sine oculis


The Six/Sine oculus family of genes

Optix is a new Drosophila member of the Six/sine oculis gene family that contains both a Six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, Optix is considered to be the orthologous gene from Drosophila, rather than sine oculis as was previously believed. Whereas Sine oculis belongs to the Six1 subclass of the Six/so gene family, Optix belongs to the Six3 subclass. Optix expression is detected in the eye, wing and haltere imaginal discs. Ectopic expression of Optix leads to the formation of ectopic eyes, suggesting that Optix has important functions in eye development. Although Optix and sine oculis both belong to the Six/so gene family and share a high degree of amino acid sequence identity, there are a number of factors that suggest that their developmental roles are different: (1) the expression patterns of Optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas Optix is expressed independent of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas Optix does not; (4) ectopic expression of Optix alone, but not of sine oculis, can induce ectopic eyes in the antennal disc. These results suggest that Optix is involved in eye morphogenesis by an eyeless-independent mechanism (Seimiya, 2000).

The vertebrate Six genes are homologs of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Two new Six genes in Drosophila, D-Six3 and D-Six4, are described that encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, the partial sequences of 12 Six gene homologs from several lower vertebrates are described. The class of Six proteins can be subdivided into three major families, each including one Drosophila member. Comparisons of the sequence identities between the homeodomain (HD) and Six domain (SD) of the individual Drosophila proteins relative to their respective vertebrate homologues show clear differences in the degree of conservation. The HDs of both so and D-Six3 are almost identical (95% and 97%) to the corresponding sequence of their murine homologs Six1/Six2 and Six3, respectively. So and D-Six3, belong to separate subclasses of Six genes, subclass Six1 and Six3 respectively (Seimiya, 2000). D-Six3 is almost identical in sequence to Optix, a second Six3 subclass protein in Drosophila (Seimiya). The similarity between the SD and so of Six1/Six2 (84%) is significantly higher than between D-Six3 and Six3 (77%). In the case of D-Six4 and its murine homolog Six4 (both members of the Six4 subclass), both the HD (82%) and SD (57%) have considerably lower sequence identities. These values show that the HDs are generally the most conserved domains of these homologous proteins, which have more variable divergences in their SDs (Seo, 1999).

The N- and C-terminal regions of the three Drosophila proteins are highly variable in length and sequence. Such differences also exist in the vertebrate homologs Six3 and Six4. One characteristic feature of the so protein is the presence of homopolymorphic regions in both the N- and C-terminal parts. Similarly, D-Six3 has a few short homopolymers in the C-terminus, whereas the D-Six4 protein does not exhibit this type of amino acid repeat (Seo, 1999).

The contents of Pro-Ser-Thr (PST) in the C-terminal regions of the three Drosophila proteins (21%-23%) is considerably lower than in many other related vertebrate Six proteins. However, the N-terminal part of D-Six3 is quite rich in PST, suggesting the presence of transactivating functions. In the case of D-Six4, which has a much larger N-terminal domain, the average PST level is significantly lower (24%). However, a subregion of 18 amino acids (residues 96-113) within the N-terminus is remarkably PST-rich (72%), and this may correspond to a transactivating domain. Another notable feature of this protein is the presence of many amino acid doublets (and a few triplets), particularly Ser-Ser and Gly-Gly, for which the significance is not known (Seo, 1999).

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, 1996a).

A sine oculis gene in the planarian Girardia tigrina (Platyhelminthes; Turbellaria; Tricladida) has been identified. The planarian sine oculis gene (Gtso) encodes a protein with a sine oculis (Six) domain and a homeodomain that shares significant sequence similarity with So proteins assigned to the Six-2 gene family. The C-terminal region comprises 229 amino acids rich in serine (14%), asparagine (13.5%), proline (7%), and threonine (8%), suggesting the presence of transactivating functions; 30% of these serines, and other less frequent residues scattered throughout the sequence, are also present in the same position of the different Sine oculis homologous proteins. Another feature of this protein is the presence of several amino acid doublets and some repeats of tetrapeptides and pentapeptides, for which the significance is not known. Gtso is expressed as a single transcript in both regenerating and fully developed eyes. Whole-mount in situ hybridization studies show exclusive expression in photoreceptor cells. Loss of function of Gtso by RNA interference during planarian regeneration inhibits eye regeneration completely. Gtso is also essential for maintenance of the differentiated state of photoreceptor cells. These results, combined with the previously demonstrated expression of Pax-6 in planarian eyes, suggest that the same basic gene regulatory circuit required for eye development in Drosophila and mouse is used in the prototypic eye spots of platyhelminthes and, therefore, is truly conserved during evolution (Pineda, 2000).

C. elegans has four members of the Six/sine oculis class of homeobox genes: ceh-32, ceh-33, ceh-34, and ceh-35. Proteins encoded by this gene family are transcription factors sharing two conserved domains, the homeodomain and the Six/sine oculis domain, both involved in DNA binding. ceh-32 expression is detected during embryogenesis in hypodermal and neuronal precursor cells and later in descendants of these cells as well as in gonadal sheath cells. RNAi inactivation studies suggest that ceh-32 plays a role in head morphogenesis, like vab-3, the C. elegans Pax-6 ortholog. ceh-32 and vab-3 are coexpressed in head hypodermal cells and ceh-32 mRNA levels are reduced in vab-3 mutants. Moreover, ectopic expression of VAB-3 in transgenic worms is able to induce ceh-32 ectopically. In addition, VAB-3 is able to bind directly to the ceh-32 upstream regulatory region in vitro and to activate reporter gene transcription in a yeast one-hybrid system. These results suggest that VAB-3 acts upstream of ceh-32 during head morphogenesis and directly induces ceh-32. Thus, ceh-32 appears to be the first target gene of VAB-3 identified so far (Dozier, 2001).

The development of visual organs is regulated in Bilateria by a network of genes where members of the Six and Pax gene families play a central role. To investigate the molecular aspects of eye evolution, the structure and expression patterns of cognate members of the Six family genes was analyzed in jellyfish (Cnidaria, Hydrozoa), representatives of a basal, non-bilaterian phylum where complex lens eyes with spherical lens, an epidermal cornea, and a retina appear for the first time in evolution. In the jellyfish Cladonema radiatum, a species with well-developed lens eyes in the tentacle bulbs, Six1/2-Cr and Six3/6-Cr, are expressed in the eye cup. Six4/5-Cr is mainly expressed in the manubrium, the feeding, and sex organ. All three Six genes are expressed in different subsets of epidermal nerve cells, possibly of the RFamide type which are part of a net connecting the different eyes with each other and the effector organs. Furthermore, expression is found in other tissues, notably in the striated muscle. During eye regeneration, expression of Six1/2-Cr and Six3/6-Cr is upregulated, but not of Six4/5-Cr. In Podocoryne carnea, a jellyfish without eyes, Six1/2-Pc and Six3/6-Pc are also expressed in the tentacle bulbs, Six1/2-Pc additionally in the manubrium and striated muscle, and Six3/6-Pc in the mechanosensory nematocytes of the tentacle. The conserved gene structure and expression patterns of all Cladonema Six genes suggest broad conservation of upstream regulatory mechanisms in eye development (Stierwald, 2004).

These data demonstrate that Cnidaria have at least one member of each of the three Six family subclasses. Therefore, the family of Six genes arose before the Urbilateria and the Cnidaria separated, but after the first big wave of gene duplications occurred, predating the Parazoa and Eumetazoa split some 980 million years ago. It is regarded as likely that after the first round of duplications and the separation of the Parazoa, sufficient genomic material was available to gradually select new developmental structures and the corresponding networks of regulatory genes. The product of this process was assumingly a non-sessile organism which had invented a muscle contraction-based locomotion, invented a gut system and consequently knew predation on fellow organisms other than prokaryotes. It had evolved an anterior-posterior body axis and an anteriorized nervous system which was used to control sensory input and directed locomotion. Since sexual development predated metazoan evolution, the putative non-sessile organism was likely of direct development. This hypothetical organism could be the source of a possible zootype. When the history of earth offered new niches, these basic cassettes of developmental genes were available as functional networks and could be co-opted to further add and refine developmental patterns and anatomical structures thus providing the base for the rapid evolution of the different phyla. The Cnidaria are thought to share with Bilateria a good part of this process. Cnidaria already have a representative of each subclass of the Six family genes and they use them correspondingly to Bilateria to differentiate eyes and mesodermal derivatives like muscle. It is noteworthy that the dual role of jellyfish Six1/2 and Six3/6 in eye formation and differentiation of mesodermal elements appears to be conserved through such a long time in evolution. This is also the case for the Six4/5 which in Drosophila is expressed in the gonads and in Cladonema in the manubrium which differentiates the gametes (Stierwald, 2004).

The Six1 subclass of the Six/Sine oculus family of genes

The recently described murine homeobox genes, Six1 and Six2, which are expressed during development in limb tendons, have also been shown to be expressed in skeletal and smooth muscle, respectively. A human SIX1 cDNA has been cloned and sequenced and shown by Northern blotting to be expressed in adult skeletal muscle. The cDNA sequence and predicted protein sequence of SIX1 and Six1 are highly homologous, with 98% similarity over the entire predicted amino acid sequence. SIX1 maps to human chromosome 14 (Boucher, 1996).

In vertebrates, limb tendons are derived from cells that migrate from the lateral plate mesoderm during early development. While some of the developmental steps leading to the formation of these tissues are known, little is known about the molecular mechanisms controlling them. Two murine homeobox-containing genes have been identified, Six 1 and Six 2: both are expressed in a complementary fashion during the development of limb tendons. Both Six 1 and Six 2 are subclass Six1 members, sharing the same Six subclass as Drosophila Sine oculus. Transcripts for both genes are found in different sets of phalangeal tendons. Six 1 and Six 2 are also expressed in skeletal and smooth muscle, respectively. These genes may participate in the patterning of the distal tendons of the limb phalanges by setting positional values along the limb axes (Oliver, 1995b).

Six genes are homeobox-containing transcription factors, many of which are expressed in head structures. A previously unknown Xenopus member of this family, XSix1, shares a high sequence homology with mouse and human Six1; during development mammalian Six1 genes are expressed in mesoderm and muscle. In contrast, XSix1 is prominently expressed in all neurogenic cephalic placodes and lateral line primordia from neurula to tadpole stages. The neurons derived from these placodes do not express XSix1, but the lateral line mechanoreceptors maintain expression. XSix1 is weakly expressed in muscle later in development (Pandur, 2000).

The murine Six gene family, homologous to Drosophila sine oculis (so) which encodes a homeodomain transcription factor, is composed of six members (Six1-6). Among the six members, only the Six2 gene has been previously shown to be expressed early in kidney development, but its function is unknown. The Six1 gene is also expressed in the kidney. In the developing kidney, Six1 is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression becomes restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, Six1 mutant mice have been generated. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1-/- kidney development, Pax2, Six2 and Sall1 expression is markedly reduced in the metanephric mesenchyme at E10.5, indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression is unaffected in Six1-/- metanephric mesenchyme at E10.5, indicating that Eya1 may function upstream of Six1. Moreover, both Eya1 and Six1 expression in the metanephric mesenchyme is preserved in Pax2-/- embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, these results show that Eya1 and Six1 genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development (Xu, 2003).

Six genes are widely expressed during vertebrate embryogenesis, suggesting that they are implicated in diverse differentiation processes. To determine the functions of the Six1 gene, Six1-deficient mice were constructed by replacing the gene's first exon by the ß-galactosidase gene. Mice lacking Six1 die at birth due to thoracic skeletal defects and severe muscle hypoplasia affecting most of the body muscles. Six1-/- neonates also lack a kidney and thymus, as well as displaying a strong disorganization of craniofacial structures, namely the inner ear, the nasal cavity, the craniofacial skeleton, and the lacrimal and parotid glands. These organ defects can be correlated with Six1 expression in the embryonic primordium structures as revealed by X-Gal staining at different stages of embryogenesis. Thus, the fetal abnormalities of Six1-/- mice appear to result from the absence of the Six1 homeoprotein during early stages of organogenesis. Interestingly, these Six1 defects are very similar to phenotypes caused by mutations of Eya1, which are responsible for the BOR syndrome in humans. Close comparison of Six1 and Eya1 deficient mice strongly suggests a functional link between these two factors. Pax gene mutations also lead to comparable phenotypes, suggesting that a regulatory network including the Pax, Six and Eya genes is required for several types of organogenesis in mammals (Laclef, 2003).

Six1 is required for mouse auditory system development. During inner ear development, Six1 expression is first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using Six1 mutant mice, it was found that all Six1+/- mice show some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1-/- mice display malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1-/- embryos arrests at the otic vesicle stage and all components of the inner ear fail to form due to increased cell death and reduced cell proliferation in the otic epithelium. Six1 expression in the otic vesicle is Eya1 dependent, but Eya1 expression is unaffected in Six1-/- otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. Several other otic markers were analyzed; the expression of Pax2 and Pax8 is unaffected in Six1-/- otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1 is required for regional specification of the otic vesicle. Finally, the data suggest that the interaction between Eya1 and Six1 is crucial for the morphogenesis of the cochlea and the posterior ampulla during inner ear development. These analyses establish a role for Six1 in early growth and patterning of the otic vesicle (Zhen, 2003).

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In Six1-deficient mice, development of the inner ear, nose, thymus, kidney and skeletal muscle is severely affected. Six1-deficient embryos are devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly begins at around E10.5 and Six1 is expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which are expressed ventrally in the wild-type otic vesicles, are abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which are expressed dorsally in the wild-type otic vesicles, expand ventrally. These results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation is reduced and apoptotic cell death is enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh are mutually independent (Ozaki, 2003).

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. Markers of pre-placodal ectoderm (PPE) (six1, eya1) have been used to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), it has been demonstrated that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains (Brugmann, 2004).

These studies predict complex in vivo interactions between six1 and other ectodermal genes. sox2 and sox3 are both induced in presumptive neural ectoderm by neural inductive signaling, and promote stabilization of a neural fate. Later, they are both expressed in neural stem cells. Endogenous six1 expression in the LNE precedes sox2/3 placodal expression, indicating that sox2/3 are not upstream of six1. six1 overexpression in the lateral neurogenic ectoderm (LNE) has no significant effect on sox2/3 neural plate expression, indicating that its effects in this border domain are cell autonomous and not due to intermediate signaling. However, when six1 is reduced in the lateral ectoderm, sox2/3 expression expands laterally. This could be a secondary result of the expansion of foxD3, which in turn expands sox2/3 and/or a mutual antagonism between six1 and sox2/3. The latter possibility is supported by the observations that six1 expression in the neural plate dramatically represses sox2/3 and that expression of sox2 in the LNE represses six1. At later stages sox2/3 are expressed in placodal domains that presumably overlap with six1 expression. How these genes interact at this later phase of placode development remains to be determined (Brugmann, 2004).

foxD3 is required for neural crest formation, and both explant and in vivo studies show that it induces neural crest and neural plate marker genes. foxD3 and six1 have a mutually antagonistic relationship; the over-expression of one gene causes the repression of the other. Conversely, the reduction of Six1 causes the foxD3 domain to expand, and the reduction of foxD3 by six1 overexpression causes expansion of other placodal markers. It is not yet known whether the interactions between foxD3 and six1 are direct or indirect: because six1 can repress both foxD3 and sox2 expression domains and foxD3 can expand sox2/3 domains, the interaction could be via sox2/3 regulation (Brugmann, 2004).

The zic1, zic2 and zic3 genes, which are likely to be functionally redundant, are first expressed throughout the entire presumptive neural epithelium and then become restricted to the lateral border of the neural plate and neural crest. The Zic genes appear to be important for the initial phase of both neural plate and neural crest development, and all three can induce ectopic expression of neural crest markers. In explants, zic1 induces foxD3 and slug, and foxD3 induces zic1 and zic2, leading to the proposal that Zic genes act upstream of foxD3 and slug to initiate neural crest fate, and that Zic gene and foxD3 expression is maintained in the neural crest by mutual interactions. A mutual positive interaction occurs in in vitro experments between zic2 and foxD3. However, the LNE foxD3 and the Zic genes do not have identical expression patterns, indicating that they also may be interacting through intermediary genes. A similarly complex interaction has been demonstrated to occur between six1 and zic2. Overexpression of six1 expands the zic2 lateral domain, but reduction of Six1 also expands it. It is proposed that the former phenotype is caused by activation/maintenance of zic2 by six1, whereas the latter phenotype is most probably caused by the expansion of foxD3, which subsequently expands the zic2 domain (Brugmann, 2004).

Members of the Dlx gene family represent some of the earliest genes expressed at the border between the neural plate and epidermis. In chick, dlx5 is expressed at the neural/non-neural border, overlapping with eya2 and six4 in the pre-placodal thickening where it is proposed to create a border zone in which lateral neurogenic fates can be expressed. In Xenopus there is a low level of dlx5/6 expression along the border of the neural plate, but the most intense stripe is adjacent to six1 expression along the anterior neural ridge, and overlapping with the lateral edge of the crescent of six1 PPE expression. In chick, dlx5 overexpression results in a weak upregulation of six4 expression, whereas in Xenopus wild-type dlx5/dlx6 both strongly reduce six1 expression, and activator Dlx constructs cause a loss of six1 expression. It is not clear whether these differences are due to species differences in the precise patterning of the embryonic ectoderm, as has been proposed for neural induction, or due to the fact that Six1 and Six4 belong to different subclasses of the Six gene family (Brugmann, 2004).

Regardless, it is clear that frog six1 has two effects on dlx5/6 expression. Most prominently, overexpression of six1 in the LNE pushes the dlx5/6 stripe laterally away from the neural plate midline. This phenotype is probably due to six1 causing an expansion of the pre-placodal ectoderm (eya1, sox11) and reduction of epidermis (keratin), resulting in the formation of a new border between the expanded LNE and the epidermis. This interpretation is consistent with the effects of six1 overexpression on keratin, and further suggests that the effect is not due to movement of the neural plate border because the sox2/3 domains do not change. Likewise, dlx5/6 negatively regulate six1 expression. A mutual regulation takes place between Dlx genes and six1: inhibition of endogenous Dlx activity relocates the six1 expression domain more laterally, whereas activation relocated it more medially. The second effect of six1 is complete repression of dlx5/6 expression in those cells expressing six1. This may result from Six1 either repressing dlx5/6 gene expression or causing changes in gene expression in the affected cells that secondarily create an environment that is not compatible with dlx5/6 expression. Interestingly, foxD3 overexpression has similar effects on dlx5/6, which could be direct, or, unlike six1, could be due to the expansion of sox2/3. Paradoxically similar dlx5/6 phenotypes occur by activator and repressor six1 construct expression and six1-MO injections. It is predicted that these can be explained by six1 effects on foxD3. The injection of six1VP16, six1-WT+eya-WT and six1-MO may indirectly reduce dlx5/6 by expansion of foxD3, whereas six1-WT alone and six1EnR constructs may directly reduce dlx5/6. These results support the proposal that dlx5/6 contribute to forming the LNE border zone, and additionally demonstrate that they do so by participating in a complex interplay with several genes expressed in adjacent domains. It will be important to determine the precise molecular interactions between these various gene pathways to fully understand their roles in specifying LNE fates (Brugmann, 2004).

Eya1 encodes a transcriptional co-activator and is expressed in cranial sensory placodes. It interacts with and functions upstream of the homeobox gene Six1 during otic placodal development. Their role in cranial sensory neurogenesis was examined. The data show that the initial cell fate determination for the vestibuloacoustic neurons and their delamination appears to be unaffected in the absence of Eya1 or Six1 as judged by the expression of the basic helix-loop-helix genes, Neurog1, which specifies the neuroblast cell lineage, and Neurod, which controls neuronal differentiation and survival. However, both genes are necessary for normal maintenance of neurogenesis. During the development of epibranchial placode-derived distal cranial sensory ganglia, while the phenotype appears less severe in Six1 than in Eya1 mutants, an early arrest of neurogenesis was observed in the mutants. The mutant epibranchial progenitor cells fail to express Neurog2, which is required for the determination of neuronal precursors, and other basic helix-loop-helix as well as the paired homeobox Phox2 genes that are essential for neural differentiation and maintenance. Failure to activate their normal differentiation program results in abnormal apoptosis of the progenitor cells. Furthermore, disruption of viable ganglion formation leads to pathfinding errors of branchial motoneurons. Finally, these results suggest that the Eya-Six regulatory hierarchy also operates in the epibranchial placodal development. These findings uncover an essential function for Eya1 and Six1 as critical determination factors in acquiring both neuronal fate and neuronal subtype identity from epibranchial placodal progenitors. These analyses define a specific role for both genes in early differentiation and survival of the placodally derived cranial sensory neurons (Zou, 2004).

The mouse cochlea emerges from the ventral pole of the otocyst to form a one and three-quarter coil. Little is known about the factors that control the growth of the cochlea. Jackson circler (jc) is a recessive mutation causing deafness resulting from a growth arrest of the cochlea duct at day 13.5 of embryonic development. This study identified the vertebrate homolog of the Drosophila Sobp (sine oculis-binding protein; Kenyon, 2005) gene (named Jxc1) in the jc locus. Jxc1 encodes a nuclear protein that has two FCS-type zinc finger domains (PS51024) and bears nuclear localization signals and highly conserved sequence motifs. Transiently expressed wild-type protein is targeted to the nucleus, but mutant isoforms were mislocalized in the cytoplasm. In jc mutants, the cellular patterning of the organ of Corti is severely disrupted, exhibiting supernumerary hair cells at the apex, showing mirror-image duplications of tunnel of Corti and inner hair cells, and expressing ectopic vestibular-like hair cells within K├Âlliker's organ. Jxc1 mRNA was detected in inner ear sensory hair cells, supporting cells, and the acoustic ganglia. Expression was also found in the developing retina, olfactory epithelium, trigeminal ganglion, and hair follicles. Collectively, these data support a role for Jxc1 in controlling a critical step in cochlear growth, cell fate, and patterning of the organ of Corti (Chen, 2008).

Planarians can regenerate any missing body part, requiring mechanisms for the production of organ systems in the adult, including their prominent tubule-based filtration excretory system called protonephridia. This study identified a set of genes, Six1/2-2, POU2/3, hunchback, Eya and Sall, that encode transcription regulatory proteins that are required for planarian protonephridia regeneration. During regeneration, planarian stem cells are induced to form a cell population in regeneration blastemas expressing Six1/2-2, POU2/3, Eya, Sall and Osr that is required for excretory system formation. POU2/3 and Six1/2-2 are essential for these precursor cells to form. Eya, Six1/2-2, Sall, Osr and POU2/3-related genes are required for vertebrate kidney development. Planarian and vertebrate excretory cells express homologous proteins involved in reabsorption and waste modification. Furthermore, novel nephridia genes were identified. These results identify a transcriptional program and cellular mechanisms for the regeneration of an excretory organ and suggest that metazoan excretory systems are regulated by genetic programs that share a common evolutionary origin (Scimone, 2011).

Six1 and myogenesis

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galactosidase transgene during embryogenesis. Mutation of one of the DNA motifs present in this region, the MEF3 motif, abolishes correct expression of this beta-galactosidase transgene. The proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin (Spitz, 1998).

Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologs of these genes, Six and Eya, respectively, show overlapping expression patterns during development. It has been hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters is observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, the intracellular distribution of Six and Eya proteins were examined in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induces nuclear translocation of Eya1, Eya2, and Eya3, which are otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 does not result in nuclear localization of any Eya proteins. Six and Eya proteins coimmunoprecipitate from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, are necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, is not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 is observed by yeast two-hybrid analysis. These results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes (Ohto, 1999).

During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs. Regulatory molecules such as receptor tyrosine kinases like c-Met and those encoded by homeobox-containing genes, including c-Met, Tbx1, Mox2, Six1 and Six2, Pitx2, Pax3 and Lbx1h (a homolog of Drosophila Ladybird genes), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevents an analysis of later functions of these genes during limb muscle development. Based on quail-chick chimaera studies, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells, leads to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect is caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. These results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles (Schafer, 1999).

A novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 has been identified. Dach2, is expressed in the developing somite prior to any myogenic genes, with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context -- myogenesis rather than eye development -- and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6 (Heanue, 1999).

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, Six1-deficient mice were constructed by replacing the gene's first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles, in particular certain hypaxial muscles. Six1-/- embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1–/– embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2 (Laclef, 2003).

Genome-wide transcription factor binding and expression profiling has been used to assemble a regulatory network controlling the myogenic differentiation program in mammalian cells. A cadre of overlapping and distinct targets of the key myogenic regulatory factors (MRFs) -- MyoD and myogenin -- and Myocyte Enhancer Factor 2 (MEF2) have been identified. MRFs and MEF2 regulate a remarkably extensive array of transcription factor genes that propagate and amplify the signals initiated by MRFs. MRFs play an unexpectedly wide-ranging role in directing the assembly and usage of the neuromuscular junction. Interestingly, these factors also prepare myoblasts to respond to diverse types of stress. Computational analyses identified novel combinations of factors that, depending on the differentiation state, might collaborate with MRFs. These studies suggest unanticipated biological insights into muscle development and highlight new directions for further studies of genes involved in muscle repair and responses to stress and damage (Blais, 2005).

One of the most striking observations was that transcription factors represent the largest cluster of MRF targets. Although consistent with a cascade model of gene activation, the markedly high number of transcription factors regulated by MRFs and MEF2 suggests that the cascade may be more extensive than expected. These analyses suggest the existence of new nodal points from which the transcriptional output of MyoD is relayed, greatly expanding the repertoire of indirect targets of MyoD. The role of MRFs in differentiation is contrasted with E2F4, a repressor that plays a role in cell cycle exit: only a handful of transcription factor genes are bound by E2F4, suggesting that gene regulatory programs involved in cell cycle control (and cell cycle exit) may be wired in fundamentally different ways from terminal differentiation (Blais, 2005).

It is proposed that transcriptional regulators (Eya1 and TEAD4/TEF-3) relay the differentiation signal initiated by MyoD. Several biochemical, computational, and genetic observations suggest that the Eya1/Six1 pathway is associated with MRF function. (1) ChIP-on-chip results indicate that Eya1 is a direct target of MyoD in growing myoblasts. Eya1 has the ability to switch the activity of Six1, a homeobox transcriptional regulator, from repressor to activator. (2) The MEF3 PWM, a binding site for Six1, is specifically enriched among myogenin target genes that are induced during differentiation. (3) Mice lacking Six1 display defects in embryonic myogenesis that are exacerbated when Eya1 function is also ablated (Blais, 2005).

In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. Six1Six4 double KO mice were generated and an aggravation of the phenotype previously reported for the single Six1 KO was demonstrated. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis (Grifone, 2005).

Osr1 acts downstream of and interacts synergistically with Six2 to maintain nephron progenitor cells during kidney organogenesis

Mammalian kidney organogenesis involves reciprocal epithelial-mesenchymal interactions that drive iterative cycles of nephron formation. Recent studies have demonstrated that the Six2 transcription factor acts cell autonomously to maintain nephron progenitor cells, whereas canonical Wnt signaling induces nephron differentiation. How Six2 maintains the nephron progenitor cells against Wnt-directed commitment is not well understood, however. This study reports that Six2 is required to maintain expression of Osr1, a homolog of the Drosophila odd-skipped zinc-finger transcription factor, in the undifferentiated cap mesenchyme. Tissue-specific inactivation of Osr1 in the cap mesenchyme causes premature depletion of nephron progenitor cells and severe renal hypoplasia. Osr1 and Six2 act synergistically to prevent premature differentiation of the cap mesenchyme. Furthermore, although both Six2 and Osr1 could form protein interaction complexes with TCF proteins, Osr1, but not Six2, enhances TCF interaction with the Groucho family transcriptional co-repressors. Loss of Osr1 was shown to result in β-catenin/TCF-mediated ectopic activation of Wnt4 enhancer-driven reporter gene expression in the undifferentiated nephron progenitor cells in vivo. Together, these data indicate that Osr1 plays crucial roles in Six2-dependent maintenance of nephron progenitors during mammalian nephrogenesis by stabilizing TCF-Groucho transcriptional repressor complexes to antagonize Wnt-directed nephrogenic differentiation (Xu, 2014).

The Six3 subclass of the Six/Sine oculus family of genes

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Six genes were examined -- sine oculis-like or optix-like (six3), retinal homeobox (rx), distal-less (dlx), ventral anterior homeobox (vax: Drosophila homolog: Empty spiracles), nkx2-1, and brain factor 1 (bf-1). These six chordate neural patterning genes are expressed within the forebrain, each with its own contour and location (Lowe, 2003).

In S. kowalevskii, the orthologs of these six genes are expressed strongly throughout the ectoderm of the prosome. Within the prosome ectoderm, the domain of each gene differs in its exact placement and contours. vax is expressed just at the anterior tip of the prosome near the apical organ. six3 and rx are expressed throughout most of the prosome. rx expression is exclusively ectodermal. rx expression is absent in the apical region of ectoderm where vax is expressed. Six3 is expressed ectodermally and at low levels mesodermally in the developing prosome, and the domain extends slightly into the mesosome ectoderm. Expression of six3 is strongest in the most anterior ectoderm and attenuates posteriorly. dlx and bf-1 are both expressed strongly in a punctate pattern of numerous individual cells or cell clusters throughout most of the prosome ectoderm and also in a diffuse pattern at a lower level throughout the prosome ectoderm. The bf-1 domain is interrupted by a band of nonexpression in the midprosome. dlx expression is seen through the proboscis and individual cells strongly positive for dlx and ectodermal cells exhibiting low-level expression are seen in both apical and basal positions. dlx is also expressed more posteriorly in a dorsal midline stripe. nkx2-1 is specifically expressed in a ventral sector of the prosome ectoderm. In chordates, nkx2-1 is expressed in the ventral (subpallial) portion of the forebrain. It is also expressed less strongly in a ring in the hemichordate pharyngeal endoderm, a domain of interest in relation to this gene's involvement in the chordate endostyle and thyroid, in the hemichordate Ptychodera flava) (Lowe, 2003).

In conclusion, these six orthologs, whose chordate cognates are expressed entirely within the forebrain, all have prominent expression domains in the prosome ectoderm of S. kowalevskii, the hemichordate's most anterior body part (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

The murine homeobox gene Six3 has regulatory functions in eye development. Two zebrafish genes, six3 and six6, have been isolated and characterized that 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 abolish 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, personal communication, 1998). 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).

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 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 not fully implements, 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).

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. An hybrid protein containing the repressor domain of Engrailed (XOptx2-Engrailed repressor) gives a similar phenotype, while an 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 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 was 16.1 cells per retinal section, while the cell number observed in control-expressing clones was 10.4. These results indicate that XOptx2 induces proliferation (Zuber, 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, 1995a).

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).

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).

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).

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, a yeast two-hybrid system was tested as a bait using the NOR-1 cDNA fragment lacking a transactivating domain. By screening of the rat fetal brain embryonic day 17 library, a rat homologue of Six3 was identified as an associated protein. NOR-1 interacts with Six3 in yeast and in vitro, and the association is 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 is not observed in yeast. In addition, cotransfection experiments with Six3 and NOR-1 indicates that Six3 has 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 is mainly detected in the rat fetal forebrain on embryonic day 18. Thereafter, the expression of both genes diminishes 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 (Ohkura, 2001).

The anterior segment of the vertebrate eye (lens, the cornea, the iris, the ciliary body, and the trabecular meshwork) consists of highly organized and specialized ocular tissues critical for normal vision. The periocular mesenchyme, originating from the neural crest, contributes extensively to the anterior segment. During chick eye morphogenesis, the homeobox gene Six3 is expressed in a subset of periocular mesenchymal cells and in differentiating anterior segment tissues. Retrovirus-mediated misexpression of Six3 causes eye anterior segment malformation, including corneal protrusion and opacification, ciliary body and iris hypoplasia, and trabecular meshwork dysgenesis. Histological and molecular marker analyses demonstrate that Six3 misexpression disrupts the integrity of the corneal endothelium and the expression of extracellular matrix components critical for corneal transparency. Six3 misexpression also leads to a reduction of the periocular mesenchymal cell population expressing Lmx1b, Pitx2, and Pax6, transcription factors critical for eye anterior segment morphogenesis. Moreover, elevated levels of Six3 attenuate proliferation of periocular mesenchymal cells in vitro and differentiating anterior segment tissues in vivo. These results suggest that, in addition to its function in eye primordium determination, Six3 plays a role in regulating the development of the vertebrate eye anterior segment (Hsieu, 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).

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, Wnt signaling was found to be sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allows the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induces Dlx2 expression and inhibits the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts direct the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling results in the induction of dT specific markers, but only in tissues that expressed Irx3 (Braun, 2003).

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, the molecular interactions among the encoded proteins was examined. Eya interacts directly with Six but never with Dach. Dach transactivates a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach is repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template is dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. These results demonstrate that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis (Ikeda, 2002).

Organogenesis in vertebrates requires the tight control of cell proliferation and differentiation. The homeobox-containing transcription factor Six3 plays a pivotal role in the proliferation of retinal precursor cells. In a yeast two-hybrid screen, the DNA replication-inhibitor geminin (See Drosophila geminin) has been identified as a partner of Six3. Geminin inhibits cell-cycle progression by sequestering Cdt1, the key component for the assembly of the pre-replication complex. Six3 efficiently competes with Cdt1 directly to bind to geminin, which reveals how Six3 can promote cell proliferation without transcription. In common with Six3 inactivation, overexpression of the geminin gene (Gem; also known as Gmn) in medaka (Oryzias latipes) induces specific forebrain and eye defects that are rescued by Six3. Conversely, loss of Gem (in common with gain of Six3) promotes retinal precursor-cell proliferation and results in expanded optic vesicles, markedly potentiating Six3 gain-of-function phenotypes. These data indicate that the transcription factor Six3 and the replication-initiation inhibitor geminin act antagonistically to control the balance between proliferation and differentiation during early vertebrate eye development (Del Bene, 2004).

The vertebrate forebrain is patterned during gastrulation into telencephalic, retinal, hypothalamic and diencephalic primordia. Specification of each of these domains requires the concerted activity of combinations of transcription factors (TFs). Paradoxically, some of these factors are widely expressed in the forebrain, which raises the question of how they can mediate regional differences. To address this issue, focus was placed on the homeobox TF Six3.2. With genomic and functional approaches it was demonstrated that, in medaka fish, Six3.2 regulates, in a concentration-dependent manner, telencephalic and retinal specification under the direct control of Sox2. Six3.2 and Sox2 have antagonistic functions in hypothalamic development. These activities are, in part, executed by Foxg1 and Rx3, which seem to be differentially and directly regulated by Six3.2 and Sox2. Together, these data delineate the mechanisms by which Six3.2 diversifies its activity in the forebrain and highlight a novel function for Sox2 as one of the main regulators of anterior forebrain development. They also demonstrate that graded levels of the same TF, probably operating in partially independent transcriptional networks, pattern the vertebrate forebrain along the anterior-posterior axis (Beccari, 2012).

The Six4 subclass of the Six/Sine oculus family of genes

SIX5 belongs to a family of highly conserved homeodomain transcription factors implicated in development and disease. The mammalian SIX5/SIX4 gene pair is likely to be involved in the development of mesodermal structures. Moreover, a variety of data have implicated human SIX5 dysfunction as a contributor to myotonic dystrophy type 1 (DM1), a condition characterized by a number of pathologies including muscle defects and testicular atrophy. However, this link remains controversial. The Drosophila gene, Six4, which is the closest homolog to SIX5 of the three Drosophila Six family members, has been investigated. Six4 is required for the normal development of muscle and the mesodermal component of the gonad. Moreover, adult males with defective Six4 genes exhibit testicular reduction. It is proposed that Six4 directly or indirectly regulates genes involved in the cell recognition events required for myoblast fusion and the germline - soma interaction. While the exact phenotypic relationship between Six4 and SIX4/5 remains to be elucidated, the defects in Six4 mutant flies suggest that human SIX5 should be more strongly considered as being responsible for the muscle wasting and testicular atrophy phenotypes in DM1 (Kirby, 2001).

A Drosophila homolog of SIX5 was isolated by screening a genomic library with a SIX5 RT-PCR probe. This homolog is identical to the published Six4 sequence, except for four silent nucleotide differences. The protein sequence of the combined Six domains and homeodomains shows that Six4 is most similar to SIX4 and SIX5 (67% and 65% identity, respectively). Moreover, all three proteins have valine in homeodomain position 5, which is a potential contributor to DNA binding specificity, whereas all other Six proteins have serine or threonine. It is likely that Six4 is derived from the common ancestor of both SIX4 and SIX5 (Kirby, 2001).

During embryogenesis, Six4 mRNA is expressed in the developing head region, mesoderm, and CNS. Mesodermal expression becomes segmental and then becomes confined weakly to the somatic gonadal precursors (SGPs, also known as follicle cell precursors) in parasegments 10-12, which subsequently form the somatic sheath that surrounds the gonad. Six4 expression then becomes strong in the SGPs after they have coalesced with the migrating germ cell precursors (pole cells) to form the immature gonad. SGPs are crucial for gonad coalescence, and germ cells remain scattered if SGPs are dysfunctional. Therefore, the requirement for Six4 in germ cell aggregation was examined using double-stranded RNA interference (dsRNAi). When embryos are injected with Six4 dsRNA, germ cells fail to coalesce. It is concluded that Six4 is required in SGPs for gonad formation (Kirby, 2001).

This information was used to identify Six4 mutations. From an EMS screen, two mutations were isolated that map to the Six4 chromosomal location, fail to complement each other, and produce embryos that exhibit gonad coalescence defects. These mutants harbor defective Six4 genes. Homozygotes of one mutation (D-Six4289) fail to hatch, and it appears to be a null or a strong hypomorph of Six4. Consistent with this, the D-Six4289 gene has a nonsense point mutation (C175 3 > T), resulting in a stop codon in place of Gln87. The second mutation (D-Six4131) is less severe. D-Six4131 mutant embryos hatch normally, although many die during larval and pupal stages. A small proportion survive to adulthood. The molecular defect of D-Six4131 is a point mutation (C2404 > T), resulting in an amino acid substitution of Cys for Arg281, which corresponds to position 102 within the Six domain. This Arg is conserved in all Six proteins, implying that it is important for structure or function of the Six domain. For some other Six proteins, including mouse Six4 and Six5, the Six domain has been shown to mediate the interaction with Eya proteins in vitro, resulting in a functional heterodimer. Drosophila Eya is coexpressed with Six4 in all the areas of the latter's expression. This suggests that Six4 is a partner of Eya in these tissues. Consistent with this, eya mutant embryos also show lack of gonad coalescence. The molecular defect in D-Six4131 is the first demonstration of an amino acid substitution in a Six domain being associated with a phenotypic effect and supports the importance of the Six domain for in vivo protein function (Kirby, 2001).

In D-Six4289 homozygous embryos, initial germ cell internalization and migration are normal, but the cells then fail to coalesce to form a gonad. The failure of gonad coalescence in Six4 mutants is consistent with Six4 expression and function in SGPs. To examine the SGPs themselves, the expression of an SGP marker, the 412 retrotransposon was analyzed. This element is expressed in the head and the mesoderm, the latter probably representing both the fat body precursors and the SGPs; expression subsequently becomes prominent in the SGPs before and after they form the gonad. In stage 10 embryos homozygous for D-Six4289, the expression of this marker is entirely abolished. Late in embryonic development, 412 expression can be observed in one to five scattered cells, which appear to be SGPs. These experiments suggest that Six4 is required for the correct pattern of gene expression within the mesoderm and SGPs. Genes involved in SGP-cell recognition are candidates for Six4 target genes (Kirby, 2001).

Germ cell coalescence is variably affected in the weaker D-Six4131 mutant. In most embryos, gonads were observed, as represented by distinct clusters of 412-expressing SGPs, but these appeared consistently smaller than wild-type. These gonads appeared to be populated by germ cells, although a proportion of the germ cells remained scattered. Given the apparent formation of gonads, the gonads of surviving D-Six4131 adults were examined. Males exhibit severe testicular reduction, although other structures of the reproductive apparatus are present (being derived from the genital imaginal disc rather than the gonad). This suggests a degree of testicular atrophy after their formation in the embryo. Females exhibit strong ovarian reduction, although highly defective ovarioles are often present (Kirby, 2001).

Somatic muscle formation was examined. In late stage wild-type embryos, an antibody to myosin reveals the regular pattern of myotubes, but in homozygous D-Six4289 embryos, the somatic muscles are strongly disrupted. Muscles appeared disorganized in their arrangement or attachment. Some muscles appeared to be entirely missing, although the number and location of such muscles varied between segments and between embryos. In Drosophila, somatic muscles are laid down by a distinct subset of myoblasts known as founder cells. Each founder cell seeds a muscle by fusing with 'generic' fusion-competent myoblasts to form a syncytial myotube. Most prominently, in D-Six4289 embryos, many isolated rounded myosin-expressing cells were scattered among the muscles. These appear to be myoblasts that have not fused with developing myotubes. Thus, there appears to be a major defect in the fusion process. In some cases, elongated unfused founder cells can still be observed attempting to form a myotube, suggesting that the mutant defect is primarily in fusion rather than in initial founder cell specification, although the latter has not been completely ruled out. Embryos homozygous for the weaker mutant, D-Six4131, show these muscle defects to a lesser extent. Homozygous D-Six4131 adult escapers usually die within a few days, but preliminary examination did not reveal any gross muscle defect. However, they have bloated abdomens, owing to a hugely distended crop, which could be a consequence of visceral muscle defects. A number of known genes are required for myoblast fusion, either for the recognition event between founder cells and fusion-competent myoblasts (such as dumbfounded) or for the events of fusion themselves (such as myoblast city). While there are many explanations for the lack of fusion, it is intriguing that Six4 may regulate cell recognition processes in both muscle and gonad formation, suggesting that there are common features to these developmental events (Kirby, 2001).

Given the functions uncovered for Six4, it seems likely that vertebrate SIX4, SIX5, or more likely a combination of both genes, will have important functions in the development of mesodermally derived tissues. Both are expressed widely, including somites, but mouse knockouts of Six4 or Six5 are viable, suggesting that there might be extensive redundancy between the two genes. Human SIX5 was originally identified (as DMAHP) as one of the genes adjacent to the CTG repeat expansion that causes DM1, and there is a variety of indirect evidence that haploinsufficiency of SIX5 is a cause of some DM1 pathologies. DM1 is a complex disease with a variety of pathologies, including myotonia, muscle wasting, testicular atrophy, and cataracts. Recent data strongly support a role in myotonia for RNA-mediated effects of the CUG repeat within the DMPK gene. Other facets of the DM1 phenotype in muscle and heart tissues may be attributed to effects mediated by DMPK mRNA, but thus far, no compelling evidence has emerged. Recent initial reports of a Six5 mouse knockout support a role for SIX5 mutation in cataract formation, but evidence for a role in mesodermally derived tissues is contradictory. The Six5 mutant mice produced by one group are reported to have no muscle or reproductive defects, while a second group reports that their Six5 mutant mice are sterile and show muscle wasting. One possibility is that SIX5 becomes the more important of the SIX4/5 gene pair later in life, while the more extreme effects of Six4 mutants reflect important early functions of a semiredundant SIX4/5 gene pair. How far the similarities between SIX5 and Six4 extend, therefore, remains to be determined, but it is a strong possibility that Six4 may exhibit similar functional relationships to its vertebrate homologs. At the least, the immediate regulatory networks may be conserved. It is an exciting possibility that specific developmental functions may also be conserved, such as regulating target genes involved in cell recognition or association. This may allow the genetic dissection of such regulatory networks and target genes using Drosophila and may illuminate the role of human SIX4/5 in development and disease (Kirby, 2001).

Members of the Six family of genes are expressed during eye formation and differentiation. In addition, most of these vertebrate genes show expression in mesodermal derivatives in adults and/or earlier stages of development. A zebrafish (Danio rerio) gene, six8, shows the greatest similarity to murine Six4 (they are both members of the Six4 subclass of Six genes). The deduced proteins of these two genes have an overall sequence identity of 41%, while the homeodomains and Six domains are highly conserved, 90% and 81%, respectively. The spatiotemporal expression pattern of six8 was analyzed by RT-PCR and in situ hybridization. Transcripts are detected in a wide range of embryonic stages and in adults. Notably, the strongest expression is observed in head mesoderm of late gastrula and early neurula stages (Seo, 1998c).

ARE (Na,K-ATPase alpha1 subunit gene regulatory element) binding protein AREC3 has four alternatively spliced forms. AREC3 has an extensive structural homology with the Drosophila sine oculis. The homologous region including a homeodomain is required for specific DNA binding to ARE. A transactivation domain has been identified in the C-terminal part of the AREC3. AREC3 localizes to the nucleus and cytoplasm of myoblast cells, and the production of AREC3 is augmented during muscle differentiation. The 115 kDa form of AREC3 protein is increased in the cytoplasmic extract, and the 67kDa form is increased both in nuclear and cytoplasmic extracts during muscle differentiation (Kawakami, 1996b).

sine oculis: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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