Retinoic acid (RA) is a vitamin A-derived, non-peptidic, small lipophilic molecule that acts as ligand for nuclear RA receptors (RARs), converting them from transcriptional repressors to activators. The distribution and levels of RA in embryonic tissues are tightly controlled by regulated synthesis through the action of specific retinol and retinaldehyde dehydrogenases and by degradation via specific cytochrome P450s (CYP26s). Recent studies indicate that RA action involves an interplay between diffusion (morphogen-like) gradients and the establishment of signalling boundaries due to RA metabolism, thereby allowing RA to finely control the differentiation and patterning of various stem/progenitor cell populations. Here, we provide an overview of the RA biosynthesis, degradation and signalling pathways and review the main functions of this molecule during embryogenesis.

Retinoic acid signalling during development

The molecular action of juvenile hormone (JH), a regulator of vital importance to insects, was until recently regarded as a mystery. The past few years have seen an explosion of studies of JH signaling, sparked by a finding that a JH-resistance gene, Methoprene-tolerant (Met), plays a critical role in insect metamorphosis. Here, we summarize the recently acquired knowledge on the capacity of Met to bind JH, which has been mapped to a particular ligand-binding domain, thus establishing this bHLH-PAS protein as a novel type of an intracellular hormone receptor. Next, we consider the significance of JH-dependent interactions of Met with other transcription factors and signaling pathways. We examine the regulation and biological roles of genes acting downstream of JH and Met in insect metamorphosis. Finally, we discuss the current gaps in our understanding of JH action and outline directions for future research.

The Juvenile Hormone Signaling Pathway in Insect Development

Mammalian spermatogenesis requires a stem cell pool, a period of amplification of cell numbers, the completion of reduction division to haploid cells (meiosis), and the morphological transformation of the haploid cells into spermatozoa (spermiogenesis). The net result of these processes is the production of massive numbers of spermatozoa over the reproductive lifetime of the animal. One study that utilized homogenization-resistant spermatids as the standard determined that human daily sperm production (dsp) was at 45 million per day per testis (60). For each human that means ∼1,000 sperm are produced per second. A key to this level of gamete production is the organization and architecture of the mammalian testes that results in continuous sperm production. The seemingly complex repetitious relationship of cells termed the "cycle of the seminiferous epithelium" is driven by the continuous commitment of undifferentiated spermatogonia to meiosis and the period of time required to form spermatozoa. This commitment termed the A to A1 transition requires the action of retinoic acid (RA) on the undifferentiated spermatogonia or prospermatogonia. In stages VII to IX of the cycle of the seminiferous epithelium, Sertoli cells and germ cells are influenced by pulses of RA. These pulses of RA move along the seminiferous tubules coincident with the spermatogenic wave, presumably undergoing constant synthesis and degradation. The RA pulse then serves as a trigger to commit undifferentiated progenitor cells to the rigidly timed pathway into meiosis and spermatid differentiation.

Spermatogenesis: The Commitment to Meiosis

Retinoic acid (RA) signalling has a central role during vertebrate development. RA synthesized in specific locations regulates transcription by interacting with nuclear RA receptors (RARs) bound to RA response elements (RAREs) near target genes. RA was first implicated in signalling on the basis of its teratogenic effects on limb development. Genetic studies later revealed that endogenous RA promotes forelimb initiation by repressing fibroblast growth factor 8 (Fgf8). Insights into RA function in the limb serve as a paradigm for understanding how RA regulates other developmental processes. In vivo studies have identified RAREs that control repression of Fgf8 during body axis extension or activation of homeobox (Hox) genes and other key regulators during neuronal differentiation and organogenesis.

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Mechanisms of retinoic acid signalling and its roles in organ and limb development

To investigate the role of extrinsic influences on the survival and growth of axotomized retinal ganglion cells (RGCs) in the mature mammalian CNS, both optic nerves (ONs) of adult rats were transected intraorbitally and, on one side, replaced by an autologous segment of peripheral nerve (PN) that had been left unconnected distally. The survival of RGCs and the regrowth of their cut axons into the PN grafts were assessed using morphometric techniques, neuroanatomic tracers, and immunologic cell markers to identify and count RGCs at times ranging from 15 d to 9 months. It was observed that (1) in the absence of a PN graft, more than 90% of the RGCs died by 1 month after axotomy; (2) between 1 and 3 months after axotomy, survival of RGCs in the PN-grafted retinas was enhanced 2-4-fold; (3) nearly 20% of the surviving RGCs regrew lengthy axons into the grafts; and (4) although the density of surviving RGCs in PN-grafted retinas decreased significantly between 1 and 3 months after axotomy, the densities of RGCs with axons extending into the graft remained relatively stable. These results confirm that in the adult rat retina, neuronal death is a major effect of axotomy near the cell soma. Although such lesions lead to the degeneration of many RGCs, we show that extrinsic influences introduced by the apposition of a PN segment at the time of severing the ON can rescue a substantial number of these neurons. Because the enhanced survival of many axotomized RGCs in the PN-grafted retinas appears to be limited to the first few weeks after injury, while those of RGCs that regenerate axons into the grafts do not show a parallel decline, it is possible that, in these experiments, neuronal viability depends on a spectrum of differently timed influences that may include the early diffusion of critical molecules arising from the graft and the subsequent establishment of more complex interactions with graft components.

https://www.jneurosci.org/content/jneuro/8/1/265.full.pdf

Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.

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Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC50 = 61.2–107.7 nM; Kd = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.

Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect

Peripheral nerve contains heterogeneous growth factors that support sensory neurons in vitro

Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby-like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3−/− embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3−/−/Shh−/− compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh−/− embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway. Developmental Dynamics 238:1140–1149, 2009. © 2009 Wiley-Liss, Inc.

Don Cameron

Papers

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis

Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect

Peripheral nerve contains heterogeneous growth factors that support sensory neurons in vitro

Tulp3 is a critical repressor of mouse hedgehog signaling