Retinoic acid (RA) is an embryonic signaling molecule regulating a wide array of target genes, thereby being a master regulator of patterning and differentiation in a variety of organs. Here we show that mouse embryos deficient for the RA-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2), if rescued from early lethality by maternal RA supplementation between E7.5 and E8.5, lack active RA signaling in the foregut region. The resulting mutants completely fail to develop lungs. Development of more posterior foregut derivatives (stomach and duodenum), as well as liver growth, is also severely affected. A primary lung bud is specified in the RA-deficient embryos, which fails to outgrow due to defective FGF10 signaling and lack of activation of FGF-target genes, such as Pea3 and Bmp4 in the epithelium. Specific Hox and Tbx genes may mediate these RA regulatory effects. Development of foregut derivatives can be partly restored in mutants by extending the RA supplementation until at least E10.5, but lung growth and branching remain defective and a hypoplastic lung develops on the right side only. Such conditions poorly restore FGF10 signaling in the lung buds. Explant culture of RALDH2-deficient foreguts show a capacity to undergo lung budding and early branching in the presence of RA or FGF10. Our data implicate RA as a regulator of gene expression in the early embryonic lung and stomach region upstream of Hox, Tbx and FGF10 signaling.

Both retinoid receptor null mutants and classic nutritional deficiency studies have demonstrated that retinoids are essential for the normal development of diverse embryonic structures (e.g. eye, heart, nervous system, urogenital tract). Detailed analysis of retinoid-modulated events is hampered by several limitations of these models, including that deficiency or null mutation is present throughout gestation, making it difficult to isolate primary effects, and preventing analysis beyond embryolethality. We developed a mammalian model in which retinoid-dependent events are documented during distinct targeted windows of embryogenesis. This was accomplished through the production of vitamin A-depleted (VAD) female rats maintained on sufficient oral retinoic acid (RA) for growth and fertility. After mating to normal males, these RA-sufficient/VAD females were given oral RA doses which allowed for gestation in an RA-sufficient state; embryogenesis proceeded normally until retinoids were withdrawn dietarily to produce a sudden, acute retinoid deficiency during a selected gestational window. In this trial, final RA doses were administered on E11.5, vehicle at E12.5, and embryos analyzed on E13.5; during this 48 hour window, the last RA dose was metabolized and embryos progressed in a retinoid-deficient state. RA-sufficient embryos were normal. Retinoid-depleted embryos exhibited specific malformations of the face, neural crest, eyes, heart, and nervous system. Some defects were phenocopies of those seen in null mutant mice for RXR alpha(-/-), RXR alpha(-/-)/RAR alpha(-/-), and RAR alpha(-/-)/RAR gamma(-/-), confirming that RA transactivation of its nuclear receptors is essential for normal embryogenesis. Other defects were unique to this deficiency model, showing that complete ligand 'knock-out' is required to see those retinoid-dependent events previously concealed by receptor functional redundancy, and reinforcing that retinoid receptors have separate yet overlapping contributions in the embryo. This model allows for precise targeting of retinoid form and deficiency to specific developmental windows, and will facilitate studies of distinct temporal events.

Asymmetric expression of Sonic hedgehog (Shh) in Hensen's node of the chicken embryo plays a key role in the genetic cascade that controls left-right asymmetry, but its involvement in left-right specification in other vertebrates remains unclear. We show that mouse embryos lacking Shh display a variety of laterality defects, including pulmonary left isomerism, alterations of heart looping, and randomization of axial turning. Expression of the left-specific gene Lefty-1 is absent in Shh(-/-) embryos, suggesting that the observed laterality defects could be the result of the lack of Lefty-1. We also demonstrate that retinoic acid (RA) controls Lefty-1 expression in a pathway downstream or parallel to Shh. Further, we provide evidence that RA controls left-right development across vertebrate species. Thus, the roles of Shh and RA in left-right specification indeed are conserved among vertebrates, and the Shh and RA pathways converge in the control of Lefty-1.

During early development of the Xenopus central nervous system (CNS), neuronal differentiation can be detected posteriorly at neural plate stages but is delayed anteriorly until after neural tube closure. A similar delay in neuronal differentiation also occurs in the anterior neural tissue that forms in vitro when isolated ectoderm is treated with the neural inducer noggin. Here we examine the factors that control the timing of neuronal differentiation both in embryos and in neural tissue induced by noggin (noggin caps). We show that the delay in neuronal differentiation that occurs in noggin caps cannot be overcome by inhibiting the activity of the neurogenic gene, X-Delta-1, which normally inhibits neuronal differentiation, suggesting that it represents a novel level of regulation. Conversely, we show that the timing of neuronal differentiation can be changed from late to early after treating noggin caps or embryos with retinoic acid (RA), a putative posteriorising agent. Concommittal with changes in the timing of neuronal differentiation, RA suppresses the expression of anterior neural genes and promotes the expression of posterior neural genes. The level of early neuronal differentiation induced by RA alone is greatly increased by the additional expression of the proneural gene, XASH3. These results indicate that early neuronal differentiation in neuralised ectoderm requires posteriorising signals, as well as signals that promote the activity of proneural genes such as XASH3. In addition, these result suggest that neuronal differentiation is controlled by anteroposterior (A-P) patterning, which exerts a temporal control on the onset of neuronal differentiation.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.

Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable:(1) for studying commitment and neuronal specification in vitro,(2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.

Somites, the segmented building blocks of the vertebrate embryo, arise one by one in a patterning process that passes wavelike along the anteroposterior axis of the presomitic mesoderm (PSM). We have studied this process in Xenopus embryos by analyzing the expression of the bHLH gene, Thylacine1, which is turned on in the PSM as cells mature and segment, in a pattern that marks both segment boundaries and polarity. Here, we show that this segmental gene expression involves a PSM enhancer that is regulated by retinoic acid (RA) signaling at two levels. RA activates Thylacine1 expression in rostral PSM directly. RA also activates Thylacine1 expression in the caudal PSM indirectly by inducing the expression of MKP3, an inhibitor of the FGF signaling pathway. RA signaling is therefore a major contributor to segmental patterning by promoting anterior segmental polarity and by interacting with the FGF signaling pathway to position segmental boundaries.

We have established a heart slice primary culture, which allows us to mechanically separate distinct cardiac cell populations and assay their relative mitogenic and trophic effects on cardiac myocyte proliferation and survival. Using this system, we have found that a signal(s) from the epicardium, but not the trabeculae and endocardium, is required in embryonic day 10 (E10) chick heart slices for continued cardiac myocyte proliferation and survival. An examination of potential epicardial growth or trophic factors has revealed that blockade of either retinoic acid (RA) or erythopoietin (epo) signaling from the epicardium inhibits cardiac myocyte proliferation and survival. The blockade of cardiac myocyte proliferation following administration of an RA antagonist can be rescued by exogenous epo. Conversely, the blockade of cardiac myocyte proliferation following administration of an anti-epo receptor antisera can be rescued by exogenous RA. Thus, our findings suggest that RA and epo signals work in parallel to support myocardial cell proliferation. In addition, we have found that these factors do not act directly on myocardial cells. Rather, they induce another soluble factor(s) in the epicardium that directly regulates proliferation of cardiac myocytes. We therefore postulate that the epicardium controls normal heart growth in ventricular segments of the embryonic chick heart by secreting a cardiac myocyte mitogen whose expression (or activity) is regulated by both RA and erythropoietin signaling.

Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.