a-quantitative-cell-migration-assay-for-murine-enteric-neural

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

UQAM

In mammals the SRY gene functions as a dominant genetic switch for testis determination (Gubbay et al.: Nature 346:1128-1135, 1990; Koopman et al.: Nature 351:117-121, 1991; Sinclair et al.: Nature 346:240-244, 1990). To study SRY transcriptional regulation within an evolutionary context, we have generated transgenic mice that express green fluorescent protein (GFP) under the control of 4.5 kb of pig SRY 5' flanking sequences (pSRYp-GFP). Autofluorescence was observed in the genital ridges of e11.5 male embryos (18-21 tail somites), and by e12.5 (27 tail somites) autofluorescence was observed within the testes cords. The expression of the transgene did not display the abrupt termination characteristic of endogenous mouse SRY, but rather showed a gradual reduction in expression characteristic of human, pig and sheep SRY. Surprisingly, no autofluorescence was observed in normal XX genital ridges, although more sensitive RT-PCR analysis detected transgene transcription. When the transgene was bred into a constitutively male line of mice (Odsex; Bishop et al.: Nat Genet 26:490-494, 2000), autofluorescence was visible in genital ridges of XX animals, in the genetic absence of Sry protein. Via RT-PCR analysis, purified autofluorescent cells from e12.5 gonadal ridges expressed mouse SRY but not Oct4 transcripts, whereas autofluorescent cells from e14.5 gonadal ridges expressed MIS but not Oct4 transcripts, in each case consistent with a pre-Sertoli cell phenotype. In vitro expression studies performed in CV-1 cells demonstrated that pig SOX9 cDNA transactivated the pig SRY promoter but that pig SRY cDNA did not. When a SOX9 potential binding site identified at -205 of the pig SRY 5' flanking sequences was mutated, the SOX9 transactivation effect was reduced by 70%. This site is conserved in the 5' flanking sequences of bovine and human SRY genes but not in the mouse gene. Gel retardation assays using this binding site showed specific binding to SOX9-enriched nuclear extracts that was competed by excess unlabelled binding site but not by mutated binding site. We suggest that pig SRY gene is responsive to a testicular environment and propose a model of feedback amplification of pig SRY transcription by SOX9.

The porcine SRY promoter is transactivated within a male genital ridge environment.

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.

Porcine SRY promoter is a target for steroidogenic factor 1.

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 Wnt3a(vt) 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.

Cdx1 autoregulation is governed by a novel Cdx1-LEF1 transcription complex.

There is considerable evidence that the Cdx gene products impact on vertebral patterning by direct regulation of Hox gene expression. Data from a number of vertebrate model systems also suggest that Cdx1, Cdx2 and Cdx4 are targets of caudalizing signals such as RA, Wnt and FGF. These observations have lead to the hypothesis that Cdx members serve to relay information from signaling pathways involved in posterior patterning to the Hox genes. Regulation of Cdx1 expression by RA and Wnt in the mouse has been well characterized; however, the means by which Cdx2 and Cdx4 are regulated is less well understood. In the present study, we present data suggesting that Cdx4 is a direct target of the canonical Wnt pathway. We found that Cdx4 responds to exogenous Wnt3a in mouse embryos ex vivo, and conversely, that its expression is down-regulated in Wnt3a(vt/vt) embryos and in embryos cultured in the presence of Wnt inhibitors. We also found that the Cdx4 promoter responds to Wnt signaling in P19 embryocarcinoma cells and have identified several putative LEF/TCF response elements mediating this effect. Consistent with these data, chromatin immunoprecipitation assays from either embryocarcinoma cells or from the tail bud of embryos revealed that LEF1 and beta-catenin co-localize with the Cdx4 promoter. Taken together, these results suggest that Cdx4, like Cdx1, is a direct Wnt target.

Cdx4 is a direct target of the canonical Wnt pathway.

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.

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

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.

The proximal Gata4 promoter directs reporter gene expression to sertoli cells during mouse gonadal development.

In mammals, the pre-Sertoli cell of the male genital ridge is the first cell type to display sex specific differentiation and differential gene expression. The genetic cascade driving the differentiation of pre-Sertoli cells and ultimately testis formation is beginning to be unravelled, but many questions remain. A better understanding of the transcriptome of pre-Sertoli cells immediately after sex determination is essential in order to further understand this differentiation process. A mouse model expressing Red Fluorescent Protein (RFP) under the control of a hybrid mouse/pig SRY promoter (HybSRYp-RFP) was used to purify cells from embryonic day 12.0 (e12.0) male genital ridges. To compare the transcriptomes of HybSRYp-RFP cell populations versus age matched whole female genital ridges, RNA was extracted and used to generate molecular probes that were hybridized onto Affymetrix Mouse Genome 430 2.0 micro-arrays. The expression of genes considered markers for pre-Sertoli cells, including Sox9, Mis, Dhh and Fgf9 were identified within the HybSRYp-RFP expressing cell population, while markers for germ cells (Oct4, SSEA-1) and endothelial cells (Ntrk3) were not identified. In contrast, markers for ovarian somatic cell expression, including Fst and Bmp2, were identified as overexpressed within the ovarian cell population. In a general fashion, genes identified as 2.5-fold over expressed in HybSRYp-RFP expressing cells coded notably for cell signalling and extra cellular proteins. The expression of Sox10, Stc2, Fgf18, Fgf13 and Wnt6 were further characterized via whole mount in situ hybridization (WISH) on male and female genital ridges between e11.5 and e14.5. Sox10, Fgf18, Fgf13 and Stc2 gene expression was detected within the male genital ridges while Wnt6 was found diffusely within both the male and female genital ridges. These data represent the earliest comprehensive microarray expression analysis of purified presumptive pre-Sertoli cells available to date.

Presumptive pre-Sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation.

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.

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

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 Gata4(Cre/+) 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.

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

The Cdx transcription factors regulate anterior-posterior (AP) vertebral patterning, at least in part, through direct regulation of Hox gene expression. Analysis of allelic series of Cdx mutant mice suggests functional overlap between these family members. However, the lack of a Cdx2 null mutant makes these analyses incomplete. Moreover, Hox proteins are sometimes redundant, making it difficult to discern whether Cdx members regulate identical Hox target genes in a redundant manner, or whether they regulate separate Hox genes which then converge on events related to vertebral patterning. To more directly assess this question, we developed a "knock in" model whereby Cdx2 was substituted for Cdx1. Consistent with functional redundancy Cdx2 "knock-in" mice exhibited perfect complementation of the Cdx1-null phenotype, as evidenced by the lack of skeletal defects or altered expression of Hox genes typically impacted by Cdx1 loss-of-function. It has been proposed that vertebral AP patterning is reliant on a gradient of the sum total of Cdx proteins, a posit that is consistent with functional redundancy between Cdx family members. To further assess this, we generated a gain-of-function model using BAC transgenesis to alter Cdx1 dosage. Cdx1 BAC transgenic mice overexpressed Cdx1 mRNA and protein, and fully complemented the Cdx1 null allele. However, gain of Cdx1 dosage via this BAC transgene in an otherwise wild type background had no discernible effects on vertebral patterning or Hox gene expression, suggesting that a moderate alteration in the Cdx protein gradient is of no consequence.

Cdx1 and Cdx2 are functionally equivalent in vertebral patterning.

GATA4 is an essential transcription factor required for the development and function of multiple tissues, including a major role in gonadogenesis. Despite its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the Gata4 gene is expressed as multiple transcripts with distinct 5' origins. These co-expressed alternative transcripts are generated by different non-coding first exons with transcripts E1a and E1b being the most prominent. Moreover, we previously showed that an Ebox element, located in Gata4 5' flanking sequences upstream of exon 1a, is important for the promoter activity of these sequences in cell lines. To confirm the importance of this element in vivo, we generated and characterized Gata4 Ebox knockout mice. Quantitative PCR analyses realized on gonads, heart and liver at three developmental stages (embryonic, pre-pubertal and adult) revealed that the Ebox mutation leads to a robust and specific decrease (up to 89%) of Gata4 E1a transcript expression in all tissues and stages examined. However, a detailed characterization of the gonads revealed normal morphology and GATA4 protein levels in these mutants. Our qPCR data further indicate that this outcome is most likely due to the presence of Gata4 E1b mRNA, whose expression levels were not decreased by the Ebox mutation. In conclusion, our work clearly confirms the importance of the proximal Ebox element and suggests that adequate GATA4 protein expression is likely protected by a compensation mechanism between Gata4 E1a and E1b transcripts operating at the translational level.

An Ebox element in the proximal Gata4 promoter is required for Gata4 expression in vivo.

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 WntÂ·Î²-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.

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

The SRY/Sry gene is expressed in pre-Sertoli cells of the male genital ridge and functions as the mammalian testis determining factor (TDF). In addition, expression of SRY/Sry outside the genital ridge has been reported, including preimplantation embryos, although the functional significance of this is not well understood.

Transgenic mouse analysis of Sry expression during the pre- and peri-implantation stage.

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal-related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R-YFP reporter mice have indicated that the Cdx2NSE-Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double-transgenic embryos has revealed that this novel Cre-driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso-ventral axis (including pre-migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non-cranial neural crest derivatives of double-transgenic embryos. Therefore, we believe that the Cdx2NSE-Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 00:1-8. Â© 2013 Wiley Periodicals, Inc.

Nicolas Pilon

Molecular Genetics of Development Laboratory,
Biological Sciences,
Molecular Genetics of Development Laboratory, Biological Sciences

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A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Nicolas Pilon

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Molecular Genetics of Development Laboratory,Biological Sciences,Molecular Genetics of Development Laboratory, Biological Sciences

UQAM

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Molecular Genetics of Development Laboratory,
Biological Sciences,
Molecular Genetics of Development Laboratory, Biological Sciences