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Results for “Homeobox Protein SIX3”

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At least 19 recordsLinked to original sources

PubMed2001

The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation.

Six3 is a vertebrate homeobox gene that is expressed in the anterior neural plate and eye anlage. We overexpressed a dominant transcriptional activator or repressor form of Six3 in zebrafish embryos to analyze their effect on eye and forebrain formation. RNA injection of the activator form of Six3 into zebrafish embryos caused reduction of the expression domains for rx2, pax2, and emx1 in the anterior neural plate, resulting in eye and forebrain hypoplasia. On the other hand, overexpression of the repressor form of Six3 or wild-type Six3 showed phenotypes opposite to those of the activator form. We found that Six3 has eh1-related motifs, motifs crucial for transcriptional repression function of Drosophila engrailed which plays a role in tethering the Groucho corepressor to the promoters. We isolated one of the zebrafish Groucho family genes, grg3, and demonstrated an interaction between Six3 and Grg3 using yeast two-hybrid analysis. Point-mutations in the eh1-related motifs in Six3 reduced both its eye and forebrain enlarging activities and its interaction with Grg3. These results strongly argue that Six3 functions as a Groucho-dependent repressor in eye and forebrain formation. Furthermore, zebrafish Six2 and Six4 also interacted with Grg3, implying a conserved function among the Six family proteins as transcriptional repressors.

Amino Acid Motifs↗

PubMed2001

The orphan nuclear receptor NOR-1 interacts with the homeobox containing protein Six3.

Neuron-derived orphan receptor 1 (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily. In order to identify cofactors that associate with NOR-1 in the fetal forebrain, we tested a yeast two-hybrid system with the NOR-1 cDNA fragment lacking a transactivating domain as a bait. By screening of the rat fetal brain embryonic day 17 library, a rat homologue of Six3 was identified as an associated protein. We demonstrated that NOR-1 interacted with Six3 in yeast and in vitro, and the association was required for the DNA binding and AF2 domains of NOR-1. Regarding the other members of the family (NGFI-B and RNR-1), association with Six3 was not observed in yeast. In addition, cotransfection experiments with Six3 and NOR-1 indicated that Six3 had a negative activity against the transactivation by NOR-1 through the NBRE response element in a dose-dependent manner. The overlap in expression of NOR-1 and Six3 was mainly detected in the rat fetal forebrain on embryonic day 18. Thereafter, the expression of both genes diminished rapidly. These results suggest that a dimer consisting of a homeobox containing protein Six3 and transcriptional factor NOR-1 might regulate gene expression during the late stage of the fetal forebrain development. This study provides, after the association of Ftz and Ftz-F1 in Drosophila, another example of a dimer formation of a homeobox protein and an orphan nuclear receptor.

Amino Acid Sequence↗

PubMedDate not supplied

[Expression of regulatory homeobox genes during retina regeneration in adult newts].

We studied molecular-genetic mechanisms of retina regeneration in amphibians and, specifically, expression of the homeobox genes Pax6, Prox1, and Six3 in normal development and during retina regeneration in the newt. Based on the structural analysis of genes in closely related amphibian species, primers were constructed that flank certain regions of these genes. PCR fragments of calculated length were obtained. The affiliation of PCR products to the above genes was confirmed by sequencing. A comparative PCR analysis of expression of Pax6, Prox1, and Six3 was carried out in the native and regenerating newt retina, which allowed estimation of the level of expression. cDNA libraries obtained from the native and regenerating retina were used as templates. The libraries were preliminary standardized according to glyceraldehydes-3-phosphate dehydrogenase, an enzyme of general cell metabolism. The genes we studied were expressed in both native and regenerating retina. The level of Pax6 and Prox1 expression increased during regeneration, while that of Six3 decreased. The decrease in the level of Six3 expression could be due to antagonistic interrelations of Prox1 and Six3. The changed level of Prox1 and Six3 expression is a new fact and requires further studies. The interactions between these and other regulatory genes and localization of their expression in the cells of native and regenerating retina will be studied using in situ hybridization and immunohistochemistry.

Age Factors↗

PubMed2001

Regulation of the human SIX3 gene promoter.

A 2-kb promoter fragment of SIX3, a human transcription factor essential for vertebrate eye development, has been characterized in a gene reporter assay system. The peak of activity implies the 2-kb sequence of SIX3, whereas 5'-deletion constructs of the promoter decreases successively to 60% of the activity starting from the entire promoter. In contrast, cutting off 300 bp of the 3' promoter extinguishes its activity completely. Coexpression experiments of different other transcription factors illuminate the regulation of SIX3 during eye development: Pax6 activates the -703/-349 SIX3 promoter threefold, and PROX1 even eightfold. In contrast, Msx2 represses the entire SIX3 promoter. Furthermore, Six3 is regulated by its own negative feedback loop. In conclusion, SIX3 expression underlies a complex regulation, which is an important part to understand the network of transcription factors during eye development.

Cells, Cultured↗

PubMed2001

Antagonistic action of Six3 and Prox1 at the gamma-crystallin promoter.

Gamma-crystallin genes are specifically expressed in the eye lens. Their promoters constitute excellent models to analyse tissue-specific gene expression. We investigated murine CRYGE/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37. Computer analysis predicts homeodomain and paired-domain binding sites for all rodent CRYGD/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of CRYGE/f promoter activity was found for PROX1. In contrast, PROX1 stimulated the CRYGF promoter 10-fold in CD5A cells without endogenous PROX1. In both cell lines Six3 repressed the CRYGF promoter to 10% of its basal activity. Our cell transfection experiments indicated that CRYG expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the CRYGD/e/f promoters. Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174. Since Prox1 and Six3 are present at the beginning of lens development, expression of CRYGD/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development.

3T3 Cells↗

PubMed2005

Agonistic and antagonistic action of AP2, Msx2, Pax6, Prox1 AND Six3 in the regulation of Sox2 expression.

Sox2 transcription factor is expressed in neural tissues and sensory epithelia from the early stages of development. Particularly, it is known to activate crystallin gene expression and to be involved in differentiation of lens and neural tissues. However, its place in the signaling cascade is not well understood. Here, we report about the response of its promoter to the presence of other transcription factors, AP2alpha, Msx2, Pax6, Prox1 and Six3, in a transient reporter gene assay using HEK293 cells as recipient cells. Taking our data together, AP2, Pax6 and PROX1 can activate the Sox2 promoter. Msx2 has an inhibitory effect, whereas Six3 does not affect the Sox2 promoter. These data indicate a common activating cascade at least for AP2, Pax6, Prox1 and Sox2.

Blotting, Western↗

PubMedDate not supplied

Regulation of proliferation, cell fate specification and differentiation by the homeodomain proteins Prox1, Six3, and Chx10 in the developing retina.

The coordination of proliferation and cell fate specification is of particular importance in the developing central nervous system, because different classes of neurons are generated at different stages of development. As a consequence, if too many or too few multipotent progenitor cells exit the cell cycle during the early stages of development, then a predictable shift in the ratio of neuronal cell types would occur. Such a perturbation could prove devastating for normal neural function. In the past several years, we have made numerous advances toward understanding the roles of cell cycle proteins in the development of the retina, a specialized region of the central nervous system. More recently, researchers have identified and characterized a number of homeodomain transcription factors that regulate the proliferation of retinal progenitor cells and the specification of cell fate. In this review, I will present recent findings about the homeodomain protein Prox1 and compare them with studies of two other homeodomain proteins that are important regulators of retinal development, Chx10 and Six3. Together, the research on the genes that encode these three proteins raises several new questions about the mechanisms underlying the coordination of proliferation and cell fate specification in the developing central nervous system.

Animals↗

PubMed2003

Cooperative action between L-Maf and Sox2 on delta-crystallin gene expression during chick lens development.

Lens development is regulated by a variety of transcription factors with distinct properties. The lens-specific transcription factor, L-Maf, is essential for lens formation and induces lens-specific markers, such as the crystallin genes. In this study, we analyzed the mechanism by which L-Maf regulates delta-crystallin expression. Misexpression of L-Maf in the head ectoderm of lens placode-forming embryos by in ovo electroporation induced delta-crystallin only in the region surrounding the lens. To define this restricted expression, we misexpressed L-Maf together with other transcription factors implicated in delta-crystallin expression. Sox2 plus L-Maf expanded the delta-crystallin-inducible domain to the entire head ectoderm and simultaneously increased the quantity of delta-crystallin mRNA expressed. In contrast, co-expression of L-Maf with other factors such as Pax6, Six3 and Prox1 had little or no effect on delta-crystallin. We also observed that L-Maf and Sox2 cooperatively enhanced the transactivation of a reporter gene bearing the delta-crystallin enhancer in ovo, implying that L-Maf and Sox2 can induce delta-crystallin through the same enhancer. In conclusion, we report here that L-Maf and Sox2 cooperatively regulate the expression of delta-crystallin during chick lens development.

Animals↗

PubMed2005

Transcriptional control of early tract formation in the embryonic chick midbrain.

The earliest step in establishing the complex neuronal networks in the vertebrate brain is the formation of a scaffold of axon tracts. How the formation of the early axon scaffold is controlled at the molecular level is unclear. Forming part of the scaffold, neurons located at the ventral midbrain-forebrain border (MFB) give rise to the medial longitudinal fascicle (mlf) and the posterior commissure (pc). We demonstrate that the homeobox genes Sax1, Six3, Emx2 and Pax6 are expressed in distinct domains in this area, suggesting that the specification of mlf and pc neurons might be controlled by the combinatorial activity of these transcription factors. We have tested this hypothesis by analysing the function of Sax1 in the embryonic chick brain. Gain-of-function experiments with Sax1 result in alterations to the early axon scaffold, most prominently an enlargement of the mlf at the expense of the pc. Ectopic expression of Sax1 also affects the expression of other ventral homeobox genes, particularly Six3 and Emx2. Our results indicate that the specification of neurons forming the early axon scaffold is governed by a homeobox code, thus resembling the mechanism of neuronal specification in the spinal cord.

Animals↗

PubMed1999

Sequence and location of SIX3, a homeobox gene expressed in the human eye.

Mouse Six3 is a homeobox gene expressed almost exclusively in the developing retina, lens, hypothalamus, and pituitary. It belongs to the same family as sine oculis, a Drosophila regulatory gene that encodes a transcription factor essential for eye development. The optix gene is its closest known Drosophila homologue, with a homeodomain that is 95% identical in sequence to the Six3 protein. We have isolated the homologous human gene, SIX3, which is expressed in the adult retina and encodes a 332 amino acid protein that is 98% identical to its mouse counterpart. The SIX3 protein coding region is interrupted by a single intron located just downstream of the homeobox. A surprising feature of the SIX3 gene is a 533 nucleotide 5' untranslated region that contains long polypyrimidine tracts with 96% identity to mouse Six3. We have used in-situ hybridization to map SIX3 to 2p21-p22, a site that is syntenic with the Six3 region of mouse chromosome 17. Large heterozygous deletions associated with human holoprosencephaly type 2 have been previously mapped to 2p21, opening the possibility that SIX3 could be involved in the development of midline structures of the head. Alternatively, the expression pattern of mouse Six3 suggests that human SIX3 could be involved in disorders of eye and pituitary development.

Adult↗

PubMed1999

Six class homeobox genes in drosophila belong to three distinct families and are involved in head development.

The vertebrate Six genes are homologues of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Here we describe two new Six genes in Drosophila, D-Six3 and D-Six4, which encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, we report the partial sequences of 12 Six gene homologues from several lower vertebrates and show that the class of Six proteins can be subdivided into three major families, each including one Drosophila member. Similar to so, both D-Six3 and D-Six4 are initially expressed at the blastoderm stage in narrow regions of the prospective head and during later stages in specific groups of head midline neurectodermal cells. D-Six3 may also be essential for development of the clypeolabrum and several head sensory organs. Thus, the major function of the ancestral Six gene probably involved specification of neural structures in the cephalic region.

Amino Acid Sequence↗

PubMed1998

Transient expression of a novel Six3-related zebrafish gene during gastrulation and eye formation.

Both the Drosophila homeobox gene sine oculis and its murine homologue Six3 have regulatory functions in eye development. In zebrafish, in addition to two previously reported homologues of murine Six3, we have identified a related gene (six7). Although the deduced Six7 protein shares less than 68% sequence identity with the other known zebrafish Six3-like proteins, the embryonic expression patterns have highly conserved features. The six7 transcripts are first detected in involuting axial mesendoderm and, subsequently, in the overlying neurectoderm from which the forebrain and optic primordia develop. Similar to the two other zebrafish Six3 homologues, the expression boundaries of six7 correspond quite closely with the edges of the optic vesicles. Hence, the partially overlapping expression domains of these three six genes probably contribute to anteroposterior specification and in defining the eye primordia.

Amino Acid Sequence↗

PubMed1996

Ectopic lens induction in fish in response to the murine homeobox gene Six3.

Recent findings show an unexpected conservation of genes involved in vertebrate and insect eye development. The Drosophila homeobox gene sine oculis is crucial for eye development. Its murine homologue, Six3 is expressed in the anterior neural plate, a region which is involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. Our findings suggest a conserved function for Six3 in metazoan eye development.

Animals↗

PubMed2002

A screen for co-factors of Six3.

The vertebrate Six3 gene a homeobox gene of the Six-family, plays a crucial role in early eye and forebrain development. Here we report the isolation of candidate factors that interact with Six3 in a yeast two-hybrid screen. Among these are two basic helix loop helix (bHLH) domain containing proteins. Biochemical analysis reveals that the bHLH proteins ATH5, ATH3, NEUROD as well as ASH1 interact specifically with XSix3. By defining the interacting domains we show that the bHLH domain of NEUROD interacts with the SIX domain of XSix3. The co-expression of the interacting molecules during late retina determination/differentiation suggests a new role for Six3 and the respective interaction partner also in these late steps of eye development.

Animals↗

PubMed1998

Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia.

The murine homeobox gene Six3 and its Drosophila homologue sine oculis both have regulatory functions in eye development. We report the isolation and characterization of two zebrafish genes, six3 and six6, that are closely related to the murine Six3 gene. Zebrafish six3 may be the structural orthologue, while the six6 gene is more similar with respect to embryonic expression. Transcripts of both zebrafish six genes are first detected in involuting axial mesendoderm and, subsequently, in the overlying anterior neural plate from which the optic vesicles and the forebrain will develop. Direct correspondence between six3/six6 expression boundaries and the optic vesicles indicate essential roles in defining the eye primordia. During later stages only the six6 gene displays similar features of expression in the eyes and rostral brain as reported previously for murine Six3.

Amino Acid Sequence↗

PubMed2003

Retinal expression of zebrafish six3.1 and its regulation by Pax6.

Homologues of the homeobox genes sine oculis (so) and eyeless (ey) are important regulators of eye development in both vertebrates and invertebrates. A Drosophila paralogue of so, optix, is an orthologue of the vertebrate Six3 gene family. Our analysis of zebrafish six3.1 demonstrated retinal expression in two separate cell layers and the ciliary marginal zone. This pattern is consistent with the observations of Six3 in other vertebrates and indicates functional conservation. We studied the 5(') flanking region of six3.1 and showed that separate enhancing elements are required for expression at different stages of eye development. This analysis also revealed specific binding of zebrafish Pax6.1 protein to an element required for six3.1 expression in ganglion cells. Furthermore, an enhancement of six3.1 transcription by Pax6.1 was observed by co-injection experiments. These results provide evidence for a direct regulatory interaction between vertebrate Pax6 and Six3 genes in eye development.

Animals↗

PubMed1998

Overexpression of the forebrain-specific homeobox gene six3 induces rostral forebrain enlargement in zebrafish.

The Drosophila homeobox gene sine oculis is expressed in the rostral region of the embryo in early development and is essential for eye and brain formation. Its murine homolog, Six3, is expressed in the anterior neural plate and eye anlage, and may have crucial functions in eye and brain development. In this study, we describe the cloning and expression of zebrafish six3, the apparent ortholog of the mouse Six3 gene. Zebrafish six3 transcripts are first seen in hypoblast cells in early gastrula embryos and are found in the anterior axial mesendoderm through gastrulation. six3 expression in the head ectoderm begins at late gastrula. Throughout the segmentation period, six3 is expressed in the rostral region of the prospective forebrain. Overexpression of six3 in zebrafish embryos induced enlargement of the rostral forebrain, enhanced expression of pax2 in the optic stalk and led to a general disorganization of the brain. Disruption of either the Six domain or the homeodomain abolish these effects, implying that these domains are essential for six3 gene function. Our results suggest that the vertebrate Six3 genes are involved in the formation of the rostral forebrain.

Amino Acid Sequence↗

PubMed2005

Lhx2 mediates the activity of Six3 in zebrafish forebrain growth.

The telencephalon shows the greatest degree of size variation in the vertebrate brain. Understanding the genetic cascade that regulates telencephalon growth is crucial to our understanding of how evolution of the normal human brain has supported such a variation in size. Here, we present a simple and quick approach to analyze this cascade that combines caged-mRNA technology and the use of antisense morpholino oligonucleotides in zebrafish embryos. Lhx2, a LIM-homeodomain protein, and Six3s (Six3b and Six3a), another homeodomain proteins, show very similar expression patterns early in forebrain development, and these are known to be involved in the growth of this part of the brain. The telencephalon of six3b and six3a double morphant (six3 morphant) embryos is markedly reduced in size due to impaired cellular proliferation. Head-specific overexpression of Lhx2 by photoactivation of a caged-lhx2 mRNA completely rescued this size reduction, whereas similar head-specific activation of Six3b could not rescue the knockdown effect of lhx2. In the forebrain of medaka embryos, Six3 facilitates cellular proliferation by sequestration of Geminin from Cdt1, a key component in the assembly of the prereplication complex. Our results suggest that Lhx2 may mediate an alternative or parallel pathway for control of cellular proliferation in the developing forebrain via Six3.

Animals↗