Five cDNA clones of the Six gene family have been identified, all of which are expressed in retina. They are Six2, Six3 alpha and Six3 beta (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity between one another in the Sina oculis-homologous region (Six domain and homeodomain) but they differ greatly in structure in some other regions. The amino acid sequence similarity of the Sina oculis-homologous region to the previously identified AREC3/Six4 is 70.1% for Six2; 57.3% for Six3 alpha and Six3 beta, and 70.3% for Six5. The expression of these genes is observed in the inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina. The So-homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC3/Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes (Kawakami, 1996).
The murine homeobox gene Six3 carries out regulatory functions in eye development. Two isolated and characterized zebrafish genes, six3 and six6, are closely related to the murine Six3 gene. Zebrafish six3 may be the structural ortholog, while the six6 gene (identical to Kobayashi's [1998] six3) 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 (Seo, 1998a).
In zebrafish, in addition to two previously reported homologs of murine Six3, a related gene (six7) has been identified. 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 homologs, 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 (Seo, 1998b).
Zebrafish six3 is 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 induces enlargement of the rostral forebrain, enhances expression of pax2 in the optic stalk and leads to a general disorganization of the brain. Disruption of either the Six domain or the homeodomain abolishes these effects, implying that these domains are essential for six3 gene function. These results suggest that the vertebrate Six3 genes are involved in the formation of the rostral forebrain (Kobayashi, 1998).
Six3 is expressed in the anterior neural plate and optic vesicles, lens, olfactory placodes and ventral forebrain. Overexpression of mouse Six3 gene in medaka fish embryos (Orvzias latipes) results in the formation of an ectopic lens, indicating that Six3 activity can trigger the genetic pathway leading to lens formation. The medaka Six3 homolog has now been isolated and its expression pattern analyzed in the medaka embryo. Medaka Six3 is phylogenetically quite distantly related to zebrafish six3/six6 (similar to zebrafish six7) (Seo, 1999). It is expressed initially in the anterior embryonic shield and later in the developing eye and prosencephalon. The early localized expression of medaka Six3 suggests a role in the regionalization of the rostral head (Loosli, 1998).
Xenopus XOptx2 (a Six3 subclass member) is a new member of the Six/sine oculis family. A characteristic distinction between the Six3 and Optx2 proteins is the length of the pre-Six domain region. All known Optx2 proteins are smaller at the amino terminus than all known Six3 proteins. XOptx mRNA expression is first detected in stage 14 embryos. At stage 15, XOptx2 is detected as a single band of expression at the most anterior edge of the developing neural plate. At approximately stage 17, expression extends laterally. By neural groove stage, this single band of expression separates into two distinct regions consistent with the location of the eye fields. As the protrusion of the eyes begins to become distinct (stage 20 to 22), XOptx2 expression appears restricted to the eyes. At stage 25, XOptx2 is also detected in the pineal gland primordia and the ventral forebrain. Expression continues to be detected in the eyes, the maturing pineal gland, and the ventral forebrain of tailbud embryos (Zuber, 1999).
Overexpression of XOptx2, a member of the family, in the Xenopus embryonic eye field results in a dramatic increase in eye size. A hybrid protein containing the repressor domain of Engrailed (XOptx2-Engrailed repressor) gives a similar phenotype, while a XOptx2-VP16 activator hybrid protein reduces eye size. XOptx2 stimulates bromodeoxyuridine incorporation, and XOptx2-induced eye enlargement is dependent on cellular proliferation. Moreover, retinoblasts transfected with XOptx2 produce clones of cells approximately twice as large as control clones. Pax6, which does not increase eye size on its own, acts synergistically with XOptx2. These results suggest that XOptx2, in combination with other genes expressed in the eye field, is crucially involved in the proliferative state of retinoblasts and thereby the size of the eye (Zuber, 1999).
Two distinct but nonexclusive mechanisms could explain the XOptx2-dependent increase in eye size. (1) Uncommitted cells not destined to be eye cells might be induced to change their fate to the eye cell lineage. This kind of cell fate conversion is common with overexpression-induced enlargements of the nervous system in Xenopus embryos. (2) Cells already destined to form an eye may have increased mitotic activity and therefore an increase in the number of cells in the optic vesicle and eye proper. One way to determine if XOptx2 causes extra cell divisions in eye field cells is the analysis of cell number in clones overexpressing this gene. Retinal cells of stage 17 embryos were cotransfected in vivo with either XOptx2 DNA and the tracer GFP or vector only and GFP. Transfected cells were detected by fluorescence in cryostat-sectioned eyes of stage 41 embryos. Although it could not be confirmed that single cells were initially transfected, there is reason to suspect that such clusters have clonal origins: (1) the clusters tended to be compact, oriented in vertical columns, and contain all cell types, as is found when single cells are injected with fluorescent or enzymatic lineage tracers; and (2) when low doses of DNA are injected, about half the eyes have no transfected cells, implying a Poisson distribution of hits, yet the average number of cells in a cluster does not decrease when the hit frequency is very low. To increase the probability that true clones were examined, the amount of DNA lipofected was minimalized so that most retinas contained either no transfected cells or single 'clones'. In addition, when more than one cluster was detected in transfected retinas, they were scored only if the clusters were well separated. Interestingly, XOptx2 has no effect on the relative proportion of cell types generated from transfected cells in these clones. This result implies that XOptx2 does not influence retinal cell fate, per se. However, XOptx2 nearly doubles the number of GFP-positive cells in retinal clones. The average clone size in XOptx2-transfected retinas is 16.1 cells per retinal section, while the cell number observed in control-expressing clones is 10.4. These results indicate that XOptx2 induces proliferation (Zuber, 1999).
Reported here is the cloning and developmental pattern of expression of xSix3, the Xenopus laevis homolog of Six3. In addition, the known sequences of vertebrate Six3 genes have been compared. xSix3 is very homologous to Six3 in other vertebrates in terms of amino acid sequence. The reported developmental pattern of expression of Six3 in chick and mouse includes not only the developing eyes and the ventral diencephalic tissue between them, but also a large, sagittally-oriented telencephalic region. The distribution of xSix3, however, is virtually restricted to the eyes and ventral diencephalon, showing only a very small territory of expression in the telencephalon (Zhou, 2000).
cSix3 is a chick homolog of the murine Six3. cSix3 transcripts are expressed from presomitic stages in the most anterior portion of the neural plate. As the neural tube folds and the optic vesicles evaginate, cSix3 is expressed in the optic vesicle and the rostroventral forebrain. At later stages, cSix3 is found in most of the structures derived from the anterior neural plate, i.e. olfactory epithelium, septum, adenohypophysis, hypothalamus and preoptic areas. During eye development, cSix3 expression is first found in the entire optic vesicle and the overlying ectoderm but soon becomes restricted to the prospective neural retina and to the lens placode. In the developing neural retina, cSix3 is expressed in the entire undifferentiated neuroepithelium but is rapidly downregulated, first in the postmitotic photoreceptors and later in the majority of retinal ganglion cells (Bovolenta, 1998).
A detailed expression analysis in chick and mouse of Six9 (Optx2), genes of the Six/sine oculis family closely related to Six3, is reported. Six9 (Optx2) is first expressed at presomitic stages in the head-fold, both in the neural plate and in the underlying axial mesoderm. Thereafter, Six9 (Optx2) is strongly expressed in the presumptive and differentiating neural retina and ventral optic stalk, in the olfactory placodes, in the hypothalamus and in the pituitary gland. This expression pattern largely overlaps with that of Six3, but several differences exist between the expression domain of the two genes. At presomitic stages, the posterior boundary of Six3 expression is at the same axial level both in the prechordal plate and in the overlying neural plate. In contrast, Six9 (Optx2) expression in the prechordal plate extends more caudal to that of the neural plate, occupying a more restricted V-shaped territory. Similarly, during the early events of eye patterning, Six3 is first expressed in the entire optic vesicle and lens placode. Only later does its expression become confined to the prospective and differentiating neural retina. Conversely, Six9 (Optx2) is never observed in the lens placode of either chick and mouse, and from early stages of optic vesicle development, Six9 (Optx2) transcripts are restrained to the prospective ventral neural retina and optic stalks (L pez-Rios, 1999).
A vertebrate member of the so/Six gene family, Six3, is expressed in the developing eye and forebrain. Injection of Six3 RNA into medaka fish embryos causes ectopic Pax6 and Rx2 expression in midbrain and cerebellum, resulting in the formation of retinal primordia at ectopic locations in the midbrain and prospective cerebellum, involving a regulatory interaction of Six3 and Pax6. Similar to the wild-type situation in the developing eye, Pax6 and Six3 are expressed in the region where the ectopic retinal primordia will subsequently form. These ectopic retinal primordia have the potential to develop into optic cups, as visualized by morphology and marker gene expression. The higher frequency of ectopic retinal primordia at early somitogenesis stages, as compared to ectopic optic cups formed at the 34-somite stage, indicates that not all ectopic retinal primordia develop into an optic cup. Thus, Six3 initiates, but does not fully implement, later stages of retinal development. Injected mouse Six3 RNA initiates ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation reveals a pivotal role for Six3 in vertebrate retina development and hints at a conserved regulatory network underlying vertebrate and invertebrate eye development (Loosli, 1999).
A murine homeobox-containing gene, Six6 (Optx2), has been isolated that shows extended identity in its coding region with Six3, the only member of the mammalian Six gene family known to be expressed in the optic primordium. Phylogenetic analysis demonstrates that Six6 and Six3 belong to a separate group of homeobox-genes that are closely related to the recently identified Drosophila Optix. Earliest Six6 expression is detected in the floor of the diencephalic portion of the primitive forebrain, a region predicted to give rise to the neurohypophysis and to the hypothalamus. Later on, Six6 mRNA is found in the primordial tissues giving rise to the mature pituitary: the Rathke's pouch and the infundibular recess. In the optic primordium, Six6 demarcates the presumptive ventral optic stalk and the ventral portion of the future neural retina. In the developing eye, Six6 expression is detected in the neural retina, the optic chiasma and optic stalk, but not in the lens. When compared to Six6, Six3 expression pattern is highly similar, but with a generally broader transcript distribution in the brain and in the visual system. Six6 does not require Pax6 for its expression in the optic primordium, suggesting that Six6 acts on a parallel and/or independent pathway with Pax6 in the genetic cascade governing early development of the eye (Jean, 1999).
Six3 from mice is now included in the new Six/sine oculis subclass of homeobox genes. Early in development Six3 expression is restricted to the anterior neural plate including areas that will later give rise to ectodermal and neural derivatives. Later, once the longitudinal axis of the brain bends, Six3 mRNA is also found in structures derived from the anterior neural plate: the ectoderm of the nasal cavity, the olfactory placode, Rathke's pouch, and also the ventral forebrain, including the region of the optic recess, hypothalamus and optic vesicles. Based on this expression pattern, Six3 appears to be one of the most anterior homeobox genes reported to date. The high sequence similarity of Six3 with Drosophila sine oculis, and its expression during eye development, suggests that this gene is the likely murine homolog. Mammals and insects share control genes such as eyeless/Pax6 and also possibly other members of the regulatory cascade required for eye morphogenesis. In Small eye (Pax6) mouse mutants, Six3 expression is not affected (Oliver, 1995).
Murine Six3 is expressed in the anterior neural plate, a region 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. These findings suggest a conserved function for Six3 in metazoan eye development (Oliver, 1996).
Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type (WT) cells in the brain, the effects of Otx on gene expression were analyzed (Rhinn, 1999).
Mutant cells result in a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2, while expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. In the forebrain, Otx2 is required to activate the expression of the homeobox gene Rpx and maintain the expression of another homeobox gene, Six3. To determine if Otx2 is required cell autonomously or non-cell autonomously to regulate expression of these genes, the forebrain of moderate chimeric embryos was analyzed in double-labelling experiments, using histochemical staining for beta-galactosidase activity to distinguish WT from Otx2 mutant cells, and whole-mount RNA in situ hybridization to characterize Rpx or Six3 expression. Rpx is expressed in the forebrain of control embryos at E8.5. In moderate chimeras, Rpx expression is absent from the patches of Otx2 mutant cells, but is present in the surrounding WT forebrain cells. At the border of the mutant cell patches, Otx2 mutant cells fail to express Rpx while neighboring WT forebrain cells maintain expression of the gene. The strict correlation at the cellular level between lack of Otx2 activity and loss of Rpx expression demonstrates that Otx2 is required cell autonomously for expression of this gene in the forebrain. In contrast, Six3, another homeobox gene expressed in the forebrain, is expressed in groups of Otx2 mutant cells as in surrounding WT cells in moderate chimeras at E8.5, indicating that Otx2 is required non-cell autonomously for maintenance of Six3 expression. Thus, Otx2 regulates expression of different regulatory genes in the forebrain through distinct pathways. Similar results were obtained for the regulation of gene expression in the mid-hindbrain region. Otx2 is required for the activation of expression of the signaling molecule Wnt1 and for the maintenance of expression of the homeobox gene En2. Wnt1 expression is observed in WT midbrain cells in control embryos and moderate chimeras but is not detected in any Otx2 mutant cells in the midbrain of moderate chimeras, including those in contact with WT cells. This result demonstrates that Otx2 is required cell autonomously in midbrain cells to activate Wnt1 expression. In contrast, En2 expression in Otx2 mutant cells in the mid-hindbrain of moderate chimeras is rescued by the presence of surrounding WT cells, demonstrating a non-cell autonomous function for Otx2 in regulating En2 expression. Therefore, Otx2 also regulates the expression of mid-hindbrain genes through different mechanisms. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 (Rhinn, 1999).
Holoprosencephaly (HPE) is a common, severe malformation of the brain that involves separation of the central nervous system into left and right halves. Mild HPE can consist of signs such as a single central incisor, hypotelorism, microcephaly, or other craniofacial findings that can be present with or without associated brain malformations. The etiology of HPE is extremely heterogeneous, with the proposed participation of a minimum of 12 HPE-associated genetic loci as well as the causal involvement of specific teratogens acting at the earliest stages of neurulation. The HPE2 locus has recently been characterized as a 1-Mb interval on human chromosome 2p21 that contains a gene associated with HPE. A minimal critical region is defined by a set of six overlapping deletions and three clustered translocations in HPE patients. The isolation and characterization of the human homeobox-containing SIX3 gene from the HPE2 minimal critical region (MCR) is described. At least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5' end of SIX3. Mutational analysis has identified four different mutations in the homeodomain of SIX3 that are predicted to interfere with transcriptional activation and are associated with HPE. It is proposed that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eye in humans (Wallis, 1999).
The Drosophila gene sine oculis (so), a nuclear homeoprotein that is required for eye development, has several vertebrate homologs (the SIX gene family). Among them, SIX3 is considered to be the functional ortholog of so because it is strongly expressed in the developing eye. However, embryonic SIX3 expression is not limited to the eye field, and SIX3 has been found to be mutated in some patients with holoprosencephaly type 2 (HPE2), suggesting that SIX3 has wide implications for head development. The cloning and characterization of SIX6, a novel human SIX gene that is the homolog of the chick Six6(Optx2) gene, is reported. SIX6 is closely related to SIX3 and is expressed in the developing and adult human retina. Data from chick and mouse suggest that the human SIX6 gene is also expressed in the hypothalamic and the pituitary regions. SIX6 spans 2567 bp of genomic DNA and is split in two exons that are transcribed into a 1393-nucleotide-long mRNA. Chromosomal mapping of SIX6 reveals that it is closely linked to SIX1 and SIX4 in human chromosome 14q22.3-q23, which provides clues about the origin and evolution of the vertebrate SIX family. Recently three independent reports have associated interstitial deletions at 14q22.3-q23 with bilateral anophthalmia and pituitary anomalies. Genomic analyses of one of these cases demonstrates SIX6 hemizygosity, strongly suggesting that SIX6 haploinsufficiency is responsible for these developmental disorders (Gallardo, 1999).
The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. The eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, and consequently organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye (Loosli, 2001).
The following model is proposed for early vertebrate retina development. Patterning of the anterior neural plate culminates in defined expression patterns of Six3 and Pax6. This anterior neural plate relies on the repression of wnt and BMP signaling, and requires the activity of the Otx transcription factors. In the region where Six3 and Pax6 expression overlap, retinal fate is specified. An Rx3-independent regulatory feedback loop of these genes then ensures the maintenance of the retinal fate. Six3 overexpression in el-mutant embryos results in dramatically enlarged retinal primordia. This expansion does not occur at the expense of forebrain tissue, suggesting that Six3 also affects cell proliferation independently of Rx3 and thereby regulates the size of the retina anlage. Consistent with the suggested role of Six3 in cell proliferation, the closely related Xenopus Optx2 gene controls the size of the optic vesicles by regulating proliferation. Under the influence of midline signaling, the retinal anlage is split into two retinal primordia. Mutations in Six3 cause holoprosencephaly in humans, indicating a requirement for Six3 in this process. The two retinal primordia then become localized to the lateral wall of the prosencephalon during neurulation (Loosli, 2001 and references therein).
Subsequent evagination of the primordia results in the formation of the optic vesicles. For this process, Rx3 function is essential. Functional studies consistently argue for a regulatory role of vertebrate Rx genes in proliferation of retinal progenitor cells in the optic vesicle, thus regulating its growth. In the absence of Rx3 function, there is no sign of morphogenesis and the specified retinal precursors do not proliferate and eventually die. Rx3 acts downstream of Six3 and Pax6, which determine the retina anlage. However, it is possible that Rx3 initially also receives input from neural plate patterning genes. Subsequent development divides the optic vesicle into specific regions that then give rise to neural retina (NR), retinal pigmented epithelium (RPE) and optic stalk. Several genes that are expressed during these later steps of retinal development require Rx3 function directly or indirectly. Interestingly, the expression of Tbx2 and Tbx3 is specifically affected in the retinal primordium, but not in the hypothalamus, where they are also co-expressed. This indicates a differential regulation of Tbx2 and Tbx3 in these tissues (Loosli, 2001 and references therein).
The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3, which is expressed exclusively in the anterior neural plate. However, 'loss of function' analysis has not been reported. Using medaka fish, it has been shown that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6 (Carl, 2002).
These experiments demonstrate that Six3 is essential for the formation of a discrete domain within the anterior neuroectoderm. In the absence of Six3 function, cells in the Six3 expression domain undergo apoptosis resulting in the absence of forebrain and eye. Conversely, overexpression of Six3 results in retinal hyperplasia, indicating that one function of Six3 is the control of proliferation in the presumptive retinal cells. In addition, Six3 functions in the determination of the naive anterior neuroectoderm as loss of function results in the absence of the respective structures, while ectopic Six3 expression leads to their ectopic formation. Gene knockdown data show that Six3 and Pax6 interact genetically at early stages of eye development. However, the morphological and molecular consequences of the loss of Six3 function are more severe. Pax6 acts on gene expression including its own only in cooperation with Six3. As seen for Pax6 morphants, small eye Pax6-/- mouse embryos initially form optic vesicles, indicating that also in the mouse Pax6 is not required for the formation of the retina anlage in the neuroectoderm (Carl, 2002).
Following its role in the determination and formation of the retina anlage, Six3 functions in proximodistal patterning of the optic vesicle. Six3 activity regulates the expression of the regional specification gene Vax1, which is required for the formation of ventral forebrain and proximal eye structures. Graded loss of Six3 function shows that the distally expressed genes Rx2 and Pax6 are less sensitive than Vax1, underscoring the proximodistal patterning activity of Six3. In addition, at this stage Six3 controls proliferation and morphogenesis by regulating the expression of Rx3, which is essential for these processes in the developing optic vesicle. Future experiments will aim to identify factors mediating the differential activity of Six3 along the proximodistal axis (Carl, 2002).
The vertebrate Six3 gene, a homeobox gene of the Six-family, plays a crucial role in early eye and forebrain development. Candidate factors have been isolate 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 it has been shown 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 (Tessmar, 2002).
Co-expression of XNeuroD or Xath5 and XSix3 in the eye starts around the time when cell fate determination is still in progress, but differentiation is initiated. At the end of differentiation, their coexpression vanishes, suggesting that their interaction with SIX3 is important for the determination and/or differentiation of distinct cell types in the retina. This finding is in accordance with the notion that in a conditional inactivation of murine Pax6 in retinal progenitor cells, co-expression of Six3 and NeuroD coincides with the exclusive generation of amacrine cells. Therefore, Six3 might permit amacrine cell fate in the presence of NeuroD (Tessmar, 2002).
Xath3 and Xath5 show expression patterns similar to XNeuroD in the developing neuroretina, suggesting that these proteins likewise form part of the determination/differentiation network of the eye. The interaction of SIX3 with a specific combination of ARPs thus may specify distinct cell types of the neuroretina. In contrast, differentiation requires both a stop of proliferation and the expression of cell type specific differentiation genes. Therefore, the ARP/XSIX3 interaction should initiate, directly or indirectly, a proliferation-stop signal. Since Six3 on its own stimulates proliferations, it is tempting to speculate that the interaction of SIX3 with XNEUROD, XATH3 or XATH5 (any of which abolish Six3's proliferative activity) promotes differentiation in those cells of the retina that co-express these atonal-related protein family members (Tessmar, 2002).
One further aspect of this study is the question of evolutionary conservation of these interactions. By performing a cross-species screening experiment, interaction partners have been selected that are presumably conserved between two different vertebrates. Domain-mapping experiments further support this conservation. Since the conserved bHLH domain of XNEUROD interacts with XSIX3, and XSIX3 interacts with XNEUROD via the conserved SIX domain, it is reasonable to speculate that similar interactions might take place in other, non-vertebrate organisms. However, the interaction between the N-terminus of NEUROD and SIX3 appears to be a specific feature of the NEUROD subfamily, since no conserved domain could be detected at the amino acid sequence level (Tessmar, 2002).
The interaction of XSIX3 with the non-Atonal related bHLH protein XASH1, but not with XESR1 or the more distantly related protein XMAX2, clearly indicates specific interactions with other non-atonal-related bHLH transcription factors, the relevance of which will be addressed in future experiments (Tessmar, 2002).
Six3 is a vertebrate homeobox gene that is expressed in the anterior neural plate and eye anlage. Dominant transcriptional activator or repressor forms of Six3 were overexpressed in zebrafish embryos to analyze their effect on eye and forebrain formation. RNA injection of the activator form of Six3 into zebrafish embryos causes reduction of the expression domains for rx2, pax2, and emx1 in the anterior neural plate, resulting in eye and forebrain hypoplasia. However, overexpression of the repressor form of Six3 or wild-type Six3 shows phenotypes the opposite of those of the activator form. 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. One of the zebrafish Groucho family genes, grg3, has been isolated and an interaction between Six3 and Grg3 has been demonstrated using yeast two-hybrid analysis. Point-mutations in the eh1-related motifs in Six3 reduce 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 interact with Grg3, implying a conserved function among the Six family proteins as transcriptional repressors (Kobayashi, 2001).
Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family (Drosophila homolog: Optix), plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the Eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6 (Drosophila homolog: Sine Oculis), which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. The interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6. Using PCR, a specific DNA motif has been identified that is bound by Six3 and it has been demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. This interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae leads to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos results in failure of lens placode invagination and production of delta-crystallin-negative cells within the placode. These specific alterations were not seen when the mutated form of Six3 cDNA was used in similar experimental approaches, indicating that Six3 interaction with Groucho proteins plays an essential role in vertebrate eye development (Zhu, 2002).
Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos, suggesting that they might be the functional counterparts of the Drosophila genes sine oculis (so) and/or optix. Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, yeast two-hybrid screens were performed, using either Six3 or Six6 as a bait. The results of the screen using Six6 is described that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domains of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish (Oryzias latipes) embryos, overlapping with the distribution of both Six3 and Six6. Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1, which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that controls eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes. Interestingly, Drosophila Optix shows similar interactions with Groucho as well as with TLE1 and AES (López-Ríos, 2003).
Although it is well established that Six3 is a crucial regulator of vertebrate eye and forebrain development, it is unknown whether this homeodomain protein has a role in the initial specification of the anterior neural plate. Exogenous Six3 can expand the anterior neural plate in both Xenopus and zebrafish, and this occurs in part through Six3-dependent transcriptional regulation of the cell cycle regulators cyclinD1 and p27Xic1, as well as the anti-neurogenic genes Zic2 and Xhairy2. However, Six3 can still expand the neural plate in the presence of cell cycle inhibitors and this is likely to be due to its ability to repress the expression of Bmp4 in ectoderm adjacent to the anterior neural plate. Furthermore, exogenous Six3 is able to restore the size of the anterior neural plate in chordino mutant zebrafish, indicating that it has the ability to promote anterior neural development by antagonising the activity of the BMP pathway. On its own, Six3 is unable to induce neural tissue in animal caps, but it can do so in combination with Otx2. These results suggest a very early role for Six3 in specification of the anterior neural plate, through the regulation of cell proliferation and the inhibition of BMP signalling (Gestri, 2005).
To elucidate whether Bmp4 and Xsix3 might antagonise each other, the effects that the overexpression of each of these genes exert on the other were analyzed. Bmp4 overexpression leads to a strong reduction of Xsix3 expression. Conversely, interfering with BMP signalling by injection of either tBR, a dominant-negative BMP receptor, or chordin mRNA induces a strong activation of Xsix3 both in animal caps and in the anterior neural plate of the embryo. Conversely, both VP16-Xsix3 and MoXsix3 injection leads to expansion of Bmp4 expression in the presumptive anterior neural plate. Additionally, TUNEL analysis shows that both Bmp4- and VP16-Xsix3-injected embryos display an anterior accumulation of apoptotic nuclei (Gestri, 2005).
To analyse whether the effects of Xsix3 loss of function are a consequence of BMP4 expansion in the anterior neural plate, whether interfering with BMP signalling can counteract the reduction of the anterior neural plate in MoXsix3-injected embryos was examined. To achieve this, the expression of Zic2 (a gene expressed both in the anterior and posterior neural plate that is strongly modulated by Xsix3), was examined in MoXsix3/tBR co-injected embryos. Injection of MoXsix3 alone represses anterior Zic2 expression. Conversely, MoXsix3/tBR co-injected embryos showed a complete or partial rescue of the Zic2 expression domain. None of the co-injected embryos showed the strong expansion of Zic2 seen for tBR alone. As a control, a similar rescue is observed when MoXsix3 is co-injected with Xsix3. Taken together, these results indicate a mutual antagonism between Xsix3 and Bmp4 (Gestri, 2005).
The vertebrate brain is anatomically and functionally asymmetric; however, the molecular mechanisms that establish left-right brain patterning are largely unknown. In zebrafish, asymmetric left-sided Nodal signaling within the developing dorsal diencephalon is required for determining the direction of epithalamic asymmetries. Six3, a transcription factor essential for forebrain formation and associated with holoprosencephaly in humans, regulates diencephalic Nodal activity during initial establishment of brain asymmetry. Reduction of Six3 function causes brain-specific deregulation of Nodal pathway activity, resulting in epithalamic laterality defects. Based on misexpression and genetic epistasis experiments, it is proposed that Six3 acts in the neuroectoderm to establish a prepattern of bilateral repression of Nodal activity. Subsequently, Nodal signaling from the left lateral plate mesoderm alleviates this repression ipsilaterally. These data reveal a Six3-dependent mechanism for establishment of correct brain laterality and provide an entry point to understanding the genetic regulation of Nodal signaling in the brain (Inbal, 2007).
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