Initial retinoid requirement for early avian development coincides with retinoid receptor coexpression in the precardiac fields and induction of normal cardiovascular development.

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities and an early death of the vitamin A-deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. We demonstrate that all-trans-, 9-cis-, 4-oxo-, and didehydroretinoic acids, and didehydroretinol and all-trans-retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor beta2 in the vitamin A-deficient quail embryo. The expression of RARbeta2 is at the same level and at the same sites where it is expressed in the normal embryo. Retinoids provided to the vitamin A-deficient embryo up to the 5-somite stage of development, but not later, completely rescue embryonic development, suggesting the 5-somite stage as a critical retinoid-sensitive time point during early avian embryogenesis. Retinoid receptors RARalpha, RARgamma, and RXRalpha are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A-deficient quail embryo, while the expression of RXRgamma is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARalpha and RARbeta. Our studies provide strong evidence for a narrow retinoid-requiring developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors is essential for a subsequent normal embryonic development.

Retinoid X receptor gamma gene transcripts are expressed by a subset of early generated retinal cells and eventually restricted to photoreceptors.

We have examined the distribution of the retinoid X receptor gamma (RXRgamma) in the developing chicken retina by using in situ hybridization and RNase protection assays. We detected RXRgamma transcripts as early as 4 days of embryonic development (d4) in central regions of the retina, spreading to more peripheral regions by d8. The first few RXRgamma-positive cells were scattered within the depth of the retinal neuroepithelium, but as they increased in number they became localized predominantly to the apical (outer, ventricular) layer. The identity of the RXRgamma-positive cells at these stages is unknown, due to the lack of cell type-specific markers. By d10, when photoreceptors and ganglion cells have been generated and begun to establish their definitive layers, RXRgamma-positive cells were virtually restricted to the photoreceptor layer, and maintained this distribution to posthatch stages. RNase protection assays were performed on whole retinae to verify the temporal pattern of in situ hybridization results and showed that between d5 and d16 there was a significant increase in the mRNA levels of the RXRgamma2 isoform. Between d16 and early posthatch stages the level of RXRgamma2 mRNA did not change significantly. Consistent with previous studies, mRNA levels of the RXRgamma1 isoform were substantially lower than mRNA levels of the RXRgamma2 isoform at all time points examined. These results demonstrate that RXRgamma mRNA is expressed in photoreceptors in the developing chicken retina and implicate RXRgamma as the earliest marker of photoreceptor differentiation documented to date.

Early developmental expression and experimental axis determination by the chicken Vg1 gene.

BACKGROUND: Genes of the transforming growth factor beta (TGF beta) superfamily have been implicated in the earliest steps of developmental patterning in vertebrates. In Xenopus, the Vg1 gene is a candidate for the initiator of axis formation: its RNA and protein are broadly but appropriately localized at the start of development, and processed Vg1 protein is a powerful inducer of organized axial tissue in blastular animal caps in vitro and when locally produced in vivo after injection of Vg1 mRNA into blastomeres. Site-specific proteolytic processing occurs ubiquitously for most TGF beta members, producing the active peptide ligand, but is tightly restricted, by unknown mechanisms, for endogenous Vg protein in Xenopus and zebrafish embryos. This restriction may be involved in the spatial localization of activity required for an organizing role. RESULTS: We have characterized an amniote (chick) orthologue of Vg1, cVg1, and examined its developmental expression. The early expression of cVg1 includes a phase broadly related to the known time and site of axis (primitive streak) initiation; the initial transcription of cVg1 is centred in the posterior marginal zone (PMZ), a region of the blastoderm known to contain the axial organizing activity at this stage. We also observed later neural and paraxial mesodermal expression of cVg1, which has not been described previously for Vg homologues in other vertebrates. We have grafted transfected COS cells, producing processed cVg1 protein, to peripheral positions around the chick early blastoderm. Such grafts initiate formation of morphologically complete primitive streaks, simulating the properties of grafts from the PMZ. CONCLUSIONS: In vertebrate development, Vg genes may be required for an evolutionarily conserved early step in positioning or induction of the axis.

Cloning, sequencing, and expressional analysis of the chick homologue of follistatin.

Follistatin, a secreted glycoprotein, has been shown to act as a potent neural inducer during early amphibian development. The function of this protein during embryogenesis in higher vertebrates is unclear, and to further our understanding of its role we have cloned, sequenced, and performed an in-depth expressional analysis of the chick homologue of follistatin. In addition we also describe the expression pattern of activin beta A and activin beta B, proteins that have previously been shown to be able to interact with follistatin. In this study we show that the expression of follistatin and the activins do not always overlap. Follistatin was first detected in Hensen's node and subsequently in the region described by Spratt [1952] as the neuralising area. In older embryos it was also expressed in a highly dynamic manner in the hindbrain as well as in the somites. We also present evidence that follistatin may have a later role in the resegmentation of the somites. We were unable to detect the expression of activin beta A during early embryogenesis, whereas activin beta B was first expressed in the extending primitive streak and subsequently in the neural folds. The results from this study are consistent with a role for follistatin in neural induction but suggest it has additional functions unrelated to its inhibitory actions on activins.

The chicken retinoid-X-receptor-α (RXR-α) gene and its expression in the developing limb.

Antero-posterior (a-p) patterning of the vertebrate limb bud is controlled by signals from the polarizing region, a group of cells in the posterior mesenchyme of the bud. Application of retinoic acid to the anterior margin of the chick wing bud induces polarizing region activity in anterior mesenchyme cells, resulting in digit duplications. Retinoic acid acts by binding to nuclear retinoid receptors, and so regulating expression of target genes. Retinoid receptors of the RXR class are essential for this activity. We have previously described a chicken RXR-γ cDNA clone (Rowe et al. 1991a). In this paper we report the isolation and characterization of a chicken RXR-α cDNA clone, and show by northern blotting that an RXR-α mRNA of approximately 5 kilobases is present in a range of tissues in embryonic and adult chickens. In situ hybridization experiments showed that RXR-α transcripts were present throughout the epithelium and mesenchyme of the chick wing bud at stages when retinoic acid can affect a-p patterning. In contrast, RXR-γ transcripts were undetectable in these cells, being restricted to peripheral nervous tissue in the bud. These data suggest that RXR-α, but not RXR-γ, could mediate the effects of locally applied retinoic acid on a-p patterning in the chick wing bud.

The chicken retinoid-X-receptor-gamma gene gives rise to two distinct species of mRNA with different patterns of expression.

Retinoids are metabolites of vitamin A that can regulate gene expression in a range of embryonic and adult cell types. They do this by binding to nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. Vertebrates possess two classes of nuclear retinoid-receptor genes, each with three members. These are the RAR-alpha, RAR-beta and RAR-gamma genes and the RXR-alpha, RXR-beta and RXR-gamma genes. In this paper we show by cDNA cloning and ribonuclease protection that the chicken RXR-gamma gene gives rise to two mRNA species (RXR-gamma 1 and RXR-gamma 2) that differ at their 5' ends. The two mRNAs have different tissue distributions in the 10-day-old chick embryo. RXR-gamma 2 mRNA was present in the eye and dorsal root ganglia but was undetectable in the liver. In contrast, RXR-gamma 1 mRNA was present in liver, was undetectable in dorsal root ganglia and was just detectable in the eye, where it was much less abundant than RXR-gamma 2 mRNA. The predicted protein products of the RXR-gamma 1 and RXR-gamma 2 mRNAs differ at their N-termini, in a region thought to modulate transcriptional transactivation by the receptor. These results show that at least one of the retinoid-X-receptor (RXR) genes gives rise to more than one protein product, a principle previously established for the retinoic acid-receptor (RAR) genes. The existence of multiple RXR protein isoforms would increase the range of heterodimers formed between RXRs and other nuclear receptors, including RARs and the receptors for thyroid hormone, vitamin D and peroxisome proliferators. This could increase the diversity of transcriptional responses mediated by these molecules.

Identification of antisense transcripts of the chicken insulin-like growth factor-II gene.

Insulin-like growth factor-II (IGF-II) is a polypeptide mitogen which is believed to play an important role in fetal development. The human and rat IGF-II genes are complex transcription units, which contain multiple promoters and polyadenylation sites and which exhibit alternate splicing of their primary transcripts. In order to study IGF-II gene expression during chick embryonic development, we screened a 10-day chick embryo cDNA library with a human IGF-II cDNA probe. We isolated a clone, designated cigf, that was comprised, in part, of sequences homologous to the second coding exon of the human, mouse and rat IGF-II genes. Comparison of the nucleotide sequence of cigf with that of the corresponding genomic clone indicated that cigf was derived from a spliced antisense transcript of the chicken IGF-II gene, which overlapped the second coding exon. Northern blotting experiments with single-stranded RNA probes synthesized using cigf DNA as a template showed that stage 22 and stage 36 chick embryos contained sense strand IGF-II transcripts of 1.4, 2.2, 4.7 and 7.0 kb and antisense strand transcripts of 0.7, 1.3, 1.8, 2.5, 4.9, 6.0 and 8.0 kb. The pattern of sense strand IGF-II transcripts was similar to that previously found in rat fetal tissues. Whilst there are precedents for the transcription of both strands of a single gene, this is the first evidence for antisense transcription of an IGF gene. The functions of the antisense transcripts remain to be determined. These findings demonstrate a further level of complexity in the IGF-II transcription unit and indicate that studies of IGF-II transcript distribution performed with double-stranded probes should be interpreted with caution. They also suggest explanations for the recent finding that IGF-II peptides are present at much lower levels in embryos than expected from the high levels of IGF-II transcripts present.