Cdx1 and Gsc distinctly regulate the transcription of BMP4 target gene ventx3.2 by directly binding to the proximal promoter region in Xenopus gastrulae.

A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide chromatin immunoprecipitation-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, the chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral marginal zone and dorsal marginal zone, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the ventral marginal zone and dorsal marginal zone by directly binding to the promoter region during Xenopus gastrulation.

Two Homeobox Transcription Factors, Goosecoid and Ventx1.1, Oppositely Regulate Chordin Transcription in Xenopus Gastrula Embryos.

The reciprocal inhibition between two signaling centers, the Spemann organizer (dorsal mesoderm) and ventral region (mesoderm and ectoderm), collectively regulate the overall development of vertebrate embryos. Each center expresses key homeobox transcription factors (TFs) that directly control target gene transcription. Goosecoid (Gsc) is an organizer (dorsal mesoderm)-specific TF known to induce dorsal fate and inhibit ventral/ectodermal specification. Ventx1.1 (downstream of Bmp signaling) induces the epidermal lineage and inhibits dorsal organizer-specific genes from the ventral region. Chordin (Chrd) is an organizer-specific secreted Bmp antagonist whose expression is primarily activated by Gsc. Alternatively, chrd expression is repressed by Bmp/Ventx1.1 in the ventral/epidermal region. However, the regulatory mechanisms underlying the transcription mediated by Gsc and Ventx1.1 remain elusive. Here, we found that the chrd promoter contained two cis-acting response elements that responded negatively to Ventx1.1 and positively to Gsc. In the ventral/ectodermal region, Ventx1.1 was directly bound to the Ventx1.1 response element (VRE) and inhibited chrd transcription. In the organizer region, Gsc was bound to the Gsc response elements (GRE) to activate chrd transcription. The Gsc-mediated positive response on the chrd promoter completely depended on another adjacent Wnt response cis-acting element (WRE), which was the TCF7 (also known as Tcf1) binding element. Site-directed mutagenesis of VRE, GRE, or WRE completely abolished the repressive or activator activity of Ventx1.1 and Gsc, respectively. The ChIP-PCR results confirmed the direct binding of Ventx1.1 and Gsc/Tcf7 to VRE and GRE/WRE, respectively. These results demonstrated that chrd expression is oppositely modulated by homeobox TFs, Ventx1.1, and Gsc/Tcf7 during the embryonic patterning of Xenopus gastrula.

SMYD2 Drives Mesendodermal Differentiation of Human Embryonic Stem Cells Through Mediating the Transcriptional Activation of Key Mesendodermal Genes.

Histone methyltransferases play a critical role in early human development, whereas their roles and precise mechanisms are less understood. SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase induced during early differentiation of human embryonic stem cells (hESCs), but little is known about its function in undifferentiated hESCs and in their early lineage fate decision as well as underlying mechanisms. Here, we explored the role of SMYD2 in the self-renewal and mesendodermal lineage commitment of hESCs. We demonstrated that the expression of SMYD2 was significantly enhanced during mesendodermal but not neuroectodermal differentiation of hESCs. SMYD2 knockout (SMYD2-/- ) did not affect self-renewal and early neuroectodermal differentiation of hESCs, whereas it blocked the mesendodermal lineage commitment. This phenotype was rescued by reintroduction of SMYD2 into the SMYD2-/- hESCs. Mechanistically, the bindings of SMYD2 at the promoter regions of critical mesendodermal transcription factor genes, namely, brachyury (T), eomesodermin (EOMES), mix paired-like homeobox (MIXL1), and goosecoid homeobox (GSC) were significantly enhanced during mesendodermal differentiation of SMYD2+/+ hESCs but totally suppressed in SMYD2-/- ones. Concomitantly, such a suppression was associated with the remarkable reduction of methylation at histone 3 lysine 4 and lysine 36 but not at histone 4 lysine 20 globally and specifically on the promoter regions of mesendodermal genes, namely, T, EOMES, MIXL1, and GSC. These results reveal that the histone methyltransferase SMYD2 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation, but it promotes the mesendodermal differentiation of hESCs through the epigenetic control of critical genes to mesendodermal lineage commitment. Stem Cells 2019;37:1401-1415.

USP21 modulates Goosecoid function through deubiquitination.

The homeobox gene Goosecoid (GSC), which is known to regulate craniofacial development, is activated by mono-ubiquitination; however, the deubiquitylase responsible for GSC deubiquitination and inhibition has yet to be identified. In the present study, we constructed the recombinant plasmid pFlag-CMV-2-GSC and the SRY (sex-determining region Y)-box 6 (Sox6) reporter gene system to identify deubiquitylases that regulate GSC expression. We demonstrate that the ubiquitin carboxyl-terminal hydrolase 21 (USP21) regulates the deubiquitination of GSC negatively, as demonstrated by its inhibition of Sox6 reporter gene transcription. USP21 interacted with GSC to promote GSC deubiquitination while having no effect on GSC protein stability. Cell viability, migration, and function in ATDC5 cells were probably influenced by USP21 through GSC. These findings suggest that USP21 modulates GSC function through deubiquitination.

Identifying biomarkers of breast cancer micrometastatic disease in bone marrow using a patient-derived xenograft mouse model.

BACKGROUND: Disseminated tumor cells (DTCs) found in the bone marrow (BM) of patients with breast cancer portend a poor prognosis and are thought to be intermediaries in the metastatic process. To assess the clinical relevance of a mouse model for identifying possible prognostic and predictive biomarkers of these cells, we have employed patient-derived xenografts (PDX) for propagating and molecularly profiling human DTCs. METHODS: Previously developed mouse xenografts from five breast cancer patients were further passaged by implantation into NOD/SCID mouse mammary fat pads. BM was collected from long bones at early, serial passages and analyzed for human-specific gene expression by qRT-PCR as a surrogate biomarker for the detection of DTCs. Microarray-based gene expression analyses were performed to compare expression profiles between primary xenografts, solid metastasis, and populations of BM DTCs. Differential patterns of gene expression were then compared to previously generated microarray data from primary human BM aspirates from patients with breast cancer and healthy volunteers. RESULTS: Human-specific gene expression of SNAI1, GSC, FOXC2, KRT19, and STAM2, presumably originating from DTCs, was detected in the BM of all xenograft mice that also developed metastatic tumors. Human-specific gene expression was undetectable in the BM of those xenograft lines with no evidence of distant metastases and in non-transplanted control mice. Comparative gene expression analysis of BM DTCs versus the primary tumor of one mouse line identified multiple gene transcripts associated with epithelial-mesenchymal transition, aggressive clinical phenotype, and metastatic disease development. Sixteen of the PDX BM associated genes also demonstrated a statistically significant difference in expression in the BM of healthy volunteers versus the BM of breast cancer patients with distant metastatic disease. CONCLUSION: Unique and reproducible patterns of differential gene expression can be identified that presumably originate from BM DTCs in mouse PDX lines. Several of these identified genes are also detected in the BM of patients with breast cancer who develop early metastases, which suggests that they may be clinically relevant biomarkers. The PDX model may also provide a clinically relevant system for analyzing and targeting these intermediaries of metastases.

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.

Smad transcription factors activated by TGF-ß or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-ß and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-ß and BMP pathways.

The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation.

Poly-ubiquitination-mediated RUNX2 degradation is an important cause of age- and inflammation-related bone loss. NEDD4 family E3 ubiquitin protein ligases are thought to be the major regulators of RUNX2 poly-ubiquitination. However, we observed a mono-ubiquitination of RUNX2 that was catalyzed by WWP2, a member of the NEDD4 family of E3 ubiquitin ligases. WWP2 has been reported to catalyze the mono-ubiquitination of Goosecoid in chondrocytes, facilitating craniofacial skeleton development. In this study, we found that osteogenic differentiation of mesenchymal stem cells promoted WWP2 expression and nuclear accumulation. Knockdown of Wwp2 in mesenchymal stem cells and osteoblasts led to significant deficiencies of osteogenesis, including decreased mineral deposition and down-regulation of osteogenic marker genes. Co-immunoprecipitation experiments showed the interaction of WWP2 with RUNX2 in vitro and in vivo Mono-ubiquitination by WWP2 leads to RUNX2 transactivation, as evidenced by the wild type of WWP2, but not its ubiquitin ligase-dead mutant, augmenting RUNX2-reponsive reporter activity. Moreover, deletion of WWP2-dependent mono-ubiquitination resulted in striking defects of RUNX2 osteoblastic activity. In addition, ectopic expression of the constitutively active type 1A bone morphogenetic protein receptor enhanced WWP2-dependent RUNX2 ubiquitination and transactivation, demonstrating a regulatory role of bone morphogenetic protein signaling in the WWP2-RUNX2 axis. Taken together, our results provide evidence that WWP2 serves as a positive regulator of osteogenesis by augmenting RUNX2 transactivation in a non-proteolytic mono-ubiquitination manner.

A novel role of the organizer gene Goosecoid as an inhibitor of Wnt/PCP-mediated convergent extension in Xenopus and mouse.

Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.

Mutational analysis of GSC, HOXA2 and PRKRA in 106 Chinese patients with microtia.

OBJECTIVE: Microtia is defined as a developmental malformation characterized by a small, abnormal shaped auricle, with atresia or stenosis of the auditory canal. Genes responsible for nonsyndromic microtia have remained elusive. We therefore report a mutational analysis of GSC, HOXA2 and PRKRA in 106 congenital microtia patients without any combined malformation to explore the relationship between GSC, HOXA2, PRKRA and nonsyndromic microtia. METHODS: A total of 106 patients with a clinical diagnosis of congenital microtia and a control group (100 unaffected controls) were recruited through the Eye and ENT Hospital of Fudan University in China. Genomic DNA was extracted following a standard protocol. DNA sequencing analysis was performed in all exons and the exon-intron borders of GSC, HOXA2 and PRKRA. RESULTS: We identified 5 genomic variants in GSC, HOXA2 and PRKRA. As to the GSC, we obtained a reported variant g.994C > T in exon 2, which resulted in no change of protein. Our results revealed that g.994C > T was also detected in 10 control cases. We also detected 2 novel variants, g.90G > A and g.114A > C, in the 5'UTR of HOXA2. No class 5 or 4 genomic variant of PRKRA was identified in our microtia patients. Additionally, two previously reported SNVs in GSC and PRKRA were also presented. CONCLUSIONS: We suggest that g.994C > T is a new SNV, which is different from the previous report. Further study is needed to prove the function of 2 novel variants in the 5'UTR of HOXA2, and to explore the possible mechanism of these variants in the occurrence of microtia.

Identification and functional analysis of a novel LHX1 mutation associated with congenital absence of the uterus and vagina.

Congenital absence of the uterus and vagina (CAUV) is the most extreme female Müllerian duct abnormality. Several researches proposed that genetic factors contributed to this disorder, whereas the precise genetic mechanism is far from full elucidation. Here, utilizing whole-exome sequencing (WES), we identified one novel missense mutation in LHX1 (NM_005568: c.G1108A, p.A370T) in one of ten unrelated patients diagnosed with CAUV. This mutation was absent from public databases and our internal database. Through the luciferase reporter analysis, we found that the mutation could change the transcriptional activity of LHX1 and its effect on the regulation of the downstream target gene GSC, which might be associated with urogenital system development. In short, we concluded that the LHX1 may be a pathogenic gene of CAUV. Our results demonstrate the power of whole exome sequencing and gene prioritization approach as diagnostic tools in clinical practice that help make genetic diagnosis of CAUV.

Spemann organizer gene Goosecoid promotes delamination of neuroblasts from the otic vesicle.

Neurons of the Statoacoustic Ganglion (SAG), which innervate the inner ear, originate as neuroblasts in the floor of the otic vesicle and subsequently delaminate and migrate toward the hindbrain before completing differentiation. In all vertebrates, locally expressed Fgf initiates SAG development by inducing expression of Neurogenin1 (Ngn1) in the floor of the otic vesicle. However, not all Ngn1-positive cells undergo delamination, nor has the mechanism controlling SAG delamination been elucidated. Here we report that Goosecoid (Gsc), best known for regulating cellular dynamics in the Spemann organizer, regulates delamination of neuroblasts in the otic vesicle. In zebrafish, Fgf coregulates expression of Gsc and Ngn1 in partially overlapping domains, with delamination occurring primarily in the zone of overlap. Loss of Gsc severely inhibits delamination, whereas overexpression of Gsc greatly increases delamination. Comisexpression of Ngn1 and Gsc induces ectopic delamination of some cells from the medial wall of the otic vesicle but with a low incidence, suggesting the action of a local inhibitor. The medial marker Pax2a is required to restrict the domain of gsc expression, and misexpression of Pax2a is sufficient to block delamination and fully suppress the effects of Gsc The opposing activities of Gsc and Pax2a correlate with repression or up-regulation, respectively, of E-cadherin (cdh1). These data resolve a genetic mechanism controlling delamination of otic neuroblasts. The data also elucidate a developmental role for Gsc consistent with a general function in promoting epithelial-to-mesenchymal transition (EMT).

DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells.

Long noncoding RNAs (lncRNAs) exhibit diverse functions, including regulation of development. Here, we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition, we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans, and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm.

Mutational Analysis of TCOF1, GSC, and HOXA2 in Patients With Treacher Collins Syndrome.

Treacher Collins syndrome is an autosomal dominant craniofacial malformation mainly caused by mutations in the TCOF1 gene. Few cases have been observed in the Chinese population. Herein, the authors report the mutational analysis of TCOF1, GSC, and HOXA2 to determine the mutational features of the 3 genes in Chinese patients with Treacher Collins syndrome. Genomic DNA of the patients and their parents was extracted from peripheral blood following a standard protocol. DNA sequencing analysis was performed on all exons and the exon-intron borders of TCOF1, GSC, and HOXA2 in addition to the 1200-bp upstream of TCOF1. Four novel single nucleotide polymorphisms were detected in TCOF1, one of which was in the promoter region. Mutations in GSC and HOXA2 were not found in the 3 patients. Our results suggest the possibility of genetic heterogeneity or different mechanisms leading to the disease. Further functional study of the alteration is necessary to obtain more definitive information.

Activation of a T-box-Otx2-Gsc gene network independent of TBP and TBP-related factors.

Embryonic development relies on activating and repressing regulatory influences that are faithfully integrated at the core promoter of individual genes. In vertebrates, the basal machinery recognizing the core promoter includes TATA-binding protein (TBP) and two TBP-related factors. In Xenopus embryos, the three TBP family factors are all essential for development and are required for expression of distinct subsets of genes. Here, we report on a non-canonical TBP family-insensitive (TFI) mechanism of transcription initiation that involves mesoderm and organizer gene expression. Using TBP family single- and triple-knockdown experiments, α-amanitin treatment, transcriptome profiling and chromatin immunoprecipitation, we found that TFI gene expression cannot be explained by functional redundancy, is supported by active transcription and shows normal recruitment of the initiating form of RNA polymerase II to the promoter. Strikingly, recruitment of Gcn5 (also known as Kat2a), a co-activator that has been implicated in transcription initiation, to TFI gene promoters is increased upon depletion of TBP family factors. TFI genes are part of a densely connected TBP family-insensitive T-box-Otx2-Gsc interaction network. The results indicate that this network of genes bound by Vegt, Eomes, Otx2 and Gsc utilizes a novel, flexible and non-canonical mechanism of transcription that does not require TBP or TBP-related factors.

c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids.

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution.

Goosecoid promotes the metastasis of hepatocellular carcinoma by modulating the epithelial-mesenchymal transition.

The homeobox gene, goosecoid (GSC), is a transcription factor that participates in cell migration during embryonic development. Because cell migration during development has characteristics similar to cell invasion during metastasis, we evaluated the potential role of GSC in the metastasis of hepatocellular carcinoma (HCC). GSC expression in HCC cell lines and tissues was evaluated, and its effects on the migration potential of HCC cells were determined by GSC knock-down and overexpression methods. In addition, the prognostic role of GSC expression in the metastasis of cancer cells in HCC patients was determined. Our data showed that GSC was highly expressed in several HCC cell lines, particularly in a highly metastatic HCC cell line. Overexpression of GSC promoted cell migration and invasion of HCC cells in vitro. Gain-of-function induced the epithelial-mesenchymal transition but not collective cell migration, whereas loss-of-function induced the reverse change. High-level expression of GSC correlated closely with poor survival and lung metastasis in HCC patients; lung metastases showed more upregulated GSC expression than the primary tumor. We conclude that GSC promotes metastasis of HCC potentially through initiating the epithelial-mesenchymal transition. GSC is also a prognostic factor for poor survival and metastasis of HCC, which suggests its potential as a therapeutic target for metastatic HCC.

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity.

Overexpression of goosecoid homeobox is associated with chemoresistance and poor prognosis in ovarian carcinoma.

Ovarian carcinoma is the most lethal cancer among all gynecological malignancies due to recurrence through chemoresistance. The aim of the present study was to identify new biomarkers to predict chemoresistance and prognosis in ovarian carcinomas. The mRNA expression by qRT-PCR was examined in 60 ovarian serous carcinomas for selected genes from the screening by PCR array focusing on apoptosis, epithelial-to-mesenchymal transition and cancer pathways. The clinical impact was assessed by analyzing the correlation between gene expression and clinicopathological variables. Further validation with immunohistochemistry was performed with 75 cases of serous carcinomas. The chemoresistance was significantly associated with high expression of FOS, GSC, SNAI1, TERT and TNFRSF10D, and low expression of CDKN1A, TNFRSF10A, TNFRSF10C and TRAF1 (p<0.05, t-test). Low expression of TRAF1 and high expression of E2F1, FOS, TERT and GSC were significantly associated with advanced clinical stage (p<0.05, χ2-test). Lymph node metastasis was significantly associated with high expression of GSC. The upregulation group of TERT, GSC, NOTCH1 and SNAI1, and downregulation group of TRAF1 were significantly associated with poor overall survival (p<0.05, log-rank test). On further validation with immunohistochemistry, overexpression of goosecoid homeobox (GSC) was associated with poor overall survival. The results suggest that GSC is the most potential biomarker of drug response and poor prognosis in ovarian serous carcinomas.

Chronic ethanol exposure increases goosecoid (GSC) expression in human embryonic carcinoma cell differentiation.

Fetal alcohol spectrum disorder (FASD) is a set of developmental malformations caused by excess alcohol consumption during pregnancy. Using an in vitro system, we examined the role that chronic ethanol (EtOH) exposure plays in gene expression changes during the early stage of embryonic differentiation. We demonstrated that EtOH affected the cell morphology, cell cycle progression and also delayed the down-regulation of OCT4 and NANOG during differentiation. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early embryogenesis. Follow-up analyzes revealed that EtOH exposure to embryoid bodies (EBs) induced the expression of an organizer-specific gene, goosecoid (GSC), in comparison to controls. Moreover, EtOH treatment altered several important genes that are involved in embryonic structure formation, nervous system development, and placental and embryonic vascularization, which are all common processes that FASD can disrupt. Specifically, EtOH treatment let to a reduction in ALDOC, ENO2 and CDH1 expression, whereas EtOH treatment induced the expression of PTCH1, EGLN1, VEGFA and DEC2 in treated EBs. We also found that folic acid (FA) treatment was able to correct the expression of the majority of genes deregulated by EtOH exposure during early embryo development. Finally, the present study identified a gene set including GSC, which was deregulated by EtOH exposure that may contribute to the etiology of fetal alcohol syndrome (FAS). We also reported that EtOH-induced GSC expression is mediated by Nodal signaling, which may provide a new avenue for analyzing the molecular mechanisms behind EtOH teratogenicity in FASD individuals.