Mesodermal and neuronal retinoids regulate the induction and maintenance of limb innervating spinal motor neurons

During embryonic development, the generation, diversification and maintenance of spinal motor neurons depend upon extrinsic signals that are tightly regulated. Retinoic acid (RA) is necessary for specifying the fates of forelimb-innervating motor neurons of the Lateral Motor Column (LMC), and the specification of LMC neurons into medial and lateral subtypes. Previous studies implicate motor neurons as the relevant source of RA for specifying lateral LMC fates at forelimb levels. However, at the time of LMC diversification, a significant amount of retinoids in the spinal cord originates from the adjacent paraxial mesoderm. Here we employ mouse genetics to show that RA derived from the paraxial mesoderm is required for lateral LMC induction at forelimb and hindlimb levels, demonstrating that mesodermally synthesized RA functions as a second source of signals to specify lateral LMC identity. Furthermore, reduced RA levels in postmitotic motor neurons result in a decrease of medial and lateral LMC neurons, and abnormal axonal projections in the limb; invoking additional roles for neuronally synthesized RA in motor neuron maintenance and survival. These findings suggest that during embryogenesis, mesodermal and neuronal retinoids act coordinately to establish and maintain appropriate cohorts of spinal motor neurons that innervate target muscles in the limb.     

Raldh expression in embryos of the direct developing frog Eleutherodactylus coqui and the conserved retinoic acid requirement for forelimb initiation

Embryos of the direct developing frog, Eleutherodactylus coqui, provide opportunities to examine frog early limb development that are not available in species with tadpoles. We cloned two retinaldehyde dehydrogenase genes, EcRaldh1 and EcRaldh2, to see which enzyme likely supplies retinoic acid for limb development. EcRaldh1 is expressed in the dorsal retina, otic vesicle, pronephros, and pronephric duct, but not in the limb. EcRaldh2 is expressed early at the blastoporal lip and then in the mesoderm in the neurula, so this expression could function in forelimb initiation. Later EcRaldh2 is expressed in the mesoderm at the base of the limbs and in the ventral spinal cord where motor neurons innervating the limbs emerge. These observations on a frog support the functional conservation of EcRaldh2 in forelimb initiation in Osteichthyans and in limb patterning and motor neuron specification in tetrapods.     

Colloquium 14: New Functions for Retinoic Acid in the Developing CNS. The role of retinoic acid in the patterning of the developing nervous system

Retinoic acid (RA) is metabolised from its precursor, retinol (vitamin A). In mammalian embryos, retinol is provided by the mother via the placenta and in birds retinol comes from the yolk. We have studied the role of RA in CNS development in quail embryos by depriving adult quails of retinol in the diet which results in them laying eggs which have no retinol stores. The resulting embryos are therefore retinol and RA deficient. The CNS of these embryos is abnormal in three regards; patterning, neural crest production and neurite outgrowth. With regard to patterning, at an early stage of development prior to somitogenesis, hindbrain patterning genes are not induced which leads to the respecification of the posterior hindbrain territory. This region is not lost from the embryo but instead becomes transformed into an enlarged anterior hindbrain. Another aspect of patterning that is abnormal in these RA deficient embryos is the dorsoventral gene expression domains in the anterior spinal cord. These domains are required for the proper specification of motor neurons, sensory neurons and various classes of interneurons. Consequently these neuronal classes are mis‐specified in the RA deficient embryos. With regard to the neural crest, these cells often fail to migrate correctly and then die in the absence of RA. With regard to neurite outgrowth, very little outgrowth seems to take place in these deficient embryos which suggests that RA is involved in neurite outgrowth. Taking these experiments into the adult to examine the role of RA in neurite regeneration, we have had success in inducing neurite outgrowth in vitro from adult mouse spinal cord by manipulating the retinoic acid receptors which transduce the RA signal at the level of the nucleus.     

Ledgerline, a novel Xenopus laevis gene, regulates differentiation of presomitic mesoderm during somitogenesis

Segmentation of the vertebrate body via the sequential formation of somites is an important process in embryogenesis. This sequential process is governed by the activation and regulation of Notch-related molecular oscillators by fibroblast growth factor and retinoic acid (RA) signaling. In this study, we identified ledgerline, a novel gene of Xenopus laevis expressed specifically in the presomitic mesoderm. Knockdown of ledgerline using antisense morpholino oligonucleotides shifted the developing somite front and altered the expression of genes that regulate molecular oscillation, including Delta2, ESR5, Hairy2a, and Thylacine1. Knockdown of ledgerline also downregulated RALDH-2 expression. Injection of RARalpha-CA, a constitutively active mutant of the RA receptor RARalpha, subsequently reduced the altered Thylacine1 expression. These results strongly suggest that ledgerline is essential for mesodermal RA activity and differentiation of the presomitic mesoderm during Xenopus somitogenesis.     

Heads or tails? Retinoic acid will decide.

A recent study (Niederreither et al. Nat Genet 1999;21:444-448 [Ref. 1]) describes the phenotype of a gene knockout for an enzyme, retinaldehyde dehydrogenase 2 (RALDH-2), that synthesizes retinoic acid (RA) in the early embryo. The effects generated by this single enzyme mutation are remarkably similar to those previously described in vitamin A-deprivation studies and compound retinoic acid receptor knockouts, which involve multiple systems of the embryo. With other data on the distribution of RA, its role in axial specification of the early embryo is considerably clarified. Surprisingly, it seems that head development is unaffected in these RALDH-2 knockout embryos; thus, the anterior of the embryo does not require RA, despite the observations that the hindbrain seems exquisitely sensitive to RA perturbation. Head development may be realised by a cytochrome P450 enzyme (CYP26), which has been described recently. Between these two opposing forces, the hindbrain develops.     

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.     

Retinoic acid in development and regeneration

Retinoids are low molecular weight, lipophilic derivatives of vitamin A which have profound effects upon the development of various embryonic systems. Here I review the effects on developing and regenerating limbs, regenerating amphibian tails and the developing central nervous system (CNS). In the regenerating amphibian limb, retinoids can proximalize, posteriorize and ventralize the axes of the blastema. In the chick limb bud retinoids can only posteriorize the tissue. In the regenerating amphibian tail retinoids can homeotically transform tail tissue into hindlimb tissue. In the developing and regenerating limb retinoic acid has been detected endogenously, confirming that this molecule plays a role in the generation of pattern and we have shown that limbs cannot develop in the absence of retinoic acid. In the developing CNS retinoic acid specifically affects the hindbrain where it causes a transformation of anterior rhombomeres into more posterior ones. Again, endogenous retinoic acid has been detected in the CNS and in the absence of retinoids the posterior hindbrain has been found to be affected. The effects of retinoids on the CNS are most likely to be mediated via theHox genes acting in the mesoderm after gastrulation. It has also been proposed that the establishment of the head-to-tail axis in the mesoderm is established by retinoic acid. These data show that retinoids play an important role in both the development and regeneration of various systems in the embryo and post-embryonically     

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.     

Retinoid receptors in chronic degeneration of the spinal cord: observations in a rat model of amyotrophic lateral sclerosis

Changes in distribution and expression of retinoid receptors may be part of a spinal cord protective response to acute injury and to chronic degeneration. In this study, we have combined RNA and protein expression analysis to characterize the expression profile of retinoid receptors in the lumbar spinal cord of the superoxide dismutase 1 G93A mutant rat model of amyotrophic lateral sclerosis, a fatal neurodegenerative disorder causing extensive motor neuron loss. We also report a nonsignificant change in RNA expression of binding proteins and metabolizing enzymes for retinol and retinoic acid in the mutant rat spinal cord at end-stage disease. Only retinoid X receptor beta (RXRbeta), and to a lesser extent retinoic acid receptor beta and alpha (RARbeta/alpha) were reliably detected in lumbar spinal cord at an early pre-symptomatic phase and throughout the disease progression. The expression of RXRbeta in lamina II neurons in the dorsal horn of transgenic and wild type (WT) animals was associated with extensive astrocyte staining in end-stage lumbar spinal cord from transgenic rats. RARbeta and RARalpha diffuse staining of large motor neurons in the pre-symptomatic transgenic and in the WT lumbar cord appear to decline in end-stage disease, when a selective and strong gamma motor neuron RARalpha staining becomes evident. As gliosis and motor neuron loss are key pathogenic features in amyotrophic lateral sclerosis, the selective expression of retinoid receptors in astrocytes and motor neurons may provide further clues to the role of retinoid signalling in neurodegeneration and suggest new treatment strategies based on retinoid-modulating agents.     

Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation

The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graft-derived cells in the chimaeric embryo following grafting of wheat germ agglutinin--gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder contributes to the neuroectoderm of the prosencephalon and mesencephalon. Cells in the distal lateral region give rise to the neuroectoderm of the rhombencephalon and the spinal cord. Embryonic ectoderm at the archenteron and adjacent to the middle region of the primitive streak contributes to the neuroepithelium of the spinal cord. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes and the cranial neural crest cells. Some labelled cells grafted to the anterior midline are found in the oral ectodermal lining, whereas cells from the archenteron are found in the notochord. With respect to mesodermal tissues, ectoderm at the archenteron and the distal-lateral region of the egg cylinder gives rise to rhombencephalic somitomeres, and the embryonic ectoderm adjacent to the primitive streak contributes to the somitic mesoderm and the lateral mesoderm. Based upon results of this and other grafting studies, a map of prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

Evidence for medial/lateral specification and positional information within the presomitic mesoderm.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.     

Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud

A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.     

Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord

During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement.