Preliminary Findings of Structure and Expression of Opioid Receptor Genes in a Peregrine Falcon ( Falco peregrinus), a Snowy Owl ( Bubo scandiacus), and a Blue-fronted Amazon Parrot ( Amazona aestiva).

To further knowledge of the physiology of opioid receptors in birds, the structure and expression of the µ-, δ-, and κ-opioid receptor genes were studied in a peregrine falcon ( Falco peregrinus), a snowy owl ( Bubo scandiacus), and a blue-fronted Amazon parrot ( Amazona aestiva). Tissue samples were obtained from birds that had been euthanatized for poor release prognosis or medical reasons. Samples were taken from the brain (telencephalon, thalamus, pituitary gland, cerebellum, pons, medulla oblongata, mesencephalon), the spinal cord and dorsal root ganglions, and plantar foot skin. Messenger RNA was recovered, and reverse transcription polymerase chain reaction (RT-PCR) was performed to generate complementary DNA (cDNA) sequences. Gene structures were documented by directly comparing cDNA sequences with recently published genomic sequences for the peregrine falcon and the blue-fronted Amazon parrot or by comparisons with genomic sequences of related species for the snowy owl. Structurally, the avian µ-opioid receptor messenger RNA (mRNA) species were complex, displaying differential splicing, alternative stop codons, and multiple polyadenylation signals. In comparison, the structure of the avian κ-receptor mRNA was relatively simple. In contrast to what is seen in humans, the avian δ-receptor mRNA structure was found to be complex, demonstrating novel 3-prime coding and noncoding exons not identified in mammals. The role of the δ-opioid receptor merits further investigation in avian species.

Male-biased aganglionic megacolon in the TashT mouse line due to perturbation of silencer elements in a large gene desert of chromosome 10.

Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line-named TashT-that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung's disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a "tipping point" of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung's disease but also provides important new insights into its male sex bias.

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

BACKGROUND: 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. RESULTS: Using Cre-mediated lineage studies and transgenic reporter mouse models, we now show that promoter sequences of human, pig and mouse SRY drive robust reporter gene expression in epiblast cells of peri-implantation embryos between embryonic day (E) 4.5 and E6.5. Analysis of endogenous Sry expression revealed that linear transcripts are produced by means of multiple polyadenylation sites in E4.5 embryos. Within the epiblast, SRY reporter expression mimics the expression seen using a Gata4 reporter model, but is dissimilar to that seen using an Oct4 reporter model. In addition, we report that overexpression of mouse Sry in embryonic stem cells leads to down-regulation of the core pluripotency markers Sox2 and Nanog. CONCLUSION: We propose that SRY/Sry may function as a male-specific maturation factor in the peri-implantation mammalian embryo, providing a genetic mechanism to help explain the observation that male embryos are developmentally more advanced compared with female embryos, and suggesting a role for SRY beyond that of TDF.

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 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.

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.