Foxd3 Expression Research Articles

Down-regulation of miR-26b induces cisplatin resistance in nasopharyngeal carcinoma by repressing JAG1.

Therapy against nasopharyngeal carcinoma (NPC) is hurdled by chemoresistance. Recent studies found that microRNA (miRNA) are important regulators of cancer resistance. In this study, we aimed to explore the role and mechanism of miR‐26b in regulating NPC cisplatin (CDDP) resistance. Real‐time PCR was used to evaluate miR‐26b levels in CDDP‐resistant and CDDP‐sensitive NPC cells, as well as human NPC tissues. MiR‐26b was ectopically overexpressed in CDDP‐resistant cells, followed by monitoring changes in cell viability and apoptosis. Interaction between JAG1 and miR‐26b was characterized by dual‐luciferase reporter assay. Furthermore, we investigated whether ectopic JAG1 expression reversed CDDP sensitivity induced by miR‐26b overexpression. The effect of FOXD3 down‐regulation on miR‐26b was also evaluated. Our results indicate that miR‐26b was lower in the CDDP‐resistant NPC cells, human NPC tissue, particularly in secondary metastases. Ectopic expression of miR‐26b sensitized NPC cells to CDDP. JAG1 is a target of miR‐26b, and its expression is inversely correlated with miR‐26b. Overexpression of JAG1 reversed the CDDP sensitivity induced by miR‐26b overexpression. FOXD3 expression was also down‐regulated in CDDP‐resistant NPC. FOXD3 promoted miR‐26b expression and down‐regulation of FOXD3 suppressed miR‐26b expression. Down‐regulation of miR‐26b is closely correlated with the CDDP resistance in NPC.

CHD7, Oct3/4, Sox2, and Nanog control FoxD3 expression during mouse neural crest-derived stem cell formation.

Neural crest-derived stem cells (NCSCs) are tissue-specific stem cells derived from multipotent neural crest cells. NCSCs are present in some adult tissues such as dorsal root ganglia, sciatic nerve, and bone marrow. However, little is known about the formation mechanisms of these cells. We have shown that BMP2/Wnt3a signaling and a chromatin remodeler, CHD7, in mice help to maintain the multipotency of neural crest cells and lead to the formation of NCSCs. In the present study, we analyzed a regulatory gene cascade in the formation of mouse NCSCs. The inhibition of FoxD3 expression significantly suppressed the expression of Sox10, which is an indispensable transcription factor for mouse NCSC formation, in the presence of BMP2/Wnt3a. CHD7, Oct3/4, Sox2, and Nanog occupied multiple conserved regions of mouse FoxD3, mE1, mE2, and mE3, in a BMP2/Wnt3a-dependent manner. Furthermore, siRNA of CHD7, Oct3/4, Sox2, and Nanog significantly suppressed FoxD3 expression. The inhibition of histone H3K4 mono- or trimethylation also repressed FoxD3 expression. The present data suggest that CHD7, Oct3/4, Sox2, and Nanog directly induce FoxD3 expression when stimulated by BMP2/Wnt3a signaling, that FoxD3 promotes Sox10 expression, and that histone H3K4 methylation plays important roles in this process of mouse NCSC formation.

Abstract 2892: FOXD3 is a novel repressor of the expression of short isoform of DCLK1 (DCLK1-S) from IntronV(β)-promoter of hDCLK1-gene, and is epigenetically silenced in hCRCs: prognostic/diagnostic implications of FOXD3/DCLK1-S expression in hCRCs

Abstract DCLK1 is a specific marker of colon and pancreatic cancers in mice, and is elevated in human colon adenocarcinomas (hCRCs). Downregulation of DCLK1, results in loss of cancer stem-cell-markers and tumorogenic-potential of human colon cancer cells (hCCCs). We recently reported a novel finding that hCRCs express short-transcripts of DCLK1 (DCLK1-S) from an alternate promoter located within IntronV of DCLK1-gene, while normal human colons express the canonical long transcript (DCLK1-L) from 5’(α)-promoter. We and others have demonstrated that 5’(α)-promoter is hypermethylated in hCRCs, resulting in epigenetic silencing and loss of expression of DCLK1-L in hCRCs. Although 5’(α)-promoter is differentially methylated in normal human colons vs hCRCs, methylation status of IntronV(β)-promoter does not change. We therefore hypothesized that differential expression of DCLK1-S in normal colons vs hCRCs is perhaps due to differences in transcriptional activity of the promoter in normal vs cancer cells. To test our hypothesis, we used several in silico and molecular biology approaches, and report for the first time that FOXD3 is a potent transcriptional inhibitor of the IntronV(β)-promoter, resulting in the absence of DCLK1-S expression in normal human colons. Our results suggest that FOXD3 gene becomes methylated during colon carcinogenesis, causing loss of FOXD3 expression, and results in the expression of DCLK1-S in hCRCs. In order to examine pathophysiological relevance of the loss of FOXD3 and gain of DCLK1-S expression in hCRCs, we measured relative levels of FOXD3/DCLK1-S by qRT-PCR in a cohort of 92 CRC patients, in relation to overall survival and clinicopathological parameters. Patients expressing high-DCLK1-S/low-FOXD3 had significantly worse overall-survival compared to patients expressing low-DCLK1-S/high-FOXD3. High expression of DCLK1-S, in conjunction with low expression of FOXD3, was a stronger independent prognostic factor than expression of high levels of DCLK1-S alone. Conclusions. Based on our studies, we report for the first time that FOXD3 is a potent repressor of IntronV(β)-promoter of hDCLK1-gene in normal cells, and that loss of FOXD3 expression due to hypermethylation and silencing of FOXD3 gene during colon carcinogenesis, results in the expression of DCLK1-S in hCRCs, representing an important biomarker of hCRCs. Our findings also suggest a prognostic/diagnostic value of measuring relative expression levels of DCLK1-S/FOXD3 in tumors of CRC patients. It is speculated that loss of DCLK1-L and FOXD3 expression, associated with increased expression of DCLK1-S can be used as an early diagnostic marker of epigenetic changes, associated with colon carcinogenesis in humans. Citation Format: Malaney R. O’Connell, Shubhashish Sarkar, Gurinder Luthra, Yoshinaga Okugawa, Yuji Toiyama, Denise Ward, Ajay Goel, Pomila Singh. FOXD3 is a novel repressor of the expression of short isoform of DCLK1 (DCLK1-S) from IntronV(β)-promoter of hDCLK1-gene, and is epigenetically silenced in hCRCs: prognostic/diagnostic implications of FOXD3/DCLK1-S expression in hCRCs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2892.

Abstract 2173: Sulforaphane suppresses the growth of triple-negative breast cancer stem cells in vitro and in vivo

Abstract Triple-negative breast cancer (TNBC) represents the poorest prognosis among the breast cancer subtypes and no current standard therapy. TNBC tumors are enriched with stem-like cells that are resistant to standard targeted drugs. Thus, it is urgent to develop new agents that are non-toxic and specifically efficacious against these cancer stem cells (CSCs) that are suggested to be responsible for the initiation and maintenance of tumors. Here, we show that sulforaphane (SFN), which is a dietary component abundant in broccoli and its sprouts, inhibits cell proliferation and sphere formation of the CSC population in TNBC cells and also reduces mammary tumor growth. To evaluate the effects of SFN in TNBC we isolated CSC population from MDA-MB-231 Luci D3H1 human cells using fluorescently activated cell sorting (FACS) analysis. The population containing CD49f+, CD24-/CD44high showed the ability to form mammospheres, which is a characteristic of stemness. When these cells were treated with natural dietary constituent SFN at the concentration of 7.5 μM, they lost the property to form mammospheres in vitro. In vivo, when BalbC/nude mice were supplemented with SFN before and after cell inoculation (daily i.p. injection of 50mg SFN/kg for 5 and 3 weeks respectively), they exhibited reduced tumor volume. The suppressive effect of SFN on the tumor volume was higher when the treatment started before tumor implantation with a thirty percent reduction for pre- (n = 20, 5 weeks) and fourteen for post-treated (n = 20, 3 weeks) group compared to respective controls (saline treated group, n = 20). These results suggest that SFN could be used as a chemopreventive agent considering its nontoxicity and efficacy. Next, we examined the effects of SFN in CSC during tumor growth using a panel of 192 stem cell markers by Nanostring mRNA analysis. The results showed that 95% of the genes were down- regulated in the pre-treatment group (n = 10) compared with the control group (n = 10). Twenty-seven genes presented p value <0.02 and fold change >1.5. We selected 8 genes for further real time validation confirming reduced gene expression of FOXD3, FDZ10, GAS1, WNT3, and NOTCH4 in pre-treatment group (n = 10) compared with control (n = 10). By real time only, we also found the same gene expression pattern for two CSC markers of our interest, CRIPTO-1 and NFkB. Our results indicated that SFN suppresses the formation of triple negative mammospheres and tumor development both in vitro (7.5uM) and in vivo (50mg/kg) possibly by targeting a CSC population. Further analysis on gene expression revealed that SFN particularly decreased the expression of stem cell markers. These results suggest that the use of SFN for chemoprevention of TNBC is plausible and warrant further clinical evaluation. Citation Format: Nadia P. Castro, Maria Cristina Rangel, Anand S. Merchant, Karen Saylor, David Salomon, Young Kim. Sulforaphane suppresses the growth of triple-negative breast cancer stem cells in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2173.

Forced expression of Nanog with mRNA synthesized in vitro to evaluate the malignancy of HeLa cells through acquiring cancer stem cell phenotypes.

Nanog is a pluripotency-related factor. It was also found to play an important role in tumorigenesis. To date, the mechanisms underlying cervical tumorigenesis still need to be elucidated. In the present study, Nanog mRNA was synthesized in vitro and transfected into HeLa cells. After mRNA transfection, the forced expressed of Nanog in HeLa cells led to markedly increased invasion, migration, resistance to chemotherapeutic agents and dedifferentiation. In a subcutaneous xenograft assay, these cells had significantly increased tumorigenic capacity. Real-time PCR indicated that Nanog‑induced dedifferentiation was associated with increased expression of endogenous Oct4, Sox2 and FoxD3. In addition, the dedifferentiated HeLa cells acquired features associated with cancer stem cells (CSCs), such as multipotent differentiation capacity, and expression of CSC markers such as CD133. These data imply that Nanog is a positive regulator of cervical cancer dedifferentiation.

Growth-Arrest-Specific 7 Gene Regulates Neural Crest Formation and Craniofacial Development in Zebrafish.

Growth-arrest-specific 7 (Gas7) is preferentially expressed in the nervous system and plays an important role during neuritogenesis in vertebrates. We recently demonstrated that gas7 is highly expressed in zebrafish neurons, where it regulates neural development. The possibility that gas7 may also regulate the development of other tissues remains to be examined. In this study, we investigate the role of Gas7 in the development of craniofacial tissues. Knockdown of gas7 using morpholino oligomers produced abnormal phenotypes in neural crest (NC) cells and their derivatives. NC-derived cartilage maturation was altered in Gas7 morphants as revealed by aberrant sox9b and dlx2 expression, a phenotype that could be rescued by coinjection of gas7 mRNA. While rhombomere morphology remained normal in Gas7 morphants, we observed reduced expression of the prechondrogenic genes sox9b and dlx2 in cells populating the posterior pharyngeal arches, but the fundamental structure of pharyngeal arches was preserved. In addition, NC cell sublineages that migrate to form neurons, glial cells, and melanocytes were altered in Gas7 morphants as revealed by aberrant expression of neurod, foxd3, and mitfa, respectively. Development of NC progenitors was also examined in Gas7 morphants at 12 hpf, and we observed that the reduction of cell precursors in Gas7 morphants was due to increased apoptosis level. These results indicate that the formation of NC progenitors and derivatives depends on Gas7 expression. Our observations also suggest that Gas7 regulates the formation of NC derivatives constituting the internal tissues of pharyngeal arches, without affecting the fundamental structure of mesodermal-derived pharyngeal arches.

A novelFoxD3Variant Is Associated With Vitiligo and Elevated Thyroid Auto-Antibodies

Vitiligo frequently coincides with autoimmune endocrinopathies, particularly Hashimoto's thyroiditis (HT). Genetic susceptibility may underlie this coincident occurrence. One candidate region is the autoimmunity susceptibility locus on chromosome 1, which encompasses forkhead transcription factor D3 (FoxD3), a gene involved in embryonal melanogenesis. We identified a promotor variant (rs78645479) in an index case of vitiligo + HT + candidiasis and evaluated its clinical and functional relevance. We genotyped 281 patients with variable autoimmune endocrinopathies: HT, Graves' disease (GD), type 1 diabetes (T1D), Addison's disease (AD), autoimmune polyglandular syndrome (APS), and/or vitiligo and 1858 controls. Furthermore, we experimentally assessed the effect of the variant on promotor activity and assessed the expression of FoxD3 in human thyroid tissue samples. Patients with vitiligo had a higher frequency of the risk allele (30%) compared with healthy controls (18.2%). In addition, the variant was associated with the incidence of elevated anti-TPO antibodies and anti-Tg antibodies, but not with TSH, FT3, or FT4 levels and also not with GD, T1D, AD, or APS. Functionally, the variant increased transcriptional activity in Jurkat and in Hek293 cells. We confirmed gene expression of FoxD3 in human thyroid tissue, which seemed elevated in thyroid tissue samples of some patients with GD and nonautoimmune goiter but not in patients with HT. In addition to a possible association of rs78645479 in FoxD3 with vitiligo, our data on the association of this FoxD3 variant with thyroid autoantibodies suggest a potential involvement of FoxD3 in thyroid immunoregulation.

Tumor suppression function of FoxD3 in lung cancer.

The transcription factor forkhead box D3 (FoxD3) is a forkhead protein and serves as a transcriptional repressor. The functions of FoxD3 in embryogenesis and in the development of neural crest cells have been diversely studied. Its role associated with tumor development has attracted attention just in recent years. However, little is known about its regulation and functions in the tumorigenesis and progression of lung cancer. We did a pioneer study to identify the function of FoxD3 in lung cancer by knocking down FoxD3 expression in lung adenocarcinoma cell lines. The result showed that silencing FoxD3 in lung cancer cell lines stimulated cell growth and inhibited cell apoptosis. Through quantified PCR analysis, we observed marked gene expression increase of insulin-like growth factor receptor 1 and BRAF under the condition of FoxD3 silencing. In contrast, cell cycle negative regulator P21 expression decreased after FoxD3 silencing. Our study is the first to identify the tumor suppressor function of FoxD3 in lung cancer.

Homeobox b5 (Hoxb5) regulates the expression of Forkhead box D3 gene (Foxd3) in neural crest

Patterning of neural crest (NC) for the formation of specific structures along the anterio-posterior (A-P) body axis is governed by a combinatorial action of Hox genes, which are expressed in the neuroepithelium at the time of NC induction. Hoxb5 was expressed in NC at both induction and migratory stages, and our previous data suggested that Hoxb5 played a role in the NC development. However, the underlying mechanisms by which Hoxb5 regulates the early NC development are largely unknown. Current study showed that both the human and mouse Foxd3 promoters were bound and trans-activated by Hoxb5 in NC-derived neuroblastoma cells. The binding of Hoxb5 to Foxd3 promoter in vivo was further confirmed in the brain and neural tube of mouse embryos. Moreover, Wnt1-Cre mediated perturbation of Hoxb5 signaling at the dorsal neural tube in mouse embryos resulted in Foxd3 down-regulation. In ovo, Foxd3 alleviated the apoptosis of neural cells induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Foxd3 expression in the chick neural tube. This study demonstrated that Hoxb5 (an A-P patterning gene) regulated the NC development by directly inducing Foxd3 (a NC specifier and survival gene).

FOXD3 modulates migration through direct transcriptional repression of TWIST1 in melanoma.

The neural crest is a multipotent, highly migratory cell population that gives rise to diverse cell types, including melanocytes. Factors regulating the development of the neural crest and emigration of its cells are likely to influence melanoma metastasis. The transcription factor FOXD3 plays an essential role in premigratory neural crest development and has been implicated in melanoma cell dormancy and response to therapeutics. FOXD3 is downregulated during the migration of the melanocyte lineage from the neural crest, and our previous work supports a role for FOXD3 in suppressing melanoma cell migration and invasion. Alternatively, TWIST1 is known to have promigratory and proinvasive roles in a number of cancers, including melanoma. Using ChIP-seq analysis, TWIST1 was identified as a potential transcriptional target of FOXD3. Mechanistically, FOXD3 directly binds to regions of the TWIST1 gene locus, leading to transcriptional repression of TWIST1 in human mutant BRAF melanoma cells. In addition, depletion of endogenous FOXD3 promotes upregulation of TWIST1 transcripts and protein. Finally, FOXD3 expression leads to a significant decrease in cell migration that can be efficiently reversed by the overexpression of TWIST1. These findings uncover the novel interplay between FOXD3 and TWIST1, which is likely to be important in the melanoma metastatic cascade. FOXD3 and TWIST1 define distinct subgroups of cells within a heterogeneous tumor.

Quantitative expression of pluripotency-related genes in parthenogenetically produced buffalo (Bubalus bubalis) embryos and in putative embryonic stem cells derived from them

Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells.

Biphasic influence of Miz1 on neural crest development by regulating cell survival and apical adhesion complex formation in the developing neural tube

Myc interacting zinc finger protein-1 (Miz1) is a transcription factor known to regulate cell cycle- and cell adhesion-related genes in cancer. Here we show that Miz1 also plays a critical role in neural crest development. In the chick, Miz1 is expressed throughout the neural plate and closing neural tube. Its morpholino-mediated knockdown affects neural crest precursor survival, leading to reduction of neural plate border and neural crest specifier genes Msx-1, Pax7, FoxD3, and Sox10. Of interest, Miz1 loss also causes marked reduction of adhesion molecules (N-cadherin, cadherin6B, and α1-catenin) with a concomitant increase of E-cadherin in the neural folds, likely leading to delayed and decreased neural crest emigration. Conversely, Miz1 overexpression results in up-regulation of cadherin6B and FoxD3 expression in the neural folds/neural tube, leading to premature neural crest emigration and increased number of migratory crest cells. Although Miz1 loss effects cell survival and proliferation throughout the neural plate, the neural progenitor marker Sox2 was unaffected, suggesting a neural crest-selective effect. The results suggest that Miz1 is important not only for survival of neural crest precursors, but also for maintenance of integrity of the neural folds and tube, via correct formation of the apical adhesion complex therein.

MicroRNA regulation of skin pigmentation in fish

Summary MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in skin color determination in fish has not been completely determined. Here, we identified that 13 miRNAs are differentially expressed between red and white skin. The analysis of miRNA spatial and temporal expression patterns suggests that miR-429 is a potential regulator of skin pigmentation. miR-429 silencing results in an obvious change in skin pigmentation. Bioinformatics analysis and a luciferase reporter assay show that miR-429 directly regulates expression of Foxd3 by targeting its 3′-untranslated (3′-UTR) region. miR-429 silencing leads to a substantial increase in the expression of Foxd3 in vivo, thereby repressing the transcription of MITF and its downstream genes, such as TYR, TYRP1 or TYRP2. These findings would provide a novel insight into the determination of skin color in fish.

Characterization of Mesenchymal Stem Cell Subpopulations from Human Amniotic Membrane with Dissimilar Osteoblastic Potential

Human fetal mesenchymal stem cells can be isolated from the amniotic membrane (AM-hMSCs) by enzymatic digestion. The biological properties of this cell population have been characterized; however, few studies have focused on the presence of stem cell subpopulations and their differentiation potential. The aim of the present study was to isolate homogeneous AM-hMSC subpopulations based on the coexpression of surface markers. In addition, we aimed to characterize stem cell subpopulations through the detection of typical stem cell markers and its differentiation potential. In this study, fluorescence-activated cell sorting (FACS) was used to positively select for the surface markers CD44, CD73, and CD105. Two subpopulations were isolated: CD44+ / CD73+ / CD105+ (CD105+), and CD44+ / CD73+ / CD105- (CD105-). To characterize the cell subpopulations, the expression of pluripotency-associated markers was analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence. Our results showed positive expression of SOX2, SOX3, PAX6, OCT3/4, and NANOG in the CD105+ and CD105(-) cell subpopulations. In contrast, we did not detect expression of SSEA4 or FOXD3 in either subpopulation. Immunophenotypes, such as mesenchymal and hematopoietic markers, were studied by FACS analyses. Our data revealed the expression of the CD49a, CD49d, CD29, integrin α9β1, CD44, CD73, and CD105 antigens in both subpopulations. In contrast, CD90, CD45, CD34, CD14, and HLA-DR expression was not detected. The ability of both subpopulations to differentiate into osteoblasts, adipocytes, and chondrocytes was evidenced using Alizarin red, Oil-Red, and Alcian blue staining, respectively. Furthermore, neuronal differentiation was demonstrated by the expression of GFAP and NEURO-D. Interestingly, we observed a dissimilar osteoblastic differentiation potential between the subpopulations. CD105- cells showed stronger expression of secreted protein acidic and rich in cysteine (SPARC) and osteonectin, which was associated with more effective calcium deposition, than CD105+ cells. In conclusion, we described a systematic method for the isolation of hMSCs that was highly reproducible and generated homogeneous cultures for osteoblast differentiation with an efficient capacity for mineralization.

MicroRNA regulation of skin pigmentation in fish

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in skin color determination in fish has not been completely determined. Here, we identified that 13 miRNAs are differentially expressed between red and white skin. The analysis of miRNA spatial and temporal expression patterns suggests that miR-429 is a potential regulator of skin pigmentation. miR-429 silencing results in an obvious change in skin pigmentation. Bioinformatics analysis and a luciferase reporter assay show that miR-429 directly regulates expression of Foxd3 by targeting its 3'-untranslated (3'-UTR) region. miR-429 silencing leads to a substantial increase in the expression of Foxd3 in vivo, thereby repressing the transcription of MITF and its downstream genes, such as TYR, TYRP1 or TYRP2. These findings would provide a novel insight into the determination of skin color in fish.

The Wnt signaling mediator tcf1 is required for expression of foxd3 during Xenopus gastrulation.

TCF1 belongs to the family of LEF1/TCF transcription factors that regulate gene expression downstream of Wnt/β-catenin signaling, which is crucial for embryonic development and is involved in adult stem cell regulation and tumor growth. In early Xenopus embryos, tcf1 plays an important role in mesoderm induction and patterning. Foxd3 emerged as a potential tcf1 target gene in a microarray analysis of gastrula stage embryos. Because foxd3 and tcf1 are coexpressed during gastrulation, we investigated whether foxd3 is regulated by tcf1. By using morpholino-mediated knockdown, we show that during gastrulation foxd3 expression is dependent on tcf1. By chromatin immunoprecipitation, we also demonstrate direct interaction of β-catenin/tcf complexes with the foxd3 gene locus. Hence, our results indicate that tcf1 acts as an essential activator of foxd3, which is critical for dorsal mesoderm formation in early embryos.

Dynamic and Differential Regulation of Stem Cell Factor FoxD3 in the Neural Crest Is Encrypted in the Genome

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

A novel FoxD3 gene trap line reveals neural crest precursor movement and a role for FoxD3 in their specification

Neural crest cells migrate extensively and contribute to diverse derivatives, including the craniofacial skeleton, peripheral neurons and glia, and pigment cells. Although several transgenic lines label neural crest subpopulations, few are suited for studying early events in neural crest development. Here, we present a zebrafish gene/protein trap line gt(foxd3-citrine)ct110a that expresses a Citrine fusion protein with FoxD3, a transcription factor expressed in premigratory and migrating neural crest cells. In this novel line, citrine expression exactly parallels endogenous foxd3 expression. High-resolution time-lapse imaging reveals the dynamic phases of precursor and migratory neural crest cell movements from the neural keel stage to times of active cell migration. In addition, Cre-recombination produces a variant line FoxD3-mCherry-pA whose homozygosis generates a FoxD3 mutant. Taking advantage of the endogenously regulated expression of FoxD3-mCherry fusion protein, we directly assess early effects of FoxD3 loss-of-function on specification and morphogenesis of dorsal root ganglia, craniofacial skeleton and melanophores. These novel lines provide new insights and useful new tools for studying specification, migration and differentiation of neural crest cells.

Defective neural crest migration revealed by a Zebrafish model of Alx1-related frontonasal dysplasia

Frontonasal dysplasia (FND) refers to a class of midline facial malformations caused by abnormal development of the facial primordia. The term encompasses a spectrum of severities but characteristic features include combinations of ocular hypertelorism, malformations of the nose and forehead and clefting of the facial midline. Several recent studies have drawn attention to the importance of Alx homeobox transcription factors during craniofacial development. Most notably, loss of Alx1 has devastating consequences resulting in severe orofacial clefting and extreme microphthalmia. In contrast, mutations of Alx3 or Alx4 cause milder forms of FND. Whilst Alx1, Alx3 and Alx4 are all known to be expressed in the facial mesenchyme of vertebrate embryos, little is known about the function of these proteins during development. Here, we report the establishment of a zebrafish model of Alx-related FND. Morpholino knock-down of zebrafish alx1 expression causes a profound craniofacial phenotype including loss of the facial cartilages and defective ocular development. We demonstrate for the first time that Alx1 plays a crucial role in regulating the migration of cranial neural crest (CNC) cells into the frontonasal primordia. Abnormal neural crest migration is coincident with aberrant expression of foxd3 and sox10, two genes previously suggested to play key roles during neural crest development, including migration, differentiation and the maintenance of progenitor cells. This novel function is specific to Alx1, and likely explains the marked clinical severity of Alx1 mutation within the spectrum of Alx-related FND.

Helicobacter pylori Causes Epigenetic Dysregulation of FOXD3 to Promote Gastric Carcinogenesis

Deregulation of forkhead box (Fox) proteins, an evolutionarily conserved family of transcriptional regulators, leads to tumorigenesis. Little is known about their regulation or functions in the pathogenesis of gastric cancer. Promoter hypermethylation occurs during Helicobacter pylori-induced gastritis. We investigated whether the deregulated genes contribute to gastric tumorigenesis. We used integrative genome-wide scans to identify concomitant hypermethylated genes in mice infected with H pylori and human gastric cancer samples. We also analyzed epigenetic gene silencing in gastric tissues from patients with H pylori infection and gastritis, intestinal metaplasia, gastric tumors, or without disease (controls). Target genes were identified by chromatin immunoprecipitation microarrays and expression and luciferase reporter analyses. Methylation profile analyses identified the promoter of FOXD3 as the only genomic region with increased methylation in mice and humans during progression of H pylori-associated gastric tumors. FOXD3 methylation also correlated with shorter survival times of patients with gastric cancer. Genome demethylation reactivated FOXD3 expression in gastric cancer cell lines. Transgenic overexpression of FOXD3 significantly inhibited gastric cancer cell proliferation and invasion, and reduced growth of xenograft tumors in mice, at least partially, by promoting tumor cell apoptosis. FOXD3 bound directly to the promoters of, and activated transcription of, genes encoding the cell death regulators CYFIP2 and RARB. Levels of FOXD3, CYFIP2, and RARB messenger RNAs were reduced in human gastric tumor samples, compared with control tissues. FOXD3-mediated transcriptional control of tumor suppressors is deregulated by H pylori infection-induced hypermethylation; this could perturb the balance between cell death and survival. These findings identify a pathway by which epigenetic changes affect gastric tumor suppression.