Six3 Expression Research Articles

Molecular Characterization of Human Retinal Progenitor Cells

To examine the molecular profile of fetal human retinal progenitor cells (hRPCs) expanded in vitro and those grown in a co-culture system with mouse retina through the analysis of protein and gene expression and neurotransmitter-stimulated calcium dynamics. hRPCS were isolated from human retina of 14 to 18 weeks gestational age (GA) and expanded in vitro. Immunoblot, microarray, and immunocytochemistry (ICC) assays were performed on undifferentiated hRPCs and those co-cultured with mouse retinas for 2 weeks. Cell function was assessed by using calcium imaging. The ICC results showed a gradual decrease in the percentages of KI67-, SOX2-, and vimentin-positive cells from passages (P) 1 to P6, whereas a sustained expression of nestin and PAX6 was observed through P6. Microarray analysis of P1 hRPCs showed the expression of early retinal developmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2, HES5, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). At P6, hRPCs continued to express VIM, KI67, NES, PAX6, SOX2, GNL3, and SIX6. On co-culture, there was a significant increase in the expression of MKI67, PAX6, SOX2, GNL3, SIX3, and RHO (rhodopsin). Calcium imaging showed a functional response to excitatory neurotransmitters. Fetal-derived hRPCs show molecular characteristics indicative of a retinal progenitor state up to P6 (latest passage studied). They show a progressive decrease in the expression of immature markers as they reach P6. These cells are functional, respond to excitatory neurotransmitters, and exhibit changes in expression patterns in response to co-culture with mouse retina.

Deficiency in Six2 during prenatal development is associated with reduced nephron number, chronic renal failure, and hypertension inBr/+ adult mice

The Br/+ mutant mouse displays decreased embryological expression of the homeobox transcription factor Six2, resulting in hertitable renal hypoplasia. The purpose of this study was to characterize the renal physiological consequences of embryonic haploinsuffiency of Six2 by analyzing renal morphology and function in the adult Br heterozygous mutant. Adult Br/+ kidneys weighed 50% less than those from wild-type mice and displayed glomerulopathy. Stereological analysis of renal glomeruli showed that Br/+ kidneys had an average of 88% fewer glomeruli than +/+ kidneys, whereas individual glomeruli in Br/+ mice maintained an average volume increase of 180% compared with normal nephrons. Immunostaining revealed increased levels of endothelin-1 (ET-1), endothelin receptors A (ET(A)) and B (ET(B)), and Na-K-ATPase were present in the dilated renal tubules of mutant mice. Physiological features of chronic renal failure (CRF) including elevated mean arterial pressure, increased plasma creatinine, and dilute urine excretion were measured in Br/+ mutant mice. Electron microscopy of the Br/+ glomeruli revealed pathological alterations such as hypercellularity, extracellular matrix accumulation, and a thick irregular glomerular basement membrane. These results indicate that adult Br/+ mice suffer from CRF associated with reduced nephron number and renal hypoplasia, as well as glomerulopathy. Defects are associated with embryological deficiencies of Six2, suggesting that proper levels of this protein during nephrogenesis are critical for normal glomerular development and adult renal function.

Misexpression of miR-196a induces eye anomaly in Xenopus laevis

miR-196a is located in the posterior trunk and plays a role in limb development. Here we show that miR-196a is able to induce eye anomaly in Xenopus laevis. Microinjection of synthetic miRNA precursor molecule for mammalian miR-196a into Xenopus embryo is sufficient for miR-196a overexpression during early development. The misexpression of miR-196a in anterior embryo led to dose-dependent eye anomalies, especially size reduction. In addition, the expression of ET, Rx1, Six3, Pax6, Lhx2, Optx2 and Ath5 in eye field or optic cup was also down-regulated. These results indicate that miR-196a can target gene(s) in the genetic network involved in eye formation, providing a potential tool for studying the mechanisms of eye development and diseases.

The LIM homeobox transcription factor Lhx2 is required to specify the retina field and synergistically cooperates with Pax6 for Six6 trans-activation

In mammals, a limited set of homeobox-containing transcription factors are expressed in the presumptive eye field and required to initiate eye development. How these factors interact together at the genetic and molecular level to coordinate this developmental process is poorly understood. We found that the Lhx2 and Pax6 transcription factors operate in a concerted manner during retinal development to promote transcriptional activation of the Six6 homeobox-gene in primitive and mature retinal progenitors. Lhx2 demarcates the presumptive retina field at the neural plate stage and Lhx2 inactivation delays initiation of Rx, Six3 and Pax6 expression in this domain. The later expressed Six6 is properly activated in the pituitary/hypothalamic axis of Lhx2 −/− embryos, but expression fails to be initiated in the optic vesicle. Lhx2 and Pax6 associate with the chromatin at several regions of Six6 in vivo and cooperate for trans-activation of Six6 regulatory elements in vitro. In retinal progenitor/stem cells, both Lhx2 and Pax6 are genetically required for proper Six6 expression and forced co-expression of Lhx2 and Pax6 can synergistically trans-activate the Six6 locus. Our work reveals how two master regulators of eye development coordinate their action to sequentially promote tissue-specific transcriptional initiation and full activation of a retinal determinant gene.

Expression of regulatory genes Px6, Otx2, Six3, and FGF2 during newt retina regeneration

Molecular-genetic mechanisms of regeneration of adult newt (Pleurodeles waltl) retina were studied. For the first time, a comparative analysis of the expression of regulatory genes Pax6, Otx2, and Six3 and Fgf2 genes encoding signal molecules was performed in the native retinal pigment epithelium (RPE) and retina and at successive stages of retina regeneration. Cell differentiation types were determined using genetic markers of cell differentiation in the RPE (RPE65) and the retina (betaII-tubulin and Rho). Activation of the expression of neurospecific genes Pax6 and Six3 and the growth factor gene Fgf2 and suppression of activation of the regulatory gene Otx2 and the RPE65 were observed at the stage of multipotent neuroblast formation in the regenerating retina. The expression of genes Pax6, Six3, and Fgf2 was retained at a later stage of retina regeneration at which the expression of retinal differentiation markers, the genes encoding betaII-tubulin (betaII-tubulin) and rhodopsin (Rho), was also detected. We assume that the above regulatory genes are multifunctional and control not only transdifferentiation of RPE cells (the key stage of retina regeneration) but also differentiation of regenerating retina cells. The results of this study, demonstrating coexpression of Pax6, Six3, Fgf2, betaII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.

Misexpression of Six2 is associated with heritable frontonasal dysplasia and renal hypoplasia in 3H1 Br mice.

A radiation-induced mouse mutant, Brachyrrhine (Br), exhibits frontonasal dysplasia and renal hypoplasia, two malformations associated with deficiencies in mesenchymal condensation. The purpose of this study was to resolve the Br locus, evaluate possible candidate genes, and identify developmental defects in the mutant chondrocranium. Linkage analysis mapped the Br mutation to a critical region distal to D17Mit76, which contains only one gene, the transcription factor Six2. Sequence analysis of the Six2 gene, including 1.5 kb of the promoter, failed to reveal the Br mutation. However, homozygous Br/Br embryos showed almost complete absence of Six2 mRNA and protein in craniofacial and renal tissues while heterozygous Br/+ embryos displayed intermediate Six2 levels. Mutant embryos displayed malformations of neural crest-derived structures of the anterior cranium where Six2 is normally expressed. These data suggest a mutation in a novel cis-acting regulatory region inhibits Six2 expression and is associated with frontonasal dysplasia and renal hypoplasia.

Six2 functions redundantly immediately downstream of Hoxa2

Hox transcription factors control morphogenesis along the head-tail axis of bilaterians. Because their direct functional targets are still poorly understood in vertebrates, it remains unclear how the positional information encoded by Hox genes is translated into morphogenetic changes. Here, we conclusively demonstrate that Six2 is a direct downstream target of Hoxa2 in vivo and show that the ectopic expression of Six2, observed in the absence of Hoxa2, contributes to the Hoxa2 mouse mutant phenotype. We propose that Six2 acts to mediate Hoxa2 control over the insulin-like growth factor pathway during branchial arch development.

Establishment of embryonic kidney and facial culture systems to evaluate the role of the transcription factor Six2 through RNAi

The Br mouse mutation results in frontonasal and renal hypodysplasia. Linkage analysis mapped the Br mutation close to the homeobox transcription factor Six2, which is normally expressed in the metanephric and facial mesenchyme during embryonic development. The purpose of this study is to evaluate Six2's role in nephrogenesis and craniofacial morphogenesis by silencing Six2 in embryonic kidney and facial prominence organ cultures. Kidney organ cultures were established from E11.5 mouse embryos, cultured for 48 hours, and immunostained to reveal ureteric bud branching patterns. Frontonasal (FNP), lateral nasal (LNP), and maxillary prominences (MXP) of E11.5 embryos were dissected and Six2 expression was measured with qRT‐PCR. Six2 expression was highest in the FNP, 3‐fold less in the MXP, and 6‐fold less in the LNP. This was confirmed by immunostaining in embryos at the same age. We evaluated five different shRNA constructs for silencing efficiency of Six2 using p19 embryonal carcinoma cells. As measured with qRT‐PCR, Six2 expression was unaffected using four of the constructs and decreased expression by 30–50% with the fifth. Using this shRNA construct, we will transfect our established culture systems, confirm Six2 expression is down‐regulated, and then evaluate differences in kidney tubule branching and FNP cell proliferation.

Secretion and Organization of a Cornea-like Tissue In Vitro by Stem Cells from Human Corneal Stroma

To investigate the potential of human corneal stromal stem cells to assume a keratocyte phenotype and to organize extracellular matrix (ECM) in vitro similar to corneal stromal tissue. Human corneal stromal stem cells (hCSSC) were isolated as side population cells by flow cytometry. Cloned hCSSC were cultured as free-floating pellets in serum-free media for 3 weeks. Gene expression was examined using gene array, quantitative RT-PCR, immunostaining, and immunoblotting. Transmission electron microscopy showed collagen fibril size and alignment. Pellet cultures of hCSSC in serum-free media upregulated the expression of keratocyte-specific genes and secreted substantial ECM containing characteristic stromal components: keratocan, keratan sulfate, collagen I, collagen V, and collagen VI. Abundant connexin 43 and cadherin 11 in pellets demonstrated cell-cell junctions typical of keratocytes in vivo. Electron microscopy of the pellet cultures revealed abundant fibrillar collagen, some of which was aligned in parallel arrays similar to those of stromal lamellae. Gene array identified expression in pellets of several genes highly expressed by keratocytes. Transcripts for these keratocyte genes -- FLJ30046, KERA, ALDH3A1, CXADR, PTGDS, PDK4, MTAC2D1, F13A1 -- were increased by as much as 100-fold in pellets compared with hCSSC. Simultaneously, expression of stem cell genes BMI1, KIT, NOTCH1, SIX2, PAX6, ABCG2, SPAG10, and OSIL was reduced by a similar factor in pellets compared with hCSSC. Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.

Ectopic Six3 expression in the dragon eye goldfish

For goldfish ( Carassius auratus), there are many varieties with different eye phenotypes due to artificial selection and adaptive evolution. Dragon eye is a variant eye characterized by a large-size eyeball protruding out of the socket similar to the eye of dragon in Chinese legends. In this study, anatomical structure of the goldfish dragon eye was compared with that of the common eye, and a stretching of the retina was observed in the enlarged dragon eye. Moreover, the homeobox-containing transcription factor Six3 cDNAs were cloned from the two types of goldfish, and the expression patterns were analyzed in both normal eye and dragon eye goldfish. No amino acid sequence differences were observed between the two deduced peptides, and the expression pattern of Six3 protein in dragon eye is quite similar to common eye during embryogenesis, but from 2 days after hatching, ectopic Six3 expression began to occur in the dragon eye, especially in the outer nuclear layer cells. With eye development, more predominant Six3 distribution was detected in the outer nuclear layer cells of dragon eye than that of normal eye, and fewer cell-layers in outer nuclear layer were observed in dragon eye retina than in normal eye retina. The highlight of this study is that higher Six3 expression occurs in dragon eye goldfish than in normal eye goldfish during retinal development of larvae.

A model of early molecular regionalization in the chicken embryonic pretectum

The pretectal region of the brain is visualized as a dorsal region of prosomere 1 in the caudal diencephalon, including derivatives from both the roof and alar plates. Its neuronal derivatives in the adult brain are known as pretectal nuclei. The literature is inconsistent about the precise anteroposterior delimitation of this region and on the number of specific histogenetic domains and subdomains that it contains. We performed a cross-correlated gene-expression map of this brain area in chicken embryos, with the aim of identifying differently fated pretectal domains on the basis of combinatorial gene expression patterns. We examined in detail Pax3, Pax6, Pax7, Tcf4, Meis1, Meis2, Nkx2.2, Lim1, Dmbx1, Dbx1, Six3, FoxP2, Zic1, Ebf1, and Shh mRNA expression, as well as PAX3 and PAX7 immunoreaction, between stages HH11 and HH28. The patterns analyzed serve to fix the cephalic and caudal boundaries of the pretectum and to define three molecularly distinct anteroposterior pretectal domains (precommissural, juxtacommissural, and commissural) and several dorsoventral subdomains. These molecular specification patterns are established step by step between stages HH10 and HH18, largely before neurogenesis begins. This set of gene-architectonic data constitutes a useful scaffold for correlations with fate maps and other experimental embryologic results and may serve as well for inquiries on homologies in this part of the brain.

Purine-mediated signalling triggers eye development

A conserved network of eye field transcription factors (EFTFs) underlies the development of the eye in vertebrates and invertebrates. To direct eye development, Pax6, a key gene in this network, interacts with genes encoding other EFTFs such as Rx1 and Six3 (refs 4-6). However, the mechanisms that control expression of the EFTFs remain unclear. Here we show that purine-mediated signalling triggers both EFTF expression and eye development in Xenopus laevis. Overexpression of ectonucleoside triphosphate diphosphohydrolase 2 (E-NTPDase2), an ectoenzyme that converts ATP to ADP, caused ectopic eye-like structures, with occasional complete duplication of the eye, and increased expression of Pax6, Rx1 and Six3. In contrast, downregulation of endogenous E-NTPDase2 decreased Rx1 and Pax6 expression. E-NTPDase2 therefore acts upstream of these EFTFs. To test whether ADP (the product of E-NTPDase2) might act to trigger eye development through P2Y1 receptors, selective in Xenopus for ADP, we simultaneously knocked down expression of the genes encoding E-NTPDase2 and the P2Y1 receptor. This could prevent the expression of Rx1 and Pax6 and eye formation completely. We next measured ATP release in the presumptive eye field, demonstrating a transient release of ATP at a time that could plausibly trigger (once converted to ADP) expression of the EFTFs. This surprising role for transient purine-mediated signalling in eye development may be widely conserved, because alterations to the locus of E-NTPDase2 on human chromosome 9 cause severe head and eye defects, including microphthalmia. Our results suggest a new mechanism for the initiation of eye development.

Repression of Six3 by a corepressor regulates rhodopsin expression

Here, we provide gain-of-function, loss-of function, and molecular evidence supporting genetic interactions between metastasis associated protein 1 (MTA1) and Six3 and between Six3 and rhodopsin. We discovered that MTA1 physically interacts with the Six3 chromatin in a histone deacetylase-dependent manner, leading to transcriptional suppression of the Six3 gene. MTA1 is also a Six3-interacting corepressor that contributes to a self-negative regulation of Six3 transcription by Six3. In contrast, deletion of the MTA1 alleles in murine embryonic fibroblasts or its knockdown in rat retinal ganglion cells stimulates Six3 expression. MTA1 inactivation in the MTA1-null mice results in an elevated Six3 level and proliferation of the retina cells with no obvious abnormities in eye formation. However, unexpectedly, we discovered an enhanced recruitment of Six3 to the rhodopsin chromatin in retina from the MTA1-null mice; Six3's homeodomain interacts with specific DNA elements in the rhodopsin promoter to stimulate its transcription, resulting in increased rhodopsin expression. Further, in holoprosencephaly patients, Six3 protein with a naturally occurring deletion mutation in the helix 3 of the homeodomain does not bind to rhodopsin DNA or stimulate rhodopsin transcription, implying a potential defective rhodopsin pathway in the affected holoprosencephaly patients. Further Six3 cooperates with Crx or NRL in stimulating transcription from the rhodopsin-luc. These findings reveal a previously unrecognized role for the MTA1 as an upstream modifier of Six3 and indicate that Six3 is a direct stimulator of rhodopsin expression, thus revealing a putative role for the MTA1/Six3/rhodopsin pathway in vertebrate eye.

A zebrafish LMO4 ortholog limits the size of the forebrain and eyes through negative regulation of six3b and rx3

The Six3 and Rx3 homeodomain proteins are essential for the specification and proliferation of forebrain and retinal precursor cells of the vertebrate brain, and the regulatory networks that control their expression are beginning to be elucidated. We identify the zebrafish lmo4b gene as a negative regulator of forebrain growth that acts via restriction of six3 and rx3 expression during early segmentation stages. Loss of lmo4b by morpholino knockdown results in enlargement of the presumptive telencephalon and optic vesicles and an expansion of the post-gastrula expression domains of six3 and rx3. Overexpression of lmo4b by mRNA injection causes complementary phenotypes, including a reduction in the amount of anterior neural tissue, especially in the telencephalic, optic and hypothalamic primordia, and a dosage-sensitive reduction in six3 and rx3 expression. We suggest that lmo4b activity is required at the neural boundary to restrict six3b expression, and later within the neural plate to for attenuation of rx3 expression independently of its effect on six3 transcription. We propose that lmo4b has an essential role in forebrain development as a modulator of six3 and rx3 expression, and thus indirectly influences neural cell fate commitment, cell proliferation and tissue growth in the anterior CNS.

Six3 Controls the Neural Progenitor Status in the Murine CNS

Six3, a homeodomain-containing transcriptional regulator belonging to the Six/so family, shows a defined spatiotemporal expression pattern in the developing murine telencephalon, suggesting that it may control the development of specific subsets of neural progenitors. We find that retrovirus-mediated misexpression of Six3 causes clonal expansion of isolated cortical progenitor cells by shortening their cell cycle and by prolonging their amplification period, while maintaining them in an immature precursor state. Our results show that the observed effects exerted by Six3 overexpression in mammalian brain depend strictly on the integrity of its DNA-binding domain, suggesting that Six3 action likely relies exclusively on its transcriptional activity. In vivo upregulation of Six3 expression in single progenitor cells of the embryonic telencephalon keeps them in an undifferentiated state. Our observations point to a role of Six3 in the control of the subtle equilibrium between proliferation and differentiation of defined precursor populations during mammalian neurogenesis.

Deficient embryonic expression of SIX2 is associated with decreased glomerular number and chronic renal failure in the adult 3H1 Br/+ mouse.

Deficient embryonic expression of SIX2 is associated with decreased glomerular number and chronic renal failure in the adult 3H1 Br/+ mouse.

Hox11 genes interact with Eya1 and Pax2 to activate Six2 and Gdnf expression during metanephric kidney induction

Hox11 genes interact with Eya1 and Pax2 to activate Six2 and Gdnf expression during metanephric kidney induction

CNBP regulates forebrain formation at organogenesis stage in chick embryos

We recently demonstrated that Cellular Nucleic acid Binding Protein ( CNBP) −/− mouse embryos exhibit forebrain truncation due to a lack of proper morphogenetic movements of the anterior visceral endoderm (AVE) during pre-gastrulation stage (Chen, W., Liang, Y., Deng, W., Shimizu, K., Ashique, A.M., Li, E., Li, Y.P., 2003. The zinc-finger protein CNBP is required for forebrain formation in the mouse, Development 130, 1367–1379). However, CNBP expression pattern in the mouse forebrain suggests that CNBP may have more direct effects during forebrain development. Our data show that CNBP is expressed in tissues of early chick embryo that are the equivalent to the mouse embryo. Using a combination of RNAi-silencing and Retrovirus-misexpression approaches, we investigated the temporal function of CNBP in the specification/development of the chick forebrain during organogenesis. The silencing of CNBP expression resulted in forebrain truncation and the absence of BF-1, Six3 and Hesx1 expression, but not Otx2 in chick embryos. Misexpression of CNBP induced the expression of BF-1, Six3 and Hesx1 in the hindbrain, but not the expression of Otx2. These results offer novel insights into the function of CNBP during organogenesis as the regulator of forebrain formation and a number of rostral head transcription factors. Moreover, CNBP and Otx2 may play roles as regulators of forebrain formation in two parallel pathways. These new insights into CNBP functions underscore the essential role of CNBP in forebrain formation during chick embryo organogenesis.

Heritable renal hypoplasia and frontonasal dysplasia is associated with misexpression of six2

Renal hypoplasia and frontonasal dysplasia are malformations associated with tissues derived from mesoderm. A radiation induced mouse mutant, called Brachyrrhine (Br) exhibits both malformations. Initial microsatellite analysis placed the mutation on distal chromsome 17 in Br. The purpose of this study was to further resolve the Br locus and determine tissue expression of candidate genes. An interspecific backcross breeding strategy was designed using 3H1 Br/+ mice crossed to Castaneus and Balb mice. Segregation analysis showed that Br was inherited as a semidominant lethal mutation when outcrossed to both inbred strains. DNA was extracted from F2 progeny and scored for recombinations. Results showed that Br localized to a region just distal to D17Mit76 that included six2. Using this microsatellite as a genotypic marker, C3H1 x Balb Br/+ F1 mice were crossed and embryos were obtained at embryonic days 10.5–11.5. The embryos were subjected to whole-mount in situ hybridization (WMISH) and quantitative RT-PCR. Results showed that homozygous Br/Br animals have significantly reduced six2 expression. Quantitative RT-PCR reflected a pattern of six2 haploinsufficiency with heterozygous mutants showing about a 50% decrease in gene dosage and homozygous mutants showing about a 10% expression. It is concluded that a mutation affecting six2 expression is responsible for mesodermal differentiation pathways in the face and kidney. Supported by R01-DK064752, Meiji Yasuda Endowment, and HS-BRIN P20 PP16467

Temporal requirement ofHoxa2in cranial neural crest skeletal morphogenesis

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.