otx and ZicL, as indicated by the fact that knockdown of any of these three genes results in loss or reduction of Twist-like1 expression. No direct binding was detected of FoxA-a to the Twist-like1 promoter, but it was found that FoxA-a binds to the upstream regions of Otx and ZicL, and that ZicL and Otx bind to the promoter of Twist-like1. Therefore, it is highly likely that FoxA-a mainly activates Twist-like1 indirectly through activating Otx and ZicL (Kubo, 2010).

Twist-like1 expression begins in B7.7 (the posterior B-line mesenchyme) at the 64-cell stage and in B8.5 (the anterior B-line mesenchyme) at the early gastrula stage. These two mesenchymal lines contribute to distinct adult tissues after metamorphosis. ZicL might be associated with the differences between these two lineages because the contribution of ZicL to Twist-like1 activation is weaker than that of Otx. To confirm this idea, a mutant Twist-like1 promoter was tested, from which a 150 bp segment containing the identified ZicL binding region was deleted. Because the Otx ChIP result indicated that the Otx binding region is distinct from the ZicL binding region, Otx was expected to bind to this mutant promoter. When introduced into fertilized eggs by electroporation, the wild-type promoter (1550 bp) drove reporter expression in 65% of the embryos, whereas the mutant promoter drove reporter expression in 36% of the embryos. In addition to the significant decrease in the number of embryos expressing the reporter, the overall fluorescence was weaker and the posterior B-line mesenchyme did not appear to express the reporter in the mutant construct. To confirm this observation, the experimental embryos were cleavage-arrested at the 110-cell stage. Cells in the arrested embryos cannot divide further, but the developmental programs proceed as in normal embryos. The mutant construct failed to drive reporter expression in the posterior B-line mesenchyme. These results suggest that ZicL contributes to the difference between these two lineages (Kubo, 2010).

A previous study showed that nine mesenchyme-specific non-regulatory genes are under the control of Twist-like1. None of these genes was identified as a direct target in the present study. Even when applied with an FDR of 1%, only one gene was identified as a direct target. Therefore, it is likely that Twist-like1 regulates the expression of mesenchyme-specific genes through its downstream regulatory gene circuit, although there is a possibility that Twist-like1 binds to the regulatory elements of these genes at later stages (Kubo, 2010).

The B6.2 and B6.4 cell pairs in the 32-cell embryo have the potential to give rise to mesenchyme and muscle. At the 64-cell stage, these cells divide, and one of the daughter cells becomes specified to give rise to the muscle cells. Previous functional assays showed that ZicL, Tbx6b and MyoD are essential for specification of muscle cells. Tbx6b begins to be expressed at the 16-cell stage, and cells expressing Tbx6b give rise not only to muscle cells but also to mesenchyme cells. Tbx6b expression declines to undetectable levels before the tailbud stage. ZicL starts to be expressed at the 32-cell stage in a variety of cells, including those with developmental fates of muscle, mesenchyme, notochord and neurons. ZicL expression in the muscle lineage disappears before the late gastrula stage. MyoD expression begins at the 44-cell stage exclusively in the muscle lineage under the control of Tbx6b and ZicL. The present study showed that ZicL, Tbx6b and MyoD constituted a tightly interconnected gene circuit that is responsible for this specification: (1) ZicL bound to the promoters of MyoD and Tbx6b; (2) Tbx6b bound to the promoters of MyoD and ZicL; and (3) MyoD bound to the promoter of Tbx6b and to its own promoter. All of these interactions, except MyoD binding to the Tbx6b promoter, have been confirmed by functional assays (Kubo, 2010).

To understand how this gene circuit regulates downstream muscle-specific genes, the promoters were examined of 13 muscle structural genes that are well annotated and known to be expressed in the larval tail muscle. Of these, ten were directly bound by MyoD and Tbx6, one by MyoD and ZicL, one by Tbx6b and ZicL, and one by MyoD alone (Kubo, 2010).

Both MyoD and Tbx6 bound to the promoters of more than three-quarters of the muscle genes examined. To test the action of this feed-forward loop comprising MyoD and Tbx6b in the regulation of muscle-specific gene expression, the expression patterns of genes under the control of this circuit were examined. Of the 155 genes under the direct control of MyoD and Tbx6b, 50 (including the above ten) were already known to be expressed in muscle cells. From the remaining 105 genes, 20 were randomly chosen, and 15 were found to be expressed in muscle cells, suggesting that this circuit is widely used for the regulation of genes expressed in muscle cells, and also that this circuit might not necessarily be sufficient for driving expression of the target (Kubo, 2010).

Brachyury is activated at the 64-cell stage exclusively in the notochord lineage, and this expression specifies the notochord fate. ZicL directly binds to the Brachyury promoter and activates its expression. It has also been shown that FoxD and FoxA-a are required for Brachyury expression, probably through regulating ZicL expression, and that FGF signaling is also required for Brachyury expression. The present assays showed that not only ZicL, but also FoxD binds to the Brachyury promoter. Although FoxD mRNA is not present in the notochord lineage at the 32-cell and 64-cell stages, when ZicL and Brachyury are activated, respectively (FoxD is expressed in the ancestors of cells in which ZicL and Brachyury are expressed), the ChIP assay indicated that FoxD binds to the promoters of ZicL and Brachyury. Because knockdown of FoxD eliminates ZicL and Brachyury expression and because the FoxD-GFP fusion protein exists in the notochord lineage at the 32-cell stage, it is likely that FoxD protein exists in these cells and binds to the promoters of ZicL and Brachyury when these two genes begin to be expressed (Kubo, 2010).

FoxA-a binding to the Brachyury promoter was not identified under 0.1% FDR. There was, however, a small peak that was counted as significant under 1% FDR. The possibility could not be ruled out that FoxA-a binds weakly to the Brachyury promoter. It is also possible that FoxA-a could bind weakly to a FoxD binding site because the FoxA-a binding peak coincided with that of FoxD. Even if this weak binding occurs in vivo, the regulation of Brachyury by FoxA-a would largely be achieved indirectly through FoxD and ZicL, since strong binding was found of FoxA-a to the promoters of FoxD and ZicL (Kubo, 2010).

Next, 14 non-regulatory genes were examined that are known to be expressed in the notochord under the control of Brachyury. Among them, 11 were identified here as direct targets of Brachyury. The present results suggest that the remaining three genes are regulated indirectly through a gene circuit under the control of Brachyury, although it cannot be ruled out that Brachyury binds to the regulatory elements of these three genes at later stages (Kubo, 2010).

The present study found many interactions between TFs and genomic DNA that were unexpected from preceding gene knockdown assays. Similar observations were also reported in preceding ChIP studies. To estimate what proportion of the binding makes a major contribution to gene regulation in Ciona embryos, MyoD mRNA or an MO against MyoD was injected into eggs, and their effects were analyzed on the expression of the same targets that were analyzed at the gastrula stage or at the tailbud stage, respectively. The mRNA levels of 14 targets, ten of which were expressed in muscle, were significantly increased (>2-fold) in embryos injected with MyoD mRNA, and MyoD MO injection significantly reduced the mRNA levels of three of these targets. The mRNA level of one target (KH.C12.38), which was weakly expressed in muscle at the tailbud stage, was significantly decreased in embryos injected with MyoD mRNA, whereas the mRNA level of one target (KH.C9.27), which was expressed in muscle at the gastrula stage, was significantly increased in embryos injected with the MyoD MO. In total, the mRNA levels of 16 targets were significantly altered by MyoD mRNA overexpression or gene suppression. The remaining four were not significantly affected, although three of these were expressed in muscle, implying that MyoD binding makes a relatively small contribution to activating these target genes. It was also found that eight of 15 Brachyury targets and seven of 12 Twist-like1 targets were significantly affected in the embryos by overexpression or knockdown of Brachyury or Twist-like1, respectively. Therefore, it is estimated that more than half of TF binding makes a major contribution to the regulatory control of gene expression (Kubo, 2010).

Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis

For over a century, muscle formation in the ascidian embryo has been representative of 'mosaic' development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, this study has identified the mechanisms through which Ciona Macho1 (Ci-Macho1, a divergent member of the Zic family) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and the cross-regulatory interactions have been unveiled between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). These representative examples were used to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos (Kugler, 2010).

Ci-macho1 postplasmic mRNA is relocated after fertilization by the cortical centrosome-attracting body (CAB). As cleavage proceeds, in both Halocynthia and Ciona Ci-macho1 mRNA becomes progressively restricted to a narrow region of the embryo, the B7.6 blastomeres; however, the Macho1 protein is generally believed to persist in an unlocalized form, and to be distributed to all descendants of the B4.1 cells. Studies in Halocynthia show that for the proper formation of other lineages that also derive from the B4.1 cells, such as mesenchyme and endoderm, the function of Macho1 needs to be actively suppressed by FGF and BMP signaling pathways. Similar mechanisms are also likely responsible for the functional suppression of zygotically expressed Ci-Macho1 in the Ciona CNS, considering that Ci-FGF16/19/20 is expressed in the Ciona CNS through tailbud stages and is required for neural development (Kugler, 2010).

Misexpression experiments described in this study suggest that no such restraining mechanism is present in notochord cells before the early tailbud stage. In fact, at early developmental stages the ectopic activation of both Ci-Tbx6b and Ci-Tbx6c was seen in notochord precursors of both lineages in Bra>macho embryos, whereas at the mid-tailbud stage only the ectopic activation of Ci-Tbx6b was observed, and it was confined to a subset of mesenchyme cells. These cells are most likely descendants of the B7.3 blastomere, a 64-cell stage precursor of both secondary notochord and mesenchyme cells. The differential competence of the notochord to respond to Ci-Macho1 might be explained by the requirement for temporally and spatially localized co-factors and/or transcriptional intermediaries. Alternatively, as in the case of the CNS, Ci-Macho1 might be functionally suppressed in the notochord of tailbud embryos by the activation of the FGF signaling pathway, as suggested by the observation that Ci-FGFR is expressed in the notochord beginning at the early tailbud stage. These mechanisms might also account for the relatively mild phenotype that was observed in embryos carrying the Bra>macho transgene, whereby the notochord is still able to form, even in transgenic embryos where mosaic incorporation is minimal (Kugler, 2010).

Using in vivo transient transgenic assays, a 2.4 kb CRM upstream of Ci-Tbx6b was identified that is able to faithfully recapitulate the muscle expression of this gene. The temporal muscle activity of the 2.4 kb CRM represents the composite read-out of early- and late-acting cis-regulatory sequences, which interpret maternal and zygotic information. The Ci-Tbx6b CRM contains a distal region which functions as the repository of the temporal information necessary to recapitulate the early expression pattern previously reported for Ci-Tbx6b. When this distal region is deleted, muscle activity is not lost, but its onset is considerably delayed. Sequence inspection and point-mutation analyses suggested that this early-acting distal region might be controlled by maternal Ci-Macho1, because three putative binding sites for this factor are present in this sequence. These sites were found to be bound in vitro by Macho1 and their concomitant mutation was found to be sufficient to cause the same delay in the onset of transcriptional activity that was observed when the entire fragment encompassing them was deleted. Together, these observations provide a mechanistic cis-regulatory explanation to the results of the misexpression assay, as well as to previous results showing that overexpression of Ci-Macho1 is sufficient to induce ectopic expression of Ci-Tbx6b and that, likewise, Hr-Macho-1 is able to ectopically induce Hr-Tbx6, among other muscle genes. It is noteworthy that in Ciona, Ci-ZicL cooperates with Ci-Macho1 to promote muscle development; this zygotic zinc-finger transcription factor is related to Ci-Macho1 and recognizes a similar consensus binding site in vitro. Interestingly, one of the three Ci-Macho1-binding sites that were characterized in the Ci-Tbx6b CRM, namely site 'C', contains permutations of the published ZicL consensus site that are compatible with binding in vitro. If this site is bound in vivo by either transcription factor, then this would explain the observation that Ci-Tbx6b is still weakly expressed in Ci-Macho1 morphant embryos, whereas its expression is no longer detectable in Ci-Macho1 and Ci-ZicL double-morphants (Kugler, 2010).

Within the 2.4 kb CRM, a 266 bp proximal region is able to direct transcription only from neurulation onwards, thus acting as a late muscle enhancer. Sequence analysis of this region revealed the presence of an imperfect CREB-binding site, a T-box-binding site (generic sequence: TNNCAC) partly matching the core consensus sequence previously reported for Ci-Tbx6b/c, and an 'AC'-core E-box. Both CREB-binding sites and AC-core E-boxes have been previously shown to be necessary for muscle activity of other muscle CRMs; however, in this case, only the T-box site substantially contributes to the muscle activity, qualitatively and quantitatively. Through EMSA, it was shown that this T-box site is bound in vitro by both Ci-Tbx6b and Ci-Tbx6c (Kugler, 2010).

Originally isolated in a subtractive screen aimed to identify genes downstream of Ci-Bra, the CiFCol1 gene attracted interest because of its sustained muscle expression, which begins around mid-gastrulation, and because its upstream region is enriched in T-box-binding sites (Kugler, 2010).

Dissection of a 2.2 kb genomic fragment located upstream of the transcription start site of CiFCol1 revealed the presence of discrete CRMs active in all the tissues where CiFCol1 is expressed. In particular, this 2.2 kb fragment harbors two distinct muscle CRMs: a distal CRM containing two generic E-boxes and depleted of T-box-binding sites and Ci-Macho1-binding sites, and a proximal CRM containing four clustered T-box-binding sites, some of which are bound weakly in vitro by the Ci-Tbx6b protein, and a low-affinity Ci-Macho1-binding site. The heterogeneity of these sequences is reflected by the temporal activity of the two CRMs, because the distal one, which does not contain any apparent T-box-binding sites, is activated later than the proximal one, which is enriched in these motifs. In particular, the distal CiFCol1 muscle CRM is active in a small subset of muscle precursors from the 110-cell stage to the neurula stage, and only by the early tailbud stage does its territory expand to encompass all muscle cells. Afterwards, it remains active in the majority of muscle cells. Therefore, the spatial range of action of this CRM in the muscle seems to be controlled by an activator(s) functioning from neurulation onwards. The presence of two E-boxes in this sequence prompted an investigation of the possible involvement of transcription factors of the bHLH family in the regulation of this CRM. It was found that neither mutation of the E-boxes nor misexpression, individual or combined, of two bHLH transcription factors, Ci-MRF and Ci-paraxis had any detectable effect, thus leaving the identification of the late activator(s) to future investigations (Kugler, 2010).

Conversely, the proximal CiFCol1 muscle CRM is ignited early in most muscle cell precursors, starting from the 32-cell stage, but its activity fades by the mid-tailbud stage. It is concluded that the additive activity of the two CRMs is probably responsible for the sustained expression of CiFCol1 in muscle cells (Kugler, 2010).

Interestingly, misexpression of Ci-Macho1, Ci-Tbx6b or Ci-Tbx6c in notochord cells all result in ectopic activation of CiFCol1 in this territory. Although it is not possible to rule out that this might be attributable to the low-affinity Ci-Macho1-binding site in the CiFCol1 early CRM, given the late onset of CiFCol1 muscle expression it seems more likely that Ci-Macho1 activates expression of CiFCol1 indirectly, through Ci-Tbx6b. To test this hypothesis the response of the CiFCol1 proximal muscle CRM to the misexpression of Ci-Tbx6b was monitored in notochord cells. It was found that misexpression of Ci-Tbx6b caused the ectopic activation of the CiFCol1 proximal muscle CRM in the notochord, whereas misexpression of Ci-Tbx6c did not have any effect. It is concluded that the ectopic activation of CiFCol1 seen in notochord cells of embryos carrying the Bra>Tbx6c construct might occur indirectly, via the activation of Ci-Tbx6b expression by Ci-Tbx6c (Kugler, 2010).

Finally, no ectopic activation was observed when the distal CiFCol1 muscle CRM was co-electroporated with either construct, consistent with the lack of Tbx6b/c-binding sites in its sequence (Kugler, 2010).

By analyzing the cis-regulatory sequences that mediate the response to Ci-Macho1 and its mediators, this study has begun to provide sharper insights into the molecular mechanisms controlling cell-autonomous muscle development in the ascidian embryo. Given the large number of genes that respond to Ci-Tbx6b and Ci-Tbx6c, it is conceivable that the mechanisms of transcriptional regulation that control the CRMs presented in this study might be shared by several other muscle genes. This hypothesis is supported by the abundance of putative Tbx6b/c-binding sites in muscle CRMs identified (Kugler, 2010).

Although the early cell-fate determination mediated by Macho-like proteins in muscle cells has been described so far as an ascidian-specific mechanism, transcription factors of the Zic family, of which Macho, ZicL and related proteins represent a diverged branch, are known to be required for shaping the body plan of widely different animals. In addition, Tbx6-related proteins in Ciona appear to be part of an evolutionarily conserved kernel that is employed for the specification and differentiation of paraxial mesoderm in several other chordates, including mouse, Xenopus and zebrafish. Hence, the elucidation of the cis-regulatory mechanisms used by these transcription factors to modulate expression of their target genes should provide insights on the inner workings of other model systems in which cis-regulatory elements are less tractable, including higher chordates (Kugler, 2010).

Differential temporal control of Foxa.a and Zic-r.b specifies brain versus notochord fate in the ascidian embryo

In embryos of an invertebrate chordate, Ciona intestinalis, two transcription factors, Foxa.a (see Drosophila Foxa) and Zic-r.b, (see Drosophila Odd-paired) are required for specification of the brain and the notochord, which are derived from distinct cell lineages. In the brain lineage, Foxa.a and Zic-r.b are expressed with no temporal overlap. In the notochord lineage, Foxa.a and Zic-r.b are expressed simultaneously. In the present study found that the temporally non-overlapping expression of Foxa.a and Zic-r.b in the brain lineage was regulated by three repressors, Prdm1-r.a and Prdm1-r.b ) (see Drosophila Hamlet) and Hes.a (see Drosophila Hairy). In morphant embryos of these three repressor genes, Foxa.a expression was not terminated at the normal time, in addition to precocious expression of Zic-r.b Consequently, Foxa.a and Zic-r.b were expressed simultaneously, which led to ectopic activation of Brachyury (see Drosophila Brachyury) and its downstream pathways for notochord differentiation. Thus, temporal controls by transcriptional repressors are essential for specifying the two distinct fates of brain and notochord by Foxa.a and Zic-r.b. Such a mechanism might enable the repeated use of a limited repertoire of transcription factors in developmental gene regulatory networks (Ikeda, 2016).

Zic2 is an enhancer-binding factor required for embryonic stem cell specification

The Zinc-finger protein of the cerebellum 2 (Zic2) is one of the vertebrate homologs of the Drosophila pair-rule gene odd-paired (opa). Molecular and biochemical studies demonstrate that Zic2 preferentially binds to transcriptional enhancers and is required for the regulation of gene expression in embryonic stem cells. Detailed genome-wide and molecular studies reveal that Zic2 can function with Mbd3/NuRD in regulating the chromatin state and transcriptional output of genes linked to differentiation. Zic2 is required for proper differentiation of embryonic stem cells (ESCs), similar to what has been previously reported for Mbd3/NuRD. This study identifies Zic2 as a key factor in the execution of transcriptional fine-tuning with Mbd3/NuRD in ESCs through interactions with enhancers. This study also points to the role of the Zic family of proteins as enhancer-specific binding factors functioning in development (Luo, 2015).

Zic proteins and neural crest

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).

Wnt signalling is required for neural crest (NC) induction; however, the direct targets of the Wnt pathway during NC induction remain unknown. This study shows that the homeobox gene Gbx2 is essential in this process and is directly activated by Wnt/beta-catenin signalling. By ChIP and transgenesis analysis it was shown that Gbx2 regulatory elements that drive expression in the NC respond directly to Wnt/beta-catenin signalling. Gbx2 has previously been implicated in posteriorization of the neural plate. This study unveils a new role for this gene in neural fold patterning. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. The NC specifier activity of Gbx2 is dependent on the interaction with Zic1 and the inhibition of preplacodal genes such as Six1. In addition, that Gbx2 is upstream of the neural fold specifiers Pax3 and Msx1. These results place Gbx2 as the earliest factor in the NC genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC specification. A new genetic cascade is proposed that operates in the distinction between anterior placodal and NC territories (Li, 2009).

Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Through a transcriptome analysis of frog microdissected neural border, this study identified an extended gene signature for the premigratory neural crest, and novel potential members of the regulatory network were defined. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, 25 Pax3 and Zic1 direct targets within this signature were uncovered. The neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). It was also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network (Plouhinec, 2014).

The intracellular domain of teneurin-2 has a nuclear function and represses zic-1-mediated transcription

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).

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, 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).

A role for Zic1 and Zic2 in Myf5 regulation and somite myogenesis

Zic genes encode a conserved family of zinc finger proteins with essential functions in neural development and axial skeletal patterning in the vertebrate embryo. Zic proteins also function as Gli co-factors in Hedgehog signaling. This study reports that Zic genes have a role in Myf5 regulation for epaxial somite myogenesis in the mouse embryo. In situ hybridization studies show that Zic1, 2, and 3 transcripts are expressed in Myf5-expressing epaxial myogenic progenitors in the dorsal medial dermomyotome of newly forming somites, and immunohistological studies show that Zic2 protein is co-localized with Myf5 and Pax3 in the dorsal medial lip of the dermomyotome, but is not expressed in the forming myotome. In functional reporter assays, Zic1 and Zic2, but not Zic3, potentiate the transactivation of Gli-dependent Myf5 epaxial somite-specific (ES) enhancer activity in 3T3 cells, and Zic1 activates endogenous Myf5 expression in 10T1/2 cells and in presomitic mesoderm explants. Zic2 also co-immunoprecipitates with Gli2, indicating that Zic2 forms complexes with Gli2 to promote Myf5 expression. Genetic studies show that, although Zic2 and Zic1 are activated normally in sonic hedgehog−/− mutant embryos, Myf5 expression in newly forming somites is deficient in both sonic hedgehog−/− and in Zic2kd/kd mutant mouse embryos, providing further evidence that these Zic genes are upstream regulators of Hedgehog-mediated Myf5 activation. Myf5 activation in newly forming somites is delayed in Zic2 mutant embryos until the time of Zic1 activation, and both Zic2 and Myf5 require noggin for their activation (Pan, 2011).

Vertebrate Zic genes, neurogenesis and neural development

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 medial telencephalon is a source of neurons that follow distinct tangential trajectories of migration to various structures such as the cerebral cortex, striatum, and olfactory bulb. This study characterized the forebrain anomalies in Zic1/Zic3 compound mutant mice. Zic1 and Zic3 were strongly expressed in the medial structures, including the septum, medial cerebral cortex, and choroid plexus. Mice homozygous for the Zic1 mutant allele together with the null Zic3 allele showed medial forebrain defects, which were not obvious in either Zic1 or Zic3 single mutants. Absence of both Zic1 and Zic3 caused hypoplasia of the hippocampus, septum, and olfactory bulb. Analysis of the cell cycle revealed that the cell cycle exit rate was increased in the septa of double mutants. Misexpression of Zic3 in the ventricular layer of the cerebral cortex inhibited neuronal differentiation. These results indicated that both Zic1 and Zic3 function in maintaining neural precursor cells in an undifferentiated state. The functions of these genes may be essential to increasing neural cell numbers regionally in the medial telencephalon and to proper mediolateral patterning of the telencephalon (Inoue, 2007).

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).

During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. This study has established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown it was showm that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, it was found that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. It was further demonstrated that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, it was shown that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and evidence is provided that these boundary signals are also required for ventricle morphogenesis. In summary, it is concluded that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain (Elsen, 2008).

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).

During neurulation, vertebrate embryos form a neural tube (NT), the rudiment of the central nervous system. In mammals and birds, a key step in cranial NT morphogenesis is dorsolateral hinge-point (DLHP) bending, which requires an apical actomyosin network. The mechanism of DLHP formation is poorly understood, although several essential genes have been identified, among them Zic2, which encodes a zinc-finger transcription factor. DLHP formation in the zebrafish midbrain was found to requires actomyosin and Zic function. Given this conservation, the zebrafish was used to study how genes encoding Zic proteins regulate DLHP formation. It was demonstrated that the ventral zic2a expression border predicts DLHP position. Using morpholino (MO) knockdown, it was shown that zic2a and zic5 are required for apical F-actin and active myosin II localization and junction integrity. Furthermore, myosin II activity can function upstream of junction integrity during DLHP formation, and canonical Wnt signaling, an activator of zic gene transcription, is necessary for apical active myosin II localization, junction integrity and DLHP formation. It is concluded that zic genes act downstream of Wnt signaling to control cytoskeletal organization, and possibly adhesion, during neurulation. This study identifies zic2a and zic5 as crucial players in the genetic network linking patterned gene expression to morphogenetic changes during neurulation, and strengthens the utility of the zebrafish midbrain as a NT morphogenesis model (Nyholm, 2009).

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. This study demonstrates that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. This study shows that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance the understanding of a key gene regulatory network that, when disrupted, causes HPE (Sanek, 2009).

Zic2-dependent axon midline avoidance controls the formation of major ipsilateral tracts in the CNS

In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. This study demonstrates that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, these data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways (Escalante, 2013).

Vertebrate Zic genes and eye development

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).

Zebrafish zic2a patterns the forebrain through modulation of Hedgehog-activated gene expression

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. This study demonstrates that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. This study shows that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance understanding of a key gene regulatory network that, when disrupted, causes HPE (Sanek, 2009).

Vertebrate Zic genes: Transcriptional targets

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).

Zic-associated holoprosencephaly

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).

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).

Holoprosencephaly (HPE) is the most frequently observed human embryonic forebrain defect. Recent evidence indicates that the two major forms of HPE, classic HPE and midline interhemispheric (MIH) HPE, are elicited by two different mechanisms. The only gene known to be associated with both forms of HPE is Zic2. The zebrafish Danio rerio was used as a model system to study Zic knockdown during midline formation by looking at the close homolog Zic1, which is expressed in an overlapping fashion with Zic2. Zic1 knockdown in zebrafish leads to a strong midline defect including partial cyclopia due to attenuated Nodal and Hedgehog signaling in the anterior ventral diencephalon. Strikingly, it was not possible to show that Zic1 is also required for maintaining early forebrain expression of the retinoic acid (RA)-degrading enzyme cyp26a1. Zic1 LOF leads to increased RA levels in the forebrain, subsequent ventralization of the optic vesicle and down-regulation of genes involved in dorsal BMP signaling. Repression of BMP signaling in dorsal forebrain has been implicated in causing MIH HPE. This work provides a mechanistical explanation at the molecular level of why Zic factors are associated with both major forms of HPE (Maurus, 2009).

Multiple developmental programs are altered by loss of Zic1 and Zic4 to cause Dandy-Walker malformation cerebellar pathogenesis

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. This study shows that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, it was shown that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1+/-;Zic4+/- background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1-/-;Zic4-/- mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlate with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype is Shh independent. In Zic1+/-;Zic4+/-;Shh+/-, normal cerebellar anlage patterning and foliation was observed. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, these data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM (Blank, 2011).


odd-paired: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.


The Interactive Fly resides on the
Society for Developmental Biology's Web server.