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.

Goosecoid Controls Neuroectoderm Specification via Dual Circuits of Direct Repression and Indirect Stimulation in Xenopus Embryos.

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.

Structures/cytotoxicity/selectivity relationship of natural steroidal saponins against GSCs and primary mechanism of tribulosaponin A.

Glioblastoma multiform (GBM) is the highly aggressive brain tumor with poor prognosis. Glioma stem cells (GSCs), small population of cancer cells that exist in GBM tissues, resistant to chemotherapy and radiotherapy and usually driving GBM recurrence, have been developed as effective therapeutic target. Steroidal saponins are one of important resources for anti-tumor agent and may be benefited to selectively clear GSCs. In this report, total of 97 natural steroidal saponins were investigated the relationship among structures/cytotoxicity/selectivity against GSCs, glioma cell lines and human untransformed cells, and revealed that tribulosaponin A was the most potent compound. Further investigation suggested that tribulosaponin A up-regulated the expression of NCF1 and NOX1 to accumulate ROS for triggering apoptosis in GSCs, but not in untransformed cells, and it was further supported by the assay that N-acetyl-l-cysteine (NAC) clearing ROS delayed GSCs apoptosis. Besides, tribulosaponin A damaged GSCs recapturing tumor spheres formation.

Wnt/ß-catenin signaling is an evolutionarily conserved determinant of chordate dorsal organizer.

Deciphering the mechanisms of axis formation in amphioxus is a key step to understanding the evolution of chordate body plan. The current view is that Nodal signaling is the only factor promoting the dorsal axis specification in the amphioxus, whereas Wnt/ß-catenin signaling plays no role in this process. Here, we re-examined the role of Wnt/ßcatenin signaling in the dorsal/ventral patterning of amphioxus embryo. We demonstrated that the spatial activity of Wnt/ß-catenin signaling is located in presumptive dorsal cells from cleavage to gastrula stage, and provided functional evidence that Wnt/ß-catenin signaling is necessary for the specification of dorsal cell fate in a stage-dependent manner. Microinjection of Wnt8 and Wnt11 mRNA induced ectopic dorsal axis in neurulae and larvae. Finally, we demonstrated that Nodal and Wnt/ß-catenin signaling cooperate to promote the dorsal-specific gene expression in amphioxus gastrula. Our study reveals high evolutionary conservation of dorsal organizer formation in the chordate lineage.

Glioma stem cell (GSC)-derived autoschizis-like products confer GSC niche properties involving M1-like tumor-associated macrophages.

Spontaneous necrosis is a defining feature of glioblastomas (GBMs), the most malignant glioma. Despite its strong correlations with poor prognosis, it remains unclear whether necrosis could be a possible cause or mere consequence of glioma progression. Here we isolated a particular fraction of necrotic products spontaneously arising from glioma cells, morphologically and biochemically defined as autoschizis-like products (ALPs). When administered to granulocyte macrophage colony-stimulating factor (GM-CSF)-primed bone marrow-derived macrophage/dendritic cells (Mφ/DCs), ALPs were found to be specifically engulfed by Mφs expressing a tumor-associated macrophage (TAM) marker CD204. ALPs from glioma stem cells (GSCs) had higher activity for the TAM development than those from non-GSCs. Of note, expression of the Il12b gene encoding a common subunit of IL-12/23 was upregulated in ALPs-educated Mφs. Furthermore, IL-12 protein evidently enhanced the sphere-forming activity of GBM patient-derived cells, although interestingly IL-12 is generally recognized as an antitumoral M1-Mφ marker. Finally, in silico analysis of The Cancer Genome Atlas (TCGA) transcriptome data of primary and recurrent GBMs revealed that higher expression of these IL-12 family genes was well correlated with more infiltration of M1-type TAMs and closely associated with poorer prognosis in recurrent GBMs. Our results highlight a role of necrosis in GSC-driven self-beneficial niche construction and glioma progression, providing important clues for developing new therapeutic strategies against gliomas.

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.

STATISTICAL COMPARISON OF AFIRMA GSC AND AFIRMA GEC OUTCOMES IN A COMMUNITY ENDOCRINE SURGICAL PRACTICE: EARLY FINDINGS.

OBJECTIVE: The Veracyte Afirma Gene Expression Classifier (GEC) has been the most widely used negative predictive value molecular classifier for indeterminate cytology thyroid nodules since January 2011. To improve the specificity and further reduce unnecessary thyroid surgeries, a second-generation assay (Afirma Genetic Sequence Classifier [GSC]) was released for clinical use in August 2017. We report 11 months of clinical outcomes experience with the GSC and compare them to our 6.5-year experience with the GEC. METHODS: We searched our practice registry for FNAB nodules with Afirma results from January 2011through June 2018. GEC versus GSC results were compared overall, in oncocytic and nononcocytic aspirates and by pathologic outcomes. RESULTS: GSC identified less indeterminate cytology nodules as suspicious (38.8%; 54/139) when compared to GEC (58.4%; 281/481). There was a decrease of in the percentage of oncocytic fine-needle aspiration thyroid biopsy (FNAB) subjects classified as suspicious in the GSC group, with 86 of 104 oncocytic indeterminates (82.7%) classified as suspicious by GEC and 12 of 34 (35.3%) classified as suspicious by GSC. The surgery rate in patients with oncocytic aspirates fell from 56% in the GEC group to 31% in the GSC-evaluated group (45%). Pathology analysis demonstrated a false-negative percentage for an incomplete surgical group of 9.5% for GEC and 1.2% for GSC. CONCLUSION: Our GSC data suggest that the GSC further reduces surgery in indeterminate thyroid nodules by improving the specificity of Afirma technology without compromising sensitivity. A primary determinant for this change is a significant improvement in the specificity of the Afirma GSC test in oncocytic FNAB aspirates. ABBREVIATIONS: FNAB = fine-needle aspiration biopsy; GEC = Gene Expression Classifier; GSC = Genetic Sequence Classifier.

Self-organization of a human organizer by combined Wnt and Nodal signalling.

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

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.

Gastrulation in an annual killifish: Molecular and cellular events during germ layer formation in Austrolebias.

BACKGROUND: Comparative studies beyond the traditional model organisms have been instrumental in enhancing our understanding of the conserved and derived features of gastrulation, a fundamental process in which the germ layers are specified and shaped to form the body axis. Here, we analyzed gastrulation in a vertebrate group with an extreme mode of early development, the annual killifish. RESULTS: Gastrulation in annual killifish of the genus Austrolebias takes place after the initially dispersed deep blastomeres congregate to form the so-called reaggregate. Cells from the early reaggregate do not appear to form part of any recognizable axial embryonic structure and are possibly extraembryonic. In contrast, later reaggregate cells become engaged in morphogenetic transformations indicative of a process of gastrulation and axis formation. The expression of brachyury and goosecoid suggests that gastrulation takes place in a compressed blastopore-like structure with an organizer region displaced to one end. No collective cell internalization proper of blastopore architecture is observed, though, and it appears that gastrulation primarily involves the reorganization of individual cells. CONCLUSIONS: The unique mode of gastrulation in annual killifish demonstrates that a process so ancient and fundamental to ontogenesis can have striking morphogenetic variations nonpredicted from the sole examination of model species. Developmental Dynamics 246:812-826, 2017. © 2017 Wiley Periodicals, Inc.

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.