SIX1 Research Articles

Six3inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon

The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3-null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.

Erratum to: Genetic approaches to human renal agenesis/hypoplasia and dysplasia

Table 2 shows some of the principal human malformation syndromes with kidney hypoplasia/dysplasia. The branchio-oto-renal syndrome is caused by mutations of EYA1, SIX1 or SIX5 genes and not SIX2. We apologize for the mistake in the article.

Single median maxillary central incisor: New data and mutation review

Single median maxillary central incisor (SMMCI) is a rare anomaly that may occur alone or associated with other conditions, frequently as part of the holoprosencephaly (HPE) spectrum. However, it has been suggested that SMMCI alone, or associated with some midline defects, may be considered a different entity from HPE (OMIM: 147250). Families with SMMCI, without HPE cases, are difficult to counsel for the risk of HPE in future generations because the same midline defects described as part of the "SMMCI syndrome" can also be part of the HPE spectrum. We screened five cases of SMMCI for mutations in three HPE genes, SHH, TGIF, and SIX3. A missense mutation c.686C>T was found in the gene SIX3 of one patient, which did not differ from the accepted 20% of known HPE gene mutations among all HPE cases. Our results and an extensive literature review of gene mutations in patients with SMMCI showed that 27/28 of them were in HPE genes: SHH (n = 21), SIX3 (n = 3), TGIF (n = 1), GLI2 (n = 1), and PTCH (n = 1), and only one in the SALL4 gene. The clinical findings in patients with SMMCI without HPE in families with mutations in HPE genes cannot be distinguished from the findings reported in the SMMCI syndrome. Therefore, persons with SMMCI and their relatives should be carefully investigated for related midline disorders, especially of the HPE spectrum, and all known HPE genes screened.

Colobomatous macrophthalmia with microcornea syndrome maps to the 2p23‐p16 region

Colobomatous macrophthalmia with microcornea syndrome (OMIM 602499) is a rare, autosomal dominant malformation characterized by microcornea, uveal coloboma, axial enlargement of the globe, and myopia. Using what is currently the largest described pedigree and candidate localization approach, we first excluded the candidate genes PAX2, PAX3, PAX6, and PAX9. Subsequently, the chromosome 14q24 region containing the CHX10, SIX1, and SIX4 genes were also excluded. Positive LOD scores were obtained with the DNA markers selected from the 2p23-p16 region. A maximum pairwise LOD score of 3.61 (Theta = 0) was noted with the DNA marker D2S1788. Haplotype analysis positioned the locus between DNA markers D2S2263 and D2S1352 within a 22 Mb physical interval. This region contains major candidate genes, such as SIX2, SIX3, and CYP1B1; however, mutation analysis did not identify a causative mutation in these genes. Macrophthalmia, colobomatous, with microcornea (MACOM) is proposed as the gene symbol for this malformation linked to 2p23-p16.

Comprehensive characterization of the cis-regulatory code responsible for the spatio-temporal expression of olSix3.2in the developing medaka forebrain

A cluster of highly conserved non-coding sequences surrounding the Six3 gene were identified in fish genomes, and transgenesis in medaka fish demonstrates that these sequences have enhancer, silencer and silencer blocker activities that are differentially combined to control the distribution of Six3.

Six3 activation of Pax6 expression is essential for mammalian lens induction and specification

The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction.

Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo

In mammals, Pax3, Six4, Six1 and Six5 genes are co-expressed with Eya1, Eya2 and Eya4 genes during mouse somitogenesis. To unravel the functions of Eya genes during muscle development, we analyzed myogenesis in Eya2-/− and in Eya1−/− embryos. A delay in limb myogenesis was observed between E10 and E13 in Eya1−/− embryos only, that is later compensated. Compound E18 Eya1−/−Eya2−/+ fetuses present a muscle phenotype comparable with that of Six1−/− fetuses; lacking a diaphragm and with a specific absence of limb muscles, suggesting either genetic epistasis between Six and Eya genes, or biochemical interactions between Six and Eya proteins. We tested these two non-exclusive possibilities. First, we show that Six proteins recruit Eya proteins to drive transcription during embryogenesis in the dermomyotomal epaxial and hypaxial lips of the somites by binding MEF3 DNA sites. Second, we show that Pax3 expression is lost in the ventrolateral (hypaxial) dermomyotomes of the somite in both Eya1−/−Eya2−/− embryos and in Six1−/−Six4−/− embryos, precluding hypaxial lip formation. This structure, from which myogenic cells delaminate to invade the limb does not form in these double mutant embryos, leading to limb buds without myogenic progenitor cells. Eya1 and Eya2, however, are still expressed in the somites of Six1Six4 double mutant and in splotch embryos, and Six1 is expressed in the somites of Eya1Eya2 double mutant embryos and in splotch embryos. Altogether these results show that Six and Eya genes lie genetically upstream of Pax3 gene in the formation of ventrolateral dermomyotome hypaxial lips. No genetic links have been characterized between Six and Eya genes, but corresponding proteins activate key muscle determination genes (Myod, Myogenin and Mrf4). These results establish a new hierarchy of genes controlling early steps of hypaxial myogenic commitment in the mouse embryo.

Deletion at 14q22‐23 indicates a contiguous gene syndrome comprising anophthalmia, pituitary hypoplasia, and ear anomalies

Anophthalmia and pituitary gland hypoplasia are both debilitating conditions where the underlying genetic defect is unknown in the majority of cases. We identified a patient with bilateral anophthalmia and absence of the optic nerves, chiasm and tracts, as well as pituitary gland hypoplasia and ear anomalies with a de novo apparently balanced chromosomal translocation, 46,XY,t(3;14)(q28;q23.2). Translocation breakpoint analysis using FISH and high-resolution microarray comparative genomic hybridization (CGH) has identified a 9.66 Mb deleted region on the long arm of chromosome 14 which includes the genes BMP4, OTX2, RTN1, SIX6, SIX1, and SIX4. Three other patients with interstitial deletions involving 14q22-23 have been described, all with bilateral anophthalmia, pituitary abnormalities, ear anomalies, and a facial phenotype similar to our patient. OTX2 is involved in ocular developmental defects, and the severity of the ocular phenotype in our patient and the other 14q22-23 deletion patients, suggests this genomic region harbors other gene/s involved in ocular development. BMP4 haploinsufficiency is predicted to contribute to the ocular phenotype on the basis of its expression pattern and observed murine mutant phenotypes. In addition, deletion of BMP4 and SIX6 is likely to contribute to the abnormal pituitary development, and SIX1 deletion may contribute to the ear and other craniofacial features. This indicates that contiguous gene deletion may contribute to the phenotypic features in the 14q22-23 deletion patients.

SIX1 Mutation Associated With Enlargement of the Vestibular Aqueduct in a Patient With Branchio‐Oto Syndrome

: The objectives of this study were to identify SIX1 gene mutations in a patient with branchio-oto syndrome (BO) and to clarify the relationship between SIX1 mutation and enlargement of the vestibular aqueduct (EVA). : A genetic study and retrospective chart review for a patient in whom EYA1 mutation had already been excluded was conducted. We studied a Japanese patient who had autosomal-dominant mixed hearing loss, a unilateral ear pit and unilateral EVA, and who was previously diagnosed as having BO. We searched for SIX1 and SLC26A4 mutations using polymerase chain reaction and direct gene sequencing. : The patient carried a heterozygous A-->G mutation at nucleotide 386 within exon 1 of SIX1 that resulted in substitution of a cysteine for a tyrosine at codon 129 (Y129C) of the gene product. Y129C is a previously identified SIX1 mutation and was not detected in any of our 164 control chromosomes. No SLC26A4 mutations were identified. : Y129C mutation in SIX1 may cause EVA as well as BO.

EYA1 and SIX1 gene mutations in Japanese patients with branchio-oto-renal (BOR) syndrome and related conditions

We isolated genomic DNA from 15 patients with branchio-oto-renal (BOR) syndrome or BOR-related conditions. Seven patients had BOR syndrome (two familial and five sporadic), and eight had deafness and renal malformations without branchial fistula (BOR-related conditions). We analyzed all exons and exon-intron boundaries of EYA1 and SIX1 using the polymerase chain reaction (PCR) direct sequencing, and characterized their mutations. In some patients, analysis of mRNA by reverse transcription (RT)-PCR was performed to examine whether the mutation affects the mRNA splicing. We identified five novel disease-causing heterozygous EYA1 mutations in five patients with BOR syndrome (two familial and three sporadic, 5/7=71%), but EYA1 and SIX1 mutations were not detected in the other two patients with BOR syndrome or any of the patients with BOR-related conditions. The detected EYA1 mutations were two nonsense mutations, two splicing acceptor-site mutations, and a point mutation (G>T) of the first base of exon 10. Analysis of mRNA by RT-PCR direct sequencing revealed that the latter point mutation led to the skipping of exon 10. In conclusion, (1) EYA1 mutations are a major cause of BOR syndrome in Japanese, (2) EYA1 and SIX1 mutations were not a major cause of BOR-related conditions, (3) we demonstrated for the first time that the point mutation (G>T) of the first base of the exon in EYA1 gene induced exon skipping.

BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.

Lens regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation. After lens removal, the pigment epithelial cells of the dorsal, but not the ventral, iris dedifferentiate and then differentiate to form a new lens. Understanding how this process is regulated might provide clues about why lens regeneration does not occur in higher vertebrates. The genes six-3 and pax-6 are known to induce ectopic lenses during embryogenesis. Here we tested these genes, as well as members of the bone morphogenetic protein (BMP) pathway that regulate establishment of the dorsal-ventral axis in embryos, for their ability to induce lens regeneration. We show that the lens can be regenerated from the ventral iris when the BMP pathway is inhibited and when the iris is transfected with six-3 and treated with retinoic acid. In intact irises, six-3 is expressed at higher levels in the ventral than in the dorsal iris. During regeneration, however, only expression in the dorsal iris is significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3 and retinoic acid or by BMP inhibitors. These data suggest that lens regeneration can be achieved in noncompetent adult tissues and that this regeneration occurs through a gene regulatory mechanism that is more complex than the dorsal expression of lens regeneration-specific genes.

Gene Amplification Is a Mechanism of Six1 Overexpression in Breast Cancer

The Six1 homeoprotein plays a critical role in expanding progenitor populations during normal development via its stimulation of proliferation and inhibition of apoptosis. Overexpression of Six1 is observed in several tumor types, suggesting that when expressed out of context, Six1 may contribute to tumorigenesis by reinstating properties normally conveyed on developing cells. Indeed, Six1 contributes to tumor cell proliferation both in breast cancer and in rhabdomyosarcomas, in which it is also implicated in metastasis. Whereas Six1 overexpression has been reported in several tumor types, the mechanism responsible for its overexpression has not previously been examined. Here we show that a change in gene dosage may contribute to Six1 mRNA overexpression. Significant Six1 gene amplification and overrepresentation occurs in numerous breast cancer cell lines as compared with normal mammary epithelial cells, and the changes in gene dosage correlate with increased Six1 mRNA levels. Of 214 human infiltrating ductal breast carcinomas examined for Six1 gene dosage, 4.7% show Six1 amplification/overrepresentation, and tumors that exhibit an increase in Six1 gene dosage overexpress Six1 mRNA. These data implicate Six1 gene amplification/overrepresentation as a mechanism of Six1 mRNA overexpression in human breast cancer.

Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo

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. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. 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.

New Perspectives on Eye Development and the Evolution of Eyes and Photoreceptors

Recent experiments on the genetic control of eye development have opened up a completely new perspective on eye evolution. The demonstration that targeted expression of one and the same master control gene, that is, Pax6 can induce the formation of ectopic eyes in both insects and vertebrates, necessitates a reconsideration of the dogma of a polyphyletic origin of the various eye types in all the animal phyla. The involvement of Pax6 and six1 and six3 genes, which encode highly conserved transcription factors, in the genetic control of eye development in organisms ranging from planarians to humans argues strongly for a monophyletic origin of the eye. Because transcription factors can control the expression of any target gene provided it contains the appropriate gene regulatory elements, the conservation of the genetic control of eye development by Pax6 among all bilaterian animals is not due to functional constraints but a consequence of its evolutionary history. The prototypic eyes postulated by Darwin to consist of two cells only, a photoreceptor and a pigment cell, were accidentally controlled by Pax6 and the subsequent evolution of the various eye types occurred by building onto this original genetic program. A hypothesis of intercalary evolution is proposed that assumes that the eye morphogenetic pathway is progressively modified by intercalation of genes between the master control genes on the top of the hierarchy and the structural genes like rhodopsin at the bottom. The recruitment of novel genes into the eye morphogenetic pathway can be due to at least two different genetic mechanisms, gene duplication and enhancer fusion.In tracing back the evolution of eyes beyond bilaterians, we find highly developed eyes in some box-jellyfish as well as in some Hydrozoans. In Hydrozoans the same orthologous six genes (six1 and six3) are required for eye regeneration as in planarians, and in the box jellyfish Tripedalia a pax B gene, which may be a precursor of Pax6, was found to be expressed in the eyes. In contrast to the adults, which have highly evolved eyes, the Planula larva of Tripedalia has single- celled photoreceptors similar to some unicellular protists. For the origin of photoreceptor cells in metazoa, I propose two hypotheses, one based on cellular differentiation and a more speculative one based on symbiosis. The former assumes that photoreceptor cells originated from a colonial protist in which all the cells were photosensitive and subsequent cellular differentiation to give rise to photoreceptor cells. The symbiont hypothesis, which I call the Russian doll model, assumes that photosensitivity arose first in photosynthetic cyanobacteria that were subsequently taken up into red algae as primary chloroplasts. The red algae in turn were taken up by dinoflagellates as secondary chloroplasts and in some species evolved into the most sophisticated eye organelles, as found, for example, in some dinoflagellates like Erythropsis and Warnovia, which lack chloroplasts. Because dinoflagellates are commonly found as symbionts in cnidarians, the dinoflagellates may have transferred their photoreceptor genes to cnidarians. In cnidarians such as Tripedalia the step from photoreceptor organelles to multicellular eyes has occurred. These two hypotheses, the cellular differentiation and the symbiont hypothesis, are not mutually exclusive and are the subject of further investigations.

Fusarium oxysporum Evades I-3-Mediated Resistance Without Altering the Matching Avirulence Gene

I-3-Mediated resistance of tomato against Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici depends on Six1, a protein that is secreted by the fungus during colonization of the xylem. Among natural isolates of F. oxysporum f. sp. lycopersici are several that are virulent on a tomato line carrying only the I-3 resistance gene. However, evasion of I-3-mediated resistance by these isolates is not correlated with mutation of the SIX1 gene. Moreover, the SIX1 gene of an I-3-virulent isolate was shown to be fully functional in that i) the gene product is secreted in xylem sap, ii) deletion leads to a further increase in virulence on the I-3 line as well as reduced virulence on susceptible lines, and iii) the gene confers full avirulence on the I-3 line when transferred to another genetic background. Remarkably, all I-3-virulent isolates were of race 1, suggesting a link between the presence of AVR1 and evasion of I-3-mediated resistance.

Evolutionary origins of vertebrate placodes: insights from developmental studies and from comparisons with other deuterostomes

Ectodermal placodes comprise the adenohypophyseal, olfactory, lens, profundal, trigeminal, otic, lateral line, and epibranchial placodes. The first part of this review presents a brief overview of placode development. Placodes give rise to a variety of cell types and contribute to many sensory organs and ganglia of the vertebrate head. While different placodes differ with respect to location and derivative cell types, all appear to originate from a common panplacodal primordium, induced at the anterior neural plate border by a combination of mesodermal and neural signals and defined by the expression of Six1, Six4, and Eya genes. Evidence from mouse and zebrafish mutants suggests that these genes promote generic placodal properties such as cell proliferation, cell shape changes, and specification of neurons. The common developmental origin of placodes suggests that all placodes may have evolved in several steps from a common precursor. The second part of this review summarizes our current knowledge of placode evolution. Although placodes (like neural crest cells) have been proposed to be evolutionary novelties of vertebrates, recent studies in ascidians and amphioxus have proposed that some placodes originated earlier in the chordate lineage. However, while the origin of several cellular and molecular components of placodes (e.g., regionalized expression domains of transcription factors and some neuronal or neurosecretory cell types) clearly predates the origin of vertebrates, there is presently little evidence that these components are integrated into placodes in protochordates. A scenario is presented according to which all placodes evolved from an adenohypophyseal-olfactory protoplacode, which may have originated in the vertebrate ancestor from the anlage of a rostral neurosecretory organ (surviving as Hatschek's pit in present-day amphioxus).

Six1 is not involved in limb tendon development, but is expressed in limb connective tissue under Shh regulation

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3 −/− mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1 −/− mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.

Identification of a NovelEYA1Splice-Site Mutation in a Danish Branchio-Oto-Renal Syndrome Family

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by variable clinical manifestations including branchial fistulae, preauricular pits, ear malformations, hearing impairment, and renal anomalies. BOR is caused by mutations in the genes EYA1 and SIX1. A Danish BOR family with five affected individuals in three generations was analyzed for mutations in all 17 exons of EYA1 using direct sequencing of polymerase chain reaction (PCR) amplified genomic DNA. A novel splice-site mutation (IVS9+1 G>C) was detected in all affected family members but not in unaffected family members or in 96 controls. We conclude that this mutation is causing BOR in the family, most likely as a result of haploinsufficiency or an abnormal protein product caused by aberrant splicing of EYA1 mRNA.

Absence ofSIX6Mutations in Microphthalmia, Anophthalmia, and Coloboma

To investigate whether 173 patients with microphthalmia, anophthalmia, and coloboma have mutations in the eye-development gene SIX6. The two exons of the SIX6 gene were amplified by PCR from patients' genomic DNA and directly sequenced to search for mutations. The PCR products of 75 patients were also analyzed by denaturing high-performance liquid chromatography (DHPLC). Six SIX6 polymorphisms were identified in the patient panel. Three of these polymorphisms change the encoded amino acid. However, all six polymorphisms were also identified in unaffected individuals. There was no statistically significant difference in genotypes between patients and control subjects. No evidence was found that SIX6 mutations underlie human congenital structural eye malformations.

Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes

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. Here, we have examined their role in cranial sensory neurogenesis. Our data show that the initial cell fate determination for the vestibuloacoustic neurons and their delamination appeared to be unaffected in the absence of Eya1 or Six1 as judged by the expression of the basic helix-loop-helix genes, Neurog1 that specifies the neuroblast cell lineage, and Neurod that 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 that 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 resulted in abnormal apoptosis of the progenitor cells. Furthermore, we show that disruption of viable ganglion formation leads to pathfinding errors of branchial motoneurons. Finally, our 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.