SIX1 Research Articles

Dissecting the Role of the Six1‐Associated Genes in Craniofacial Development and Disease

Genetic variants of SIX1, a transcription factor implicated in many cellular processes during craniofacial development, and of its co‐factor EYA1 underlie 50% of Branchio‐oto‐renal syndrome (BOR) cases. BOR is an autosomal dominant disorder characterized by various craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. Interestingly, some BOR variants in SIX1 also underlie craniosynostosis (CRS) which indicates that SIX1 has a crucial role in craniofacial bone development. One important aspect of SIX1 function is that it interacts with co‐factors that can induce or repress its transcriptional activity. In this work, we expand our knowledge of the Six1 gene regulatory network with respect to craniofacial development. We first used a Six1 knockout mouse line to assess gene expression profiles of dissected E10.5 mandibular arches which give rise to the middle ear ossicles and lower jaw, via RNA‐sequencing. Our transcriptomic analysis led to the identification of 122 genes, including components of the FGF and WNT signaling pathways, known contributors to craniofacial bone development. In parallel, we used in vitro studies to characterize three novel co‐factors of Six1: Sobp, Mcrs1 and Pa2g4 that were recently identified in Xenopus. Our findings indicate that Sobp and Mcrs1 are bona fide Six1 co‐factors in mouse. We are now studying the genes identified in the RNA‐seq and the novel Six1 co‐factors using in vitro culture of mouse pre‐osteoblastic cells (MC3T3‐E1) to identify their role in bone formation. Altogether, our results identified novel Six1‐associated genes that are potential candidate genes for the unresolved cases of BOR and CRS.

Investigating the effects of the marine drug Manzamine‐A related to craniofacial development in vitro

Manzamine‐A is a marine‐derived alkaloid which has anti‐viral and anti‐proliferative properties and is currently being investigated for its efficacy in the treatment of certain viruses (malaria, herpes, HIV‐1) and cancers (breast, cervical, colorectal). Manzamine‐A has been found to exert effects via modulation of SIX1 gene expression, a gene critical to craniofacial development via the WNT, NOTCH, and PI3K/AKTpathways. To date little work has focused on Manzamine‐A and how its use may effect craniofacial development. We hypothesize that Manzamine‐A, through SIX1, alters bone cell activity important to craniofacial skeletal development. As Manzamine‐A is a drug that has great potential in a variety of therapeutics, it is critical that we asses potential side effects that this drug could have on both patients and any developmental effects that may occur during use by expectant mothers. We aimed to assess the effects of Manzamine‐A on four cell types, pre‐osteoblasts, osteoblasts, pre‐osteoclasts, and osteoclasts. PCR, qrtPCR, MTS cell viability assays and Caspase 3/7 apoptosis assay were used to test the effects of Manzamine‐A on these cells. We also performed alkaline phosphatase (ALP) and Tartrate Resistant Acid Phosphatase (TRAP) assays to test the function of osteoblasts and osteoclasts respectively. To date we have found that Six‐1 is highly expressed in osteoblasts and their progenitors. In contrast Six‐1 has low expression levels in osteoclasts and their progenitors. Further, osteoblast progenitor cells exhibit great sensitivity to Manzamine‐A treatment exhibited by a significant decrease in cell viability, increase in cellular apoptosis, and decrease in ALP activity. Studies are ongoing for osteoclast lineage cells. Overall our preliminary data suggests Manzamine‐A may have great effects on bone health overall and may disrupt skeletal development if exposed during pregnancy or postnatal development.

A human coronavirus OC43-derived polypeptide causes neuropathic pain.

Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV‐OC43 p65‐like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP‐derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV‐OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception‐, immune‐, and cellular energy‐related activities. Affinity capture shows the association of OC43p with an Na+/K+‐transporting ATPase, providing a potential direct target and mechanistic insight into virus‐induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV‐OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus‐induced neuropathic pain.

Regulation of miR-30b in cancer development, apoptosis, and drug resistance

miR-30b, which is encoded by the gene located on chromosome 8q24.22, plays an important role in a variety of diseases. In most types of tumors, miR-30b significantly inhibits the proliferation, migration, and invasion of cancer cells through the regulation of target genes. Moreover, miR-30b can inhibit the PI3K/AKT signaling pathway through the regulation of EGFR, AKT, Derlin-1, GNA13, SIX1, and other target genes, thus inhibiting the EMT process of tumor cells and promoting apoptosis. In addition, miR-30 plays a significant role in alleviating drug resistance in tumor cells. Although the use of miR-30b as a clinical diagnostic indicator or anticancer drug is still facing great difficulties in the short term, with the deepening of research, the potential application of miR-30b is emerging.

Deletion of the homeodomain gene Six3 from kisspeptin neurons causes subfertility in female mice

The homeodomain transcription factor SIX3 is a known regulator of eye, nose, and forebrain development, and has recently been implicated in female reproduction. Germline heterozygosity of SIX3 is sufficient to cause subfertility, but the cell populations that mediate this role are unknown. The neuropeptide kisspeptin is a critical component of the reproductive axis and plays roles in sexual maturation, ovulation, and the maintenance of gonadotropin secretion. We used Cre-Lox technology to remove Six3 specifically from kisspeptin neurons in mice to test the hypothesis that SIX3 in kisspeptin neurons is required for reproduction. We found that loss of Six3 in kisspeptin neurons causes subfertility and estrous cycle irregularities in females, but no effect in males. Overall, we find that SIX3 expression in kisspeptin neurons is an important contributor to female fertility.

Genetic research progress in branchio-oto syndrome/ branchio-oto-renal syndrome.

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.

Re-Examining the Cochlea in Branchio-Oto-Renal Syndrome: Genotype-Phenotype Correlation.

Temporal bone imaging plays an important role in the work-up of branchio-oto-renal syndrome. Previous reports have suggested that the unwound or offset cochlea is a highly characteristic marker for branchio-oto-renal syndrome. Our goals were to examine the prevalence of this finding in a branchio-oto-renal syndrome cohort and analyze genetic-phenotypic associations not previously established. This multicenter retrospective study included 38 ears in 19 unrelated individuals with clinically diagnosed branchio-oto-renal syndrome and confirmed mutations in the EYA1 or SIX1 genes. Two blinded neuroradiologists independently reviewed and documented temporal bone imaging findings in 13 categories for each ear. Imaging phenotypes were correlated with genotypes. There was excellent interrater agreement for all 13 phenotypic categories (κ ≥ 0.80). Of these, 9 categories showed statistically significant differences between patients with EYA1-branchio-oto-renal syndrome and SIX1-branchio-oto-renal syndrome. Cochlear offset was present in 100% of patients with EYA1-branchio-oto-renal syndrome, but in only 1 ear (12.5%) among patients with SIX1-branchio-oto-renal syndrome. A short thorny appearance of the cochlear apical turn was observed in most patients with SIX1-branchio-oto-renal syndrome. An offset cochlea is associated with the EYA1-branchio-oto-renal syndrome genotype. The SIX1-branchio-oto-renal syndrome genotype is associated with a different cochlear phenotype that almost always is without offset and has a short thorny tip as the apical turn. Therefore, cochlear offset is not a characteristic marker for all patients with branchio-oto-renal syndrome. The lack of a cochlear offset in a patient with clinically suspected branchio-oto-renal syndrome does not exclude the diagnosis and, in fact, may be predictive of the SIX1 genotype.

Targeted myostatin loss-of-function mutation increases type II muscle fibers in Meishan pigs

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. The skeletal muscle in MSTN−/– mice is significantly hypertrophied, with muscle fiber type II increasing significantly while muscle fiber type I decreasing. However, it is still not clear how the skeletal muscle types change in MSTN−/– pigs, and how the mechanism for MSTN regulates fiber types, especially in large animals like pigs. This study conducted a comprehensive analysis of the composition of skeletal muscle fibers in MSTN−/– pigs produced in our laboratory. It was observed that, compared with wild-type (WT) pigs, both the total mass of skeletal muscle and type IIb muscle fibers increased significantly (P<0.01), while the type I and type IIa muscle fibers decreased significantly (P<0.01), in MSTN−/– Meishan pigs. In addition, to explore the influence of MSTN on muscle fiber type and its regulation mechanism in the embryonic stage, this study selected a few genes (Myf5, Mef2d, MyoD and Six1) associated with muscle fiber type and validated their expression by quantitative RT-PCR. Herein, it was found that Myh7, Myh2, Myh4 and Myh1 can be detected in the skeletal muscle of pigs at 65 days of gestation (dg). Compared with WT pigs, in MSTN−/– Meishan pigs, Myh7 decreased significantly (P<0.01), while Myh4 (P<0.001) and Myh1 (P<0.05) increased significantly. Meanwhile, the increased expression of Myf5 (P<0.05), Mef2d (P<0.01) and Six1 (P<0.05) in MSTN−/– Meishan pigs suggested that MSTN should regulate the directional development of muscle fiber types in the early stage of embryonic development. Thus, at the embryonic stage, the type II muscle fibers began to increase in MSTN−/– pigs. These results can provide valuable information not only for pig meat quality improvement, but also for the study of human skeletal muscle development and disease treatment.

Genetic and Phenotypic Variability in Chinese Patients With Branchio-Oto-Renal or Branchio-Oto Syndrome.

Background: Branchio-oto-renal syndrome (BOR) and branchio-oto syndrome (BOS) are rare autosomal dominant disorders defined by varying combinations of branchial, otic, and renal anomalies. Here, we characterized the clinical features and genetic etiology of BOR/BOS in several Chinese families and then explored the genotypes and phenotypes of BOR/BOS-related genes, as well as the outcomes of auditory rehabilitation in different modalities. Materials and Methods: Probands and all affected family members underwent detailed clinical examinations. Their DNA was subjected to whole-exome sequencing to explore the underlying molecular etiology of BOR/BOS; candidate variants were validated using Sanger sequencing and interpreted in accordance with the American College of Medical Genetics guidelines. In addition, a literature review concerning EYA1 and SIX1 alterations was performed to explore the genotypes and phenotypes of BOR/BOS-related genes. Results: Genetic testing identified the novel deletion (c.1425delC, p(Asp476Thrfs*4); NM_000,503.6), a nonsense variant (c.889C > T, p(Arg297*)), and two splicing variants in the EYA1 gene (c.1050+1G > T and c.1140+1G > A); it also identified one novel missense variant in the SIX1 gene (c.316G > A, p(Val106Met); NM_005,982.4). All cases exhibited a degree of phenotypic variability between or within families. Middle ear surgeries for improving bone-conduction component hearing loss had unsuccessful outcomes; cochlear implantation (CI) contributed to hearing gains. Conclusion: This is the first report of BOR/BOS caused by the SIX1 variant in China. Our findings increase the numbers of known EYA1 and SIX1 variants. They also emphasize the usefulness of genetic testing in the diagnosis and prevention of BOR/BOS while demonstrating that CI for auditory rehabilitation is a feasible option in some BOR/BOS patients.

A SIX1/EYA2 Inhibitor Impairs CALM-AF10 and Jurkat Leukemia Cell Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. We demonstrated that CALM-AF10 increases Six1 expression and localizes to the Six1 locus, as it does the Hoxa genes. SIX1, like the Hoxa genes, is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. In addition, SIX1 and its cofactor EYA2 are overexpressed in numerous solid tumors, and an inhibitor of the SIX1/EYA2 complex (Compound 8430) has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored.Objective: Evaluate the effect of a SIX1/EYA2 complex inhibitor on leukemia cell proliferation.Design/Methods: SIX1 gene and protein expression were assessed in CALM-AF10, Jurkat (T-ALL) and NOMO1 (AML) leukemia cell lines via Western Blot and RT-qPCR. CALM-AF10 leukemias were derived from murine models in our lab, Jurkat and NOMO1 cell lines were obtained from ATCC. The effect of compound 8430 - an inhibitor of the Six1/Eya2 interaction - on cell proliferation was evaluated using Cell-Titer-Glo Assays and liquid culture proliferation assays. In addition, we used the the CRM1 Nuclear Export Inhibitor KPT-330 alone and in combination with 8430 in these cell lines. SynergyFinder2 (https://synergyfinder.fimm.fi/) was used to assess synergy of 8430 and KPT-330. δ-score is a calculated value that indicates synergistic drug interaction, with a higher δ-score indicative of a synergistic effect of the drugs.Results: SIX1 gene and protein expression are increased in CALM-AF10 leukemia cell lines and Jurkat T-ALL cells, but not NOMO1 cells. Compound 8430 decreases cell proliferation in CALM-AF10 leukemias and Jurkat leukemia cell lines, however it did not affect the AML line NOMO1. Correspondingly, liquid cultures showed that 8430 alone slowed the proliferation of CALM-AF10 leukemia and the Jurkat cells, but not NOMO1 cells. The addition of KPT-330 to 8430 was synergistic in CALM-AF10 leukemia cells with a KPT-330 dose of 60 nM and multiple dose levels of 8430 (δ-scores 17-19) while in the Jurkat leukemia cells a dose of 30 nM of KPT-330 was synergistic at multiple dose levels of 8430 (δ-score 6-8) (Figure 1).Conclusions: The SIX1 homeobox gene is highly expressed during development, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. A role for Six1 in CALM-AF10 leukemogenesis is further supported by the ability of a SIX1/EYA2 inhibitor to slow the proliferation of CALM-AF10 leukemia cells. Importantly, based on our observation that 8430 slows proliferation of Jurkat cells, SIX1 inhibition may be relevant in other leukemias. Finally, our demonstration that 8430 synergizes with KPT-330, a Nuclear Export Inhibitor, suggests the possibility of a novel therapeutic approach for CALM-AF10 and other leukemias. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Draft Genome Resource of a Novel Virulent Fusarium oxysporum f. sp. cubense Race 1 Strain (VCG 0124) Infecting Cavendish (AAA) Group of Banana in India.

Fusarium oxysporum f. sp. cubense is one of the most destructive soilborne fungi causing Fusarium wilt disease in banana. Generally, F. oxysporum f. sp. cubense race 1 (R1) severely affects most of the banana varieties, except Cavendish banana (AAA). Here, we present the draft genome of an isolate of VCG 0124, a novel virulent R1 strain that severely affects the Cavendish group of banana isolated from the Theni district of Tamil Nadu, India. The genome assembly of R1 comprises 61,471,473 bp with 88 contigs and 18,377 protein-coding regions. The genome contains homologs of F. oxysporum f. sp. cubense race-specific secreted-in-xylem (SIX) genes SIX1, SIX5, SIX9, and SIX13. The absence of SIX4 and SIX6 and deletion of a peptide in SIX1 virulence factor genes in the R1 (VCG 0124) strain might be the contributing factor for strains infecting Cavendish banana in India.

Identification of Photoperiod-Induced LncRNAs and mRNAs in Pituitary Pars Tuberalis of Sheep.

The pituitary pars tuberalis (PT) is the regulating center of seasonal reproduction, which can sense the melatonin signal and eventually cause downstream changes of GnRH secretion through TSHβ. Recently, lncRNAs have been identified in animal reproductive-related tissues, and they play important roles in reproductive regulation. Therefore, in this study, we expect to identify photoperiod-induced lncRNAs and genes in pituitary PT of sheep by comparison of expression profiles between short photoperiod (SP) and long photoperiod (LP). Through RNA-Seq, a total of 55,472 lncRNAs were identified in pituitary PT of Sunite ewes. The number of differentially expressed (DE) genes and lncRNAs between SP and LP increased gradually with the extension of LP (from LP7 to LP42). The notable LP-induced candidate genes included EYA3, TSHB, SIX1, DCT, VMO1, AREG, SUV39H2, and EZH2, and SP-induced genes involved ENSOARG00000012585, CHGA, FOS, SOCS3, and TH. In enriched pathways for DE genes and lncRNA target genes between SP and LP, the reproduction- and circadian-related pathways were highlighted. In addition, the interactome analysis of lncRNAs and their targets implied that MSTRG.209166 and its trans-target TSHB, MSTRG.288068 and its cis-target SIX1, and ENSOARG00000026131 and its cis-target TH might participate in regulation of seasonal reproduction. Together, these results will help to determine important photoperiod-induced lncRNAs and genes and give us some new insights into the epigenetic regulation of seasonal reproduction in sheep.

BRANCHIO-OTO-RENAL (BOR) SYNDROME

Introduction. This work is devoted to the problem of embryology of the ears and kidneys. First ear and kidney abnormalities were reported in 1946 by Edith Potter’s association of crumpled and flattened ears with bilateral kidney agenesis. Ear malformations are associated with an increased frequency of clinically significant structural renal anomalies compared with the general population. These include specific multiple congenital anomaly syndromes, Townes-Brocks syndrome, branchio-oto-renal syndrome.The link can be explained by structural and functional similarities between tissues in the inner ear and in the kidney. Also, toxins that accumulate in kidney failure can damage nerves, including those in the inner ear.Goal. To study the causes, clinical manifestations of Branchio-oto-renal (BOR) syndrome. Materials and Methods. Review of modern and foreign literary sources; methods - description, analysis, abstracting.Results and discussion. Mutations in three genes, EYA1, SIX1, and SIX5, have been reported in people with BOR/BO syndrome. About 40 percent of people with this condition have a mutation in the EYA1 gene. SIX1 gene mutations are a much less common cause of the disorder. SIX5 gene mutations have been found in a small number of people with BOR syndrome, although researchers question whether mutations in this gene cause the condition. Some affected individuals originally reported to have SIX5 gene mutations were later found to have EYA1 gene mutations as well, and researchers suspect that the EYA1 gene mutations may be the actual cause of the condition in these people.The proteins produced from the EYA1, SIX1, and SIX5 genes play important roles in development before birth. The EYA1 protein interacts with several other proteins, including SIX1 and SIX5, to regulate the activity of genes involved in many aspects of embryonic development. Research suggests that these protein interactions are essential for the normal formation of many organs and tissues, including the second branchial arch, ears, and kidneys. Mutations in the EYA1, SIX1, or SIX5 gene may disrupt the proteins’ ability to interact with one another and regulate gene activity.Conclusions. The link between ear anomalies and kidney function can be explained by structural and functional similarities between tissues in the inner ear and in the kidney. Additionally, toxins that accumulate in kidney failure can damage nerves, including those in the inner ear.

Analysis of a child with holoprosencephaly due to variant of SIX3 gene

To explore the genetic basis of a child with holoprosencephaly. Genomic DNA of the child was extracted and subjected to whole exome sequencing. Suspected variant was verified by Sanger sequencing of her family members. Cranial MRI suggested lobulated holoprosencephaly with partial absence of corpus callosum. Genetic testing revealed that she has carried a heterozygous c.517C>G (p.His173Asp) variant of the SIX3 gene, for which both of her parents were of wild type. Based on the American College of Medical Genetics and Genomics guidelines, the c.517C>G variant of SIX3 gene was predicted to be pathogenic (PS2+PM1+PM2+PM5+PP3). The SIX3 gene c.517C>G variant probably underlay the multiple malformations in this child. Above finding has enabled her definite diagnosis.

New SHH and Known SIX3 Variants in a Series of Latin American Patients with Holoprosencephaly

Holoprosencephaly (HPE) is the failure of the embryonic forebrain to develop into 2 hemispheres promoting midline cerebral and facial defects. The wide phenotypic variability and causal heterogeneity make genetic counseling difficult. Heterozygous variants with incomplete penetrance and variable expressivity in the SHH, SIX3, ZIC2, and TGIF1 genes explain ∼25% of the known causes of nonchromosomal HPE. We studied these 4 genes and clinically described 27 Latin American families presenting with nonchromosomal HPE. Three new SHH variants and a third known SIX3 likely pathogenic variant found by Sanger sequencing explained 15% of our cases. Genotype-phenotype correlation in these 4 families and published families with identical or similar driver gene, mutated domain, conservation of residue in other species, and the type of variant explain the pathogenicity but not the phenotypic variability. Nine patients, including 2 with SHH pathogenic variants, presented benign variants of the SHH, SIX3, ZIC2, and TGIF1 genes with potential alteration of splicing, a causal proposition in need of further studies. Finding more families with the same SIX3 variant may allow further identification of genetic or environmental modifiers explaining its variable phenotypic expression.

Homeobox Gene Six3 is Required for the Differentiation of D2-Type Medium Spiny Neurons

Medium spiny neurons (MSNs) in the striatum, which can be divided into D1 and D2 MSNs, originate from the lateral ganglionic eminence (LGE). Previously, we reported that Six3 is a downstream target of Sp8/Sp9 in the transcriptional regulatory cascade of D2 MSN development and that conditionally knocking out Six3 leads to a severe loss of D2 MSNs. Here, we showed that Six3 mainly functions in D2 MSN precursor cells and gradually loses its function as D2 MSNs mature. Conditional deletion of Six3 had little effect on cell proliferation but blocked the differentiation of D2 MSN precursor cells. In addition, conditional overexpression of Six3 promoted the differentiation of precursor cells in the LGE. We measured an increase of apoptosis in the postnatal striatum of conditional Six3-knockout mice. This suggests that, in the absence of Six3, abnormally differentiated D2 MSNs are eliminated by programmed cell death. These results further identify Six3 as an important regulatory element during D2 MSN differentiation.

Sobp is a novel Six1 co‐factor during inner ear development

According to the CDC, the prevalence of hearing loss is 1.4 per 1000 babies screened in 2009 in the USA. Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by hearing loss, branchial fistulas/cysts, and renal anomalies in about a third of patients. Mutations in the SIX1 gene and the SIX1 co-factor EYA1 gene are present in half of BOR patients. We are using Xenopus laevis and mouse as models to discover novel genes involved in this syndrome. Based on a screen in fly of proteins shown to interact with So (Drosophila Six1 homologue), sobp was identified as potential Six1 co-factor. Expression data show that this gene is expressed in patterns that overlap with six1 during craniofacial development. Co-IP and immunofluorescence experiments demonstrate that Sobp binds to and co-localizes with Six1 in the cell nucleus. Additionally, Six1 is able to bind and translocate to the cell nucleus an Sobp construct lacking its nuclear localization signal further demonstrating interaction. Luciferase assays to test if this factor is able to modify Six1 function show that Sobp represses Six1+Eya1 transcriptional activity. Knockdown experiments using morpholino antisense oligonucleotides and CRISPR/Cas9 followed by ISH and qPCR analyses of embryos at late neural plate stages show that Sobp is required for neural crest (foxd3) and placode (six1) formation. These changes are accompanied by an expansion of the neural plate (sox2) and reduction of the embryonic epidermis (krt12.4). Sobp overexpression confirmed that Sobp is required for neural crest (foxd3) and placode (six1) formation; changes in those two genes were accompanied by a slight expansion of the krt12.4-expressing domain. With further development, Sobp knockdown and overexpression disrupt otocyst development shown by disrupted expression of dlx5, pax2, irx1 and sox9 at tailbud stages, and cause severe craniofacial defects in the tadpole. These results indicate that Sobp is required for proper craniofacial development, and that it modifies Six1 function, suggesting this protein may be a potential candidate gene for BOR and other craniofacial defects.

Zmym2 and Zmym4 Act as Co‐Factors of Six1 During Craniofacial Development

Branchio-oto-renal syndrome (BOR) is an autosomal dominant condition that presents with variable cranial, otic, and renal malformations. However, only half of BOR cases have a known genetic cause: mutations in the genes encoding Six1 and its co-factor Eya1. Therefore, it is important to identify other proteins that may interact with either Six1 or Eya1 that could be involved in the craniofacial phenotypes involved in BOR patients. In a screen for potential new cofactors, we identified Zinc Finger MYM-containing 2 (Zmym2) and Zmym4, whose expression overlaps with Six1 the pre-placodal ectoderm and otic vesicle; they also are expressed in the cranial neural crest and branchial arches. Knockdown of Zmym2 or Zmym4 resulted in a broader but less intense expression of msx1, pax3 and tfap2 in the neural border zone at neural plate stages, and expanded neural crest (foxD3, sox9) and placode (six1) genes at neurula stages. At larval stages, dlx5 expression in the neural crest and otic vesicles were reduced. Luciferase assays demonstrate that Zmym2 reduces Six1-Eya1 transcriptional activation whereas Zmym4 enhances it. These data indicate that Zmym2 and Zmym4 serve an important role in the development of craniofacial tissue, including the otic vesicle, and suggest they may have a role in BOR.

Achillea santolina and Raphanus sativus extracts downregulated NOTCH1, SIX1 and WNT1 developmental gene expressions in breast and colon cancer cell lines: Thesis Abstract

Thesis Abstract Background: Although anticancer treatment with chemotherapy is effective, cancer response occurs due mainly to reduction of cancer stem cells. The development of cancer stem cell is regulated by several genes including NOTCH1, SIX1 and WNT1. Aim: we have found recently that treatment of Caco2, HepG2 and MCF-7 with Achillia santolina or Rahanus sativus results antitumor effect. In this study we aimed to evaluate the effect of these natural product on the gene expressions NOTCH1, SIX1 and WNT1 in Caco2 and MCF-7. Materials and Methods: Colon adenocarcinoma cell line (Caco2) and Breast cancer cell line (MCF-7) as well as normal amniotic cell line (WISH) were treated with 110 IC50 concentration of Achillea santolina and Raphanus sativus extracts to compared to 110 IC50 of cisplatin as a reference chemotherapy drug. Total RNA was extracted after 72 hours of treatment and incubation then cDNA was prepared for reverse transcriptase PCR for NOCH1, WNT1, and SIX1. Results: R. sativus treatment highly decreased NOTCH1 expression in MCF7 and Caco2 cell lines, while decreased SIX1 gene expression in Caco2 cell line and WNT1 in MCF7 cell line. On the other hand, A. santolina observably decreased the WNT1 gene expression in MCF7 cell line. Conclusion: The crude extracts of R. sativus decreased the developmental gene expression of NOTCH1, SIX1 in Caco2 cell line as same as on NOTCH1 and WNT1 in MCF-7 cell line more than cisplatin chemotherapy. While the A. santolina crude extract decrease the expression of WNT1 in MCF-7 cell line more than cisplatin chemotherapy.

Molecular mechanisms of hearing loss in Nager syndrome

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.