Nicolas Pilon

Cdx1 Autoregulation Is Governed by a Novel Cdx1-LEF1 Transcription Complex

ABSTRACT The Cdx1 gene product is essential for normal anterior-posterior vertebral patterning. Expression of Cdx1 is regulated by several pathways implicated in anterior-posterior patterning events, including retinoid and Wnt signaling. We have previously shown that retinoic acid plays a key role in early stages of Cdx1 expression at embryonic day 7.5 (E7.5), while both Wnt3a signaling and an autoregulatory loop, dependent on Cdx1 itself, are involved in later stages of expression (E8.5 to E9.5). This autoregulation is reflected by the ability of Cdx1 to affect expression from proximal Cdx1 promoter sequences in tissue culture. However, this region is devoid of a demonstrable Cdx response element(s). We have now found that Cdx1 and LEF1, a nuclear effector of Wnt signaling, synergize to induce expression from the Cdx1 promoter through previously documented LEF/T-cell factor response elements. We also found a direct physical interaction between the homeodomain of Cdx1 and the B box of LEF1, suggesting a basis for this synergy. Consistent with these observations, analysis of Cdx1 Wnt3avt compound mutants demonstrated that Wnt and Cdx1 converged on Cdx1 expression and vertebral patterning in vivo. Further data suggest that Cdx-high-mobility group box interactions might be involved in a number of additional pathways.

Wnt signaling is a key mediator of Cdx1 expression in vivo.

In the mouse, Cdx1 is essential for normal anteroposterior vertebral patterning through regulation of a subset of Hox genes. Retinoic acid (RA) and certain Wnts have also been implicated in vertebral patterning, although the relationship between these signaling pathways and the regulation of mesodermal Hox gene expression is not fully understood. Prior work has shown that Cdx1 is a direct target of both Wnt and retinoid signaling pathways, and might therefore act to relay these signals to the Hox genes. Wnt and RA are believed to impact on Cdx1 through an atypical RA-response element (RARE) and Lef/Tcf-response elements (LRE), respectively, in the proximal promoter. To address the roles of these regulatory motifs and pathways, we derived mice mutated for the LRE or the LRE plus the RARE. In contrast to RARE-null mutants, which exhibit limited vertebral defects, LRE-null and LRE+RARE-null mutants exhibited vertebral malformations affecting the entire cervical region that closely phenocopied the malformations seen in Cdx1-null mutants. Mutation of the LRE also greatly reduced induction of Cdx1 by RA, demonstrating a requirement for Wnt signaling in the regulation of this gene by retinoids. LRE and LRE+RARE mutants also exhibited vertebral fusions, suggesting a defect in somitogenesis. As Wnt signaling is implicated in somitogenesis upstream of the Notch pathway, it is conceivable that Cdx1 might play a role in this process. However, none of the Notch pathway genes assessed was overtly affected.

Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation.

We have generated complete complementary DNA (cDNA) sequences for the porcine steroidogenic acute regulatory protein (StAR) gene, using a combination of genomic PCR amplification and reverse transcription-PCR amplification of pig ovarian cDNA. Porcine StAR cDNA consists of 855 bp and shares 90.2%, 87.3%, 84.3%, and 83.9% homologies with bovine, human, mouse, and rat StAR cDNA at the nucleotide level, and 89.1%, 88.8%, 86.7%, and 86.3% homologies with bovine, human, mouse, and rat StAR protein at the deduced amino acid level. Northern analysis of porcine StAR showed that it is expressed in adult and fetal steroidogenic tissues, including adult testes and ovaries and adult adrenal glands as well as steroidogenic tissues of pregnancy, including developing fetal testes, corpus luteum, and pregnancy, but not the fetal ovary. Major hybridizing bands of 1.8 and 1.1 kilobases were demonstrated. In contrast to human StAR, porcine StAR was not expressed in adult or fetal kidneys. Expression of porcine StAR by the p...

Porcine SRY Promoter Is a Target for Steroidogenic Factor 1

Abstract To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated a 4.5-kilobase (kb) pig SRY 5′ flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at −661 base pairs (bps) 5′ from the translation initiation site. Deletion studies of the SRY 5′ flanking sequences in PGR 9E11 cells demonstrated that −1.4 kb of 5′ flanking sequences retained full transcriptional activity compared with the −4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, because deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the −1.4 kb fragment identified two potential binding sites for SF-1, at −1369 and at −290 from the ATG. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse SF-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at −1369 bp and at −290 bp, and that the site at −1369 bp is quantitatively the most important.

The Proximal Gata4 Promoter Directs Reporter Gene Expression to Sertoli Cells During Mouse Gonadal Development

Abstract The GATA4 transcription factor is an important developmental determinant for many organs, such as the heart, gut, and testis. Despite this pivotal role, our understanding of the transcriptional mechanisms that control the proper spatiotemporal expression of the GATA4 gene remains limited. We have generated transgenic mice expressing a green fluorescent protein (GFP) marker under the control of rat Gata4 5′ flanking sequences. Several GATA4-expressing organs displayed GFP fluorescence, including the heart, intestine, and pancreas. In the gonads, while GATA4 is expressed in pregranulosa, granulosa, and theca ovarian cells, and Sertoli, Leydig, and peritubular testicular cells, the first 5 kb of Gata4 regulatory sequences immediately upstream of exon 1 were sufficient to direct GFP reporter expression only in testis and, specifically, in Sertoli cells. Onset of GFP expression occurred after Sertoli cell commitment and was maintained in these cells throughout development to adulthood. In vitro studies revealed that the first 118 bp of the Gata4 promoter is sufficient for full basal activity in several GATA4-expressing cell lines. Promoter mutagenesis and DNA-binding experiments identified two GC-box motifs and, particularly, one E-box element within this −118-bp region that are crucial for its activity. Further analysis revealed that members of the USF family of transcription factors, especially USF2, bind to and activate the Gata4 promoter via this critical E-box motif.

Novel pre- and post-gastrulation expression of Gata4 within cells of the inner cell mass and migratory neural crest cells

GATA4 is a transcription factor known to be important for the development of many organs such as the heart, intestine, and gonads. However, information regarding the control of its expression is only now beginning to emerge. To further understand the regulation of Gata4 expression during mouse embryonic development, we have generated a novel knockin allele allowing expression of the Cre recombinase under the control of Gata4 regulatory sequences. When these Gata4Cre/+ mice were crossed with the Cre reporter mouse R26R‐YFP, we surprisingly found widespread mosaic YFP expression in e10.0 embryos. This particular expression pattern was traced back to the e5.5 stage via a cell lineage study, suggesting activation of transcription at the Gata4 locus around the blastocyst stage. In accordance with this hypothesis, we found that Gata4 is expressed in cultured embryonic stem (ES) cells and within the inner cell mass (ICM) of e4.5 blastocysts. Interestingly, such early Gata4 transcription can be recapitulated in transgenic reporter studies using 5 kb of the proximal rat Gata4 promoter. During mouse development, these 5‐kb regulatory sequences were previously reported to direct reporter gene expression to Sertoli cells of the testes [Mazaud Guittot et al. ( 2007 ) Biol Reprod 76:85–95]. We now show that these regulatory sequences can also drive robust fluorescent reporter gene expression in migratory neural crest cells. Comparisons to Wnt1‐Cre‐mediated YFP labelling of neural crest cells suggest that most of the migratory neural crest cells are labelled in e9.5 to e11.5 Gata4p[5kb]‐RFP or ‐GFP embryos. Analysis of GFP transcription via whole‐mount in situ hybridization in e10.5 and e11.5 embryos demonstrated that the 5‐kb Gata4 promoter is preferentially active in cells of the boundary caps at the dorsal root entry zone and motor exit points flanking the neural tube. RT‐PCR gene expression analysis of FACS‐purified GFP‐positive cells from e9.5 Gata4p[5kb]‐GFP embryos revealed co‐expression of Gata4 with many neural crest stem cell markers. Together with sphere‐forming and differentiation cell culture assays, our results indicate that the Gata4 promoter is active within at least a subset of the neural crest stem cells. Taken altogether, our studies have revealed new Gata4 expression patterns during mouse embryonic development, which are controlled by its 5‐kb proximal 5′ flanking sequences. Developmental Dynamics 237:1133–1143, 2008.

The developmental genetics of Hirschsprung's disease

Hirschsprungs disease (HSCR), also known as aganglionic megacolon, derives from a congenital malformation of the enteric nervous system (ENS). It displays an incidence of 1 in 5000 live births with a 4:1 male to female sex ratio. Clinical signs include severe constipation and distended bowel due to a non‐motile colon. If left untreated, aganglionic megacolon is lethal. This severe congenital condition is caused by the absence of colonic neural ganglia and thus lack of intrinsic innervation of the colon due in turn to improper colonization of the developing intestines by ENS progenitor cells. These progenitor cells are derived from a transient stem cell population called neural crest cells (NCC). The genetics of HSCR is complex and can involve mutations in multiple genes. However, it is estimated that mutations in known genes account for less than half of the cases of HSCR observed clinically. The male sex bias is currently unexplained. The objective of this review is to provide an overview of the pathophysiology and genetics of HSCR, within the context of our current knowledge of NCC development, sex chromosome genetics and laboratory models.

A collagen VI–dependent pathogenic mechanism for Hirschsprung’s disease

Hirschsprungs disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR--named Holstein--that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.

Caudal-related homeobox (Cdx) protein-dependent integration of canonical Wnt signaling on paired-box 3 (Pax3) neural crest enhancer.

Background: Cdx transcription factors are known to convey the posteriorizing signals from the canonical Wnt pathway. Results: Cdx proteins integrate canonical Wnt signals on a Pax3 neural crest enhancer. Conclusion: Cdx proteins are involved in Wnt-mediated induction of Pax3 at the neural plate border. Significance: Our data suggest that Cdx proteins are important novel players within the neural crest gene regulatory network. One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

Human and pig SRY 5′ flanking sequences can direct reporter transgene expression to the genital ridge and to migrating neural crest cells

Mechanisms for sex determination vary greatly between animal groups, and include chromosome dosage and haploid–diploid mechanisms as seen in insects, temperature and environmental cues as seen in fish and reptiles, and gene‐based mechanisms as seen in birds and mammals. In eutherian mammals, sex determination is genetic, and SRY is the Y chromosome located gene representing the dominant testes determining factor. How SRY took over this function from ancestral mechanisms is not known, nor is it known what those ancestral mechanisms were. What is known is that SRY is haploid and thus poorly protected from mutations, and consequently is poorly conserved between mammalian species. To functionally compare SRY promoter sequences, we have generated transgenic mice with fluorescent reporter genes under the control of various lengths of human and pig SRY 5′ flanking sequences. Human SRY 5′ flanking sequences (5 Kb) supported reporter transgene expression within the genital ridge of male embryos at the time of sex determination and also supported expression within migrating truncal neural crest cells of both male and female embryos. The 4.6 Kb of pig SRY 5′ flanking sequences supported reporter transgene expression within the male genital ridge but not within the neural crest; however, 2.6 Kb and 1.6 Kb of pig SRY 5′ flanking sequences retained male genital ridge expression and now supported extensive expression within cells of the neural crest in embryos of both sexes. When 2 Kb of mouse SRY 5′ flanking sequences (−3 to −1 Kb) were placed in front of the 1.6 Kb of pig SRY 5′ flanking sequences and this transgene was introduced into mice, reporter transgene expression within the male genital ridge was retained but neural crest expression was lost. These observations suggest that SRY 5′ flanking sequences from at least two mammalian species contain elements that can support transgene expression within cells of the migrating neural crest and that additional SRY 5′ flanking sequences can extinguish this expression. Developmental Dynamics 235:623–632, 2006.