The enteric nervous system of C. elegans is specified by the Sine Oculis-like homeobox gene ceh-34

Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode C. elegans that resides within the foregut (pharynx) of the worm. A Caenorhabditis elegans homolog of the Drosophila Sine Oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly, without affecting panneuronal features. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine Oculis homolog, our findings invite speculations about the early evolution of nervous systems.

The SIX Family of Transcription Factors: Common Themes Integrating Developmental and Cancer Biology

The sine oculis (SIX) family of transcription factors are key regulators of developmental processes during embryogenesis. Members of this family control gene expression to promote self-renewal of progenitor cell populations and govern mechanisms of cell differentiation. When the function of SIX genes becomes disrupted, distinct congenital defects develops both in animal models and humans. In addition to the embryonic setting, members of the SIX family have been found to be critical regulators of tumorigenesis, promoting cell proliferation, epithelial-to-mesenchymal transition, and metastasis. Research in both the fields of developmental biology and cancer research have provided an extensive understanding of SIX family transcription factor functions. Here we review recent progress in elucidating the role of SIX family genes in congenital disease as well as in the promotion of cancer. Common themes arise when comparing SIX transcription factor function during embryonic and cancer development. We highlight the complementary nature of these two fields and how knowledge in one area can open new aspects of experimentation in the other.

Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease

The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors play critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of branchio-oto-renal syndrome (BOR), which is characterized by moderate to severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckel's, ceratohyal and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.

SIX1 Down-Regulation Influence Drug Resistance, Epithelial-Mesenchymal Transcription Factors and Self-Renewal Capacity in Hepatocellular Carcinoma

Sine oculis homeoprotein 1 (SIX1) was discovered to exert an essential role in embryonic development and it was also identified to be re-activated in various types of mammalian cancer. Immunohistochemical and SIX1 gene expression analyses were performed to determine the prognostic role of SIX1 expression. SIX1 expression was suppressed by short hairpin RNA transduction in the SNU398 HCC cell line. The effects of SIX1 on proliferation, epithelial-mesenchymal transition, apoptosis, drug resistance, and sphere formation were assessed in SIX1 knock-down cells. The upregulated expression levels of SIX1 were revealed to be correlated with the stage of the disease in breast, colon and liver cancer, with liver cancer exhibiting the highest expression profile. SIX1 knockdown significantly affected the cell morphology, proliferation, downregulated the protein expression levels of ZEB1, ZEB2 and SNAI1 and upregulated the expression levels of TWIST1 in hepatocellular carcinoma cells. Furthermore, SIX1 knockdown cells were more sensitive to sorafenib treatment; however, the expression profile analysis of the drug resistance genes ABCB1, ABCC1 and ABCG2 did not explain this sensitivity. Finally, SIX1 knockdown cells were identified to have decreased CD90 levels and lost their sphere-forming ability, which is essential for cancer stem cell properties. Overall, these results indicated that SIX1 expression may be useful as a diagnostic marker for patients with HCC.

SIX6 is differentially expressed in the tumors of breast cancer patients treated with trastuzumab.

Trastuzumab (Herceptin) is a monoclonal antibody targeting the extracellular domain of the human epidermal growth factor receptor 2 (HER2) (1) utilized for the treatment of adjuvant and metastatic breast cancer (2) in the United States and worldwide. We mined public microarray data (3, 4) to discover in an unbiased manner the most significant transcriptional changes associated with trastuzumab treatment. We identified the sine oculis family homeobox transcription factor SIX6 as among the genes most differentially expressed in the primary tumors of patients with breast cancer treated with trastuzumab. A single dose of trastuzumab was sufficient to result in differential expression of SIX6 in the primary tumors of patients with breast cancer, suggesting that transcriptional induction of SIX6, a transcription factor with functions in specification of the ventral stalk from the neural retina (5), is a direct result of trastuzumab administration.

Systemic paralogy and function of retinal determination network homologs in arachnids

Background Arachnids are important components of cave ecosystems and display many examples of troglomorphisms, such as blindness, depigmentation, and elongate appendages. Little is known about how the eyes of arachnids are specified genetically, let alone the mechanisms for eye reduction and loss in troglomorphic arachnids. Additionally, duplication of Retinal Determination Gene Network (RDGN) homologs in spiders has convoluted functional inferences extrapolated from single-copy homologs in pancrustacean models. Results We investigated a sister species pair of Israeli cave whip spiders, Charinus ioanniticus and C. israelensis (Arachnopulmonata, Amblypygi), of which one species has reduced eyes. We generated embryonic transcriptomes for both Amblypygi species, and discovered that several RDGN homologs exhibit duplications. We show that duplication of RDGN homologs is systemic across arachnopulmonates (arachnid orders that bear book lungs), rather than being a spider-specific phenomenon. A differential gene expression (DGE) analysis comparing the expression of RDGN genes in field-collected embryos of both species identified candidate RDGN genes involved in the formation and reduction of eyes in whip spiders. To ground bioinformatic inference of expression patterns with functional experiments, we interrogated the function of three candidate RDGN genes identified from DGE using RNAi in the spider Parasteatoda tepidariorum. We provide functional evidence that one of these paralogs, sine oculis/Six1 A (soA), is necessary for the development of all arachnid eye types. Conclusions Our work establishes a foundation to investigate the genetics of troglomorphic adaptations in cave arachnids, and links differential gene expression to an arthropod eye phenotype for the first time outside of Pancrustacea. Our results support the conservation of at least one RDGN component across Arthropoda and provide a framework for identifying the role of gene duplications in generating arachnid eye diversity.

SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis

ABSTRACTPax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.

How spiders make their eyes: Systemic paralogy and function of retinal determination network homologs in arachnids

Arachnids are important components of cave ecosystems and display many examples of troglomorphisms, such as blindness, depigmentation, and elongate appendages. Little is known about how the eyes of arachnids are specified genetically, let alone the mechanisms for eye reduction and loss in troglomorphic arachnids. Additionally, paralogy of Retinal Determination Gene Network (RDGN) homologs in spiders has convoluted functional inferences extrapolated from single-copy homologs in pancrustacean models. Here, we investigated a sister species pair of Israeli cave whip spiders (Arachnopulmonata, Amblypygi, Charinus) of which one species has reduced eyes. We generated the first embryonic transcriptomes for Amblypygi, and discovered that several RDGN homologs exhibit duplications. We show that paralogy of RDGN homologs is systemic across arachnopulmonates (arachnid orders that bear book lungs), rather than being a spider-specific phenomenon. A differential gene expression (DGE) analysis comparing the expression of RDGN genes in field-collected embryos of both species identified candidate RDGN genes involved in the formation and reduction of eyes in whip spiders. To ground bioinformatic inference of expression patterns with functional experiments, we interrogated the function of three candidate RDGN genes identified from DGE in a spider, using RNAi in the spider Parasteatoda tepidariorum. We provide functional evidence that one of these paralogs, sine oculis/Six1 A (soA), is necessary for the development of all arachnid eye types. Our results support the conservation of at least one RDGN component across Arthropoda and establish a framework for investigating the role of gene duplications in arachnid eye diversity.

MiR-203a-3p regulates TGF-β1-induced epithelial–mesenchymal transition (EMT) in asthma by regulating Smad3 pathway through SIX1

Asthma is a common chronic airway disease with increasing prevalence. MicroRNAs act as vital regulators in cell progressions and have been identified to play crucial roles in asthma. The objective of the present study is to clarify the molecular mechanism of miR-203a-3p in the development of asthma. The expression of miR-203a-3p and Sine oculis homeobox homolog 1 (SIX1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of SIX1, fibronectin, E-cadherin, vimentin, phosphorylated-drosophila mothers against decapentaplegic 3 (p-Smad3) and Smad3 were measured by Western blot. The interaction between miR-203a-3p and SIX1 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. MiR-203a-3p was down-regulated and SIX1 was up-regulated in asthma serums, respectively. Transforming growth factor-β1 (TGF-β1) treatment induced the reduction of miR-203a-3p and the enhancement of SIX1 in BEAS-2B and 16HBE cells in a time-dependent manner. Subsequently, functional experiments showed the promotion of epithelial–mesenchymal transition (EMT) induced by TGF-β1 treatment could be reversed by miR-203a-3p re-expression or SIX1 deletion in BEAS-2B and 16HBE cells. SIX1 was identified as a target of miR-203a-3p and negatively regulated by miR-203a-3p. Then rescue assay indicated that overexpressed miR-203a-3p ameliorated TGF-β1 induced EMT by regulating SIX1 in BEAS-2B and 16HBE cells. Moreover, miR-203a-3p/SIX1 axis regulated TGF-β1 mediated EMT process in bronchial epithelial cells through phosphorylating Smad3. These results demonstrated that MiR-203a-3p modulated TGF-β1-induced EMT in asthma by regulating Smad3 pathway through targeting SIX1.