Cholinesterases preceding major tracts in vertebrate neurogenesis

The role of acetylcholinesterase (AChE) in neurotransmission is well known. But long before synapses are formed in vertebrates, AChE is expressed in young postmitotic neuroblasts that are about to extend the first long tracts. AChE histochemistry can thus be used to map primary steps of brain differentiation. Preceding and possibly inducing AChE in avian brains, the closely related butyrylcholinesterase (BChE) spatially foreshadows AChE-positive cell areas and the course of their axons. In particular, before spinal motor axons grow, their corresponding rostral sclerotomes and myotomes express BChE, and both their neuronal source and myotomal target cells express AChE. Since axon growth has been found inhibited by acetylcholine, it is postulated that both cholinesterases can attract neurite growth cones by neutralizing the inhibitor. Thus, the early expression of both cholinesterases that is at least partially independent from classical cholinergic synaptogenesis, sheds new light on the developmental and medical significance of these enzymes.     

Position fine-tuning of caudal primary motoneurons in the zebrafish spinal cord

In zebrafish embryos, each myotome is typically innervated by three primary motoneurons (PMNs): the caudal primary (CaP), middle primary (MiP) and rostral primary (RoP). PMN axons first exit the spinal cord through a single exit point located at the midpoint of the overlying somite, which is formed beneath the CaP cell body and is pioneered by the CaP axon. However, the placement of CaP cell bodies with respect to corresponding somites is poorly understood. Here, we determined the early events in CaP cell positioning using neuropilin 1a (nrp1a):gfp transgenic embryos in which CaPs were specifically labeled with GFP. CaP cell bodies first exhibit an irregular pattern in presence of newly formed corresponding somites and then migrate to achieve their proper positions by axonogenesis stages. CaPs are generated in excess compared with the number of somites, and two CaPs often overlap at the same position through this process. Next, we showed that CaP cell bodies remain in the initial irregular positions after knockdown of Neuropilin1a, a component of the class III semaphorin receptor. Irregular CaP position frequently results in aberrant double exit points of motor axons, and secondary motor axons form aberrant exit points following CaP axons. Its expression pattern suggests that sema3ab regulates the CaP position. Indeed, irregular CaP positions and exit points are induced by Sema3ab knockdown, whose ectopic expression can alter the position of CaP cell bodies. Results suggest that Semaphorin-Neuropilin signaling plays an important role in position fine-tuning of CaP cell bodies to ensure proper exit points of motor axons.     

An evaluation of myelomeres and segmentation of the chick embryo spinal cord.

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of K?llén (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.     

Cholinesterases during development of the avian nervous system

1. Long before onset of synaptogenesis in the chicken neural tube, the closely related enzymes butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are expressed in a mutually exclusive manner. Accordingly, neuroblasts on the ventricular side of the neural tube transiently express BChE before they abruptly accumulate AChE while approaching the outer brain surface. 2. By exploiting AChE as a sensitive and early histochemical differentiation marker, we have demonstrated complex polycentric waves of differentiation spreading upon the cranial part of the chicken neural tube but a smooth rostrocaudal wave along the spinal cord. Shortly after expression of AChE, these cells extend long projecting neurites. In particular, segmented spinal motor axons originate from AChE-positive motoneurones; they navigate through a BChE-active zone within the rostral half of the sclerotomes before contacting BChE/AChE-positive myotome cells. At synaptogenetic stages, cholinesterases additionally are detectable in neurofibrillar laminae foreshadowing the establishment of cholinergic synapses. 3. In order to elucidate the functional significance of cholinesterases at early stages, we have investigated specific cholinesterase molecules and their mechanisms of action in vivo and in vitro. A developmental shift from the low molecular weight forms to the tetramers of both enzymes has been determined. In vitro, the addition of a selective BChE inhibitor leads to a reduction of AChE gene expression. Thus, in vivo and in vitro data suggest roles of cholinesterases in the regulation of cell proliferation and neurite growth. 4. Future research has to show whether neurogenetic functioning of cholinesterases can help to understand their reported alterations in neural tube defects, mental retardations, dementias and in some tumours.     

T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo.

Trunk neural crest cells and motor axons move in a segmental fashion through the rostral (anterior) half of each somitic sclerotome, avoiding the caudal (posterior) half. This metameric migration pattern is thought to be caused by molecular differences between the rostral and caudal portions of the somite. Here, we describe the distribution of T-cadherin (truncated-cadherin) during trunk neural crest cell migration. T-cadherin, a novel member of the cadherin family of cell adhesion molecules was selectively expressed in the caudal half of each sclerotome at all times examined. T-cadherin immunostaining appeared graded along the rostrocaudal axis, with increasing levels of reactivity in the caudal halves of progressively more mature (rostral) somites. The earliest T-cadherin expression was detected in a small population of cells in the caudal portion of the somite three segments rostral to last-formed somite. This initial T-cadherin expression was observed concomitant with the invasion of the first neural crest cells into the rostral portion of the same somite in stage 16 embryos. When neural crest cells were ablated surgically prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that the metameric T-cadherin distribution occurs independent of neural crest cell signals. This expression pattern is consistent with the possibility that T-cadherin plays a role in influencing the pattern of neural crest cell migration and in maintaining somite polarity.     

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.     

J1/tenascin-related molecules are not responsible for the segmented pattern of neural crest cells or motor axons in the chick embryo

It has been suggested that substrate adhesion molecules of the tenascin family may be responsible for the segmented outgrowth of motor axons and neural crest cells during formation of the peripheral nervous system. We have used two monoclonal antibodies (M1B4 and 578) and an antiserum [KAF9(1)] to study the expression of J1/tenascin-related molecules within the somites of the chick embryo. Neural crest cells were identified with monoclonal antibodies HNK-1 and 20B4. Young somites are surrounded by J1/tenascin immunoreactive material, while old sclerotomes are immunoreactive predominantly in their rostral halves, as described by other authors (Tan et al. 1987--Proc. natn. Acad. Sci. U.S.A. 84, 7977; Mackie et al. 1988--Development 102, 237). At intermediate stages of development, however, immunoreactivity is found mainly in the caudal half of each sclerotome. After ablation of the neural crest, the pattern of immunoreactivity is no longer localised to the rostral halves of the older, neural-crest-free sclerotomes. SDS-polyacrylamide gel electrophoresis of affinity-purified somite tissue, extracted using M1B4 antibody, shows a characteristic set of bands, including one of about 230 x 10(3), as described for cytotactin, J1-200/220 and the monomeric form of tenascin. Affinity-purified somite material obtained from neural-crest-ablated somites reveals some of the bands seen in older control embryos, but the high molecular weight components (120-230 x 10(3] are missing. Young epithelial somites also lack the higher molecular mass components. The neural crest may therefore participate in the expression of J1/tenascin-related molecules in the chick embryo. These results suggest that these molecules are not directly responsible for the segmented outgrowth of precursors of the peripheral nervous system.     

The roles of cell migration and myofiber intercalation in patterning formation of the postmitotic myotome

We have previously found that the postmitotic myotome is formed by two successive waves of myoblasts. A first wave of pioneer cells is generated from the dorsomedial region of epithelial somites. A second wave originates from all four edges of the dermomyotome but cells enter the myotome only from the rostral and caudal lips. We provide new evidence for the existence of these distinctive waves. We show for the first time that when the somite dissociates, pioneer myotomal progenitors migrate as mesenchymal cells from the medial side towards the rostral edge of the segment. Subsequently, they generate myofibers that elongate caudally. Pioneer myofiber differentiation then progresses in a medial-to-lateral direction with fibers reaching the lateralmost region of each segment. At later stages, pioneers participate in the formation of multinucleated fibers during secondary myogenesis by fusing with younger cells. We also demonstrate that subsequent to primary myotome formation by pioneers, growth occurs by uniform cell addition along the dorsoventral myotome. At this stage, the contributing cells arise from multiple sources as the myotome keeps growing even in the absence of the dorsomedial lip. Moreover, as opposed to suggestions that myotome growth is driven primarily and directly by the medial and lateral edges, we demonstrate that there is no direct fiber generation from the dorsomedial lip. Instead, we find that added fibers elongate from the extreme edges. Altogether, the integration between both myogenic waves results in an even pattern of dorsoventral growth of the myotome which is accounted for by progressive cell intercalation of second wave cells between preexisting pioneer fibers.     

Temporal and spatial expression of TGF-beta2 in chicken somites during early embryonic development

A multifunctional growth and differentiation factor TGF-beta is expressed at various developmental stages, and its principle role may be involvement in organogenesis. The present study was performed to evaluate the temporal and spatial expression of TGF-beta2 mRNA in developing somites of chicken embryos during their early developmental periods. TGF-betas were expressed in various tissues of the whole embryo obtained at stage 26 (5 days of incubation) as revealed by whole-mount in situ hybridization. TGF-beta2 mRNA was predominantly expressed in somites as well as the head, branchial arch, wing buds, and leg buds. TGF-beta2 mRNA first appeared in the rostral somites on E4, and its expression sites expanded to the middle range of somites at stage 26. At stages 29-31 (6-7 days), expression in the rostral somites disappeared, and it appeared in the caudal somites. TGF-beta2 expression was also analyzed in sections of the embryo by in situ hybridization. The expression sites of TGF-beta2 were clearly observed in the myotomal somite tips as well as the neural tube. RT-PCR analysis showed that TGF-beta2 expression was very low in the blastocyte stage embryo and thereafter increased linearly in the whole trunk until stage 26. These data indicate that TGF-beta2 may be a regulatory factor participating in the somitogenesis of chicken embryos.     

Segmentation of sensory and sympathetic ganglia: interactions between neural crest and somite cells.

The segmental pattern of peripheral ganglia in higher vertebrates is generated by interactions between neural crest and somite cells. Each mesodermal somite is subdivided into at least two distinct domains represented by its rostral and caudal halves. Most migratory pathways taken by neural crest cells in trunk regions of the axis, as well as the outgrowth of motoneuron fibers are restricted to the rostral domain of each somite. Experimental modification of the somites, achieved by constructing a mesoderm composed of multiple rostral half-somites, results in the formation of continuous and unsegmented nerves, dorsal root ganglia (DRG) and sympathetic ganglia (SG). In contrast, both neurites and crest cells are absent from a mesoderm composed of multiple-caudal half somites. However, the mechanisms responsible for gangliogenesis within the rostral half of the somite, appear to be different for DRG and SG. Vertebral development from the somites is also segmental. In implants of either multiple rostral or caudal somite-halves, the grafted mesoderm dissociates normally into sclerotome and dermomyotome. However, the morphogenetic capabilities of each somitic half differ. The lateral vertebral arch is continuous in the presence of caudal half-somite grafts and is virtually absent in rostral half-somite implants. Therefore, the rostrocaudal subdivision of the sclerotome determines the segmental pattern of neural development and is also important for the proper metameric development of the vertebrae.     

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.     

Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos.

We report the cloning of two new quail myogenic cDNAs, quail myogenic factor 2 (qmf2) and qmf3, which encode helix-loop-helix proteins homologous to mammalian myogenic factors myogenin and myf-5. In situ hybridization has been used to investigate the developmental expression of qmf2 and qmf3, as well as qmf1, the quail homologue to mammalian MyoD1, during the formation of the brachial somites. These studies show that qmf1 and qmf3 are activated sequentially in medially localized somite cells, immediately following somite formation but prior to myotome formation. qmf1, qmf2, and qmf3 are expressed in the myotome of compartmentalized somites. These findings suggest that determination of the myogenic cell lineage in quail somites is a progressive process controlled by influences of the neural tube on the expression of the qmf regulatory genes in newly forming somites.     

Amphibian (urodele) myotomes display transitory anterior/posterior and medial/lateral differentiation patterns

Myotome differentiation during Mexican axolotl (Ambystoma mexicanum) somitogenesis was analyzed by employing anti-actin and anti-myosin monoclonal antibodies as molecular probes. Myotome differentiation occurs after segmentation and proceeds in the cranial-to-caudal direction along the somite file. Within individual somites myotome differentiation displays distinct polarities. Examination of the somite file at the tailbud stage revealed that soon after segmentation, actin/myosin accumulate predominantly in the anterior and medial region of the myotome initially. Subsequently, cells within the myotome differentiate in an anterior-to-posterior and medial-to-lateral direction. Experimental analysis of presomitic paraxial mesoderm grafts before segmentation revealed that this transient myotome polarity is autonomous. Comparative analyses indicate that this myotome differentiation pattern is urodele specific. Cynops pyrrhogaster undergoes myotome differentiation like the axolotl, while two anurans, Xenopus laevis and Bombina orientalis, do not.     

Differential Distribution of Retinoic Acid Synthesis in the Chicken Embryo as Determined by Immunolocalization of the Retinoic Acid Synthetic Enzyme, RALDH-2

Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid generating enzyme in the early embryo. Here we report the immunolocalization of this enzyme (RALDH-2-IR) in stage 6-29 chicken embryos; we also show that tissues that exhibit strong RALDH-2-IR in the embryo contain RALDH-2 and synthesize retinoic acid. RALDH-2-IR indicates dynamic and discrete patterns of retinoic acid synthesis in the embryo, particularly within the somitic mesoderm, lateral mesoderm, kidney, heart, and spinal motor neurons. Prior to somitogenesis, RALDH-2-IR is present in the paraxial mesoderm with a rostral boundary at the level of the presumptive first somite; as the somites form, they exhibit strong RALDH-2-IR. Cervical presomitic mesoderm exhibits RALDH-2-IR but thoracic presomitic mesoderm does not. Neural crest cells do not express detectable levels of RALDH-2, but migrating crest cells are associated with RALDH-2 expressing mesoderm. The developing limb mesoderm expresses little RALDH-2-IR; however, RALDH-2-IR is strongly expressed in tissues adjacent to the limb. The most lateral, earliest-projecting motor neurons at all levels of the spinal cord exhibit RALDH-2-IR. Subsequently, many additional motor neurons in the brachial and lumbar cord regions express RALDH-2-IR. Motor neuronal expression of RALDH-2-IR is present in the growing axons as they extend to the periphery, indicating a potential role of retinoic acid in nerve influences on peripheral differentiation. With the exception of a transient expression in the facial/vestibulocochlear nucleus, cranial motor neurons do not express detectable levels of RALDH-2-IR.     

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

Determination of epithelial half-somites in skeletal morphogenesis.

The segmental body plan of vertebrates arises from the metameric organization of the paraxial mesoderm into somites. Each mesodermal somite is subdivided into at least two distinct domains: rostral and caudal. The segmental pattern of dorsal root ganglia, sympathetic ganglia and nerves is imposed by differential properties of either somitic domain. In the present work, we have extended these studies by investigating the contribution of rostral or caudal-half somites to vertebral development using grafts of multiple somite halves. In both rostral and caudal somitic implants, the grafted mesoderm dissociates normally into sclerotome and dermomyotome, and the sclerotome further develops into vertebrae. However, the morphogenetic capabilities of each somitic half differ. The pedicle of the vertebral arch is almost continuous in caudal half-somite grafts and is virtually absent in rostral half-somite implants. Similarly, the intervertebral disk is present in rostral half-somite chimeras, and much reduced or virtually absent in caudal somite chimeras. Thus, only the caudal half cells are committed to give rise to the vertebral pedicle, and only the rostral half cells are committed to give rise to the fibrocartilage of the intervertebral disk. Each vertebra is therefore composed of a pedicle-containing area, apparently formed by the caudal half-somite, followed by a pedicle-free zone, the intervertebral foramen, derived from the rostral somite. These data directly support the hypothesis of resegmentation, in which vertebrae arise by fusion of the caudal and rostral halves of two consecutive somites.     

Disruption of segmental neural crest migration and ephrin expression in delta-1 null mice

Neural crest cells migrate segmentally through the rostral half of each trunk somite due to inhibitory influences of ephrins and other molecules present in the caudal-half of somites. To examine the potential role of Notch/Delta signaling in establishing the segmental distribution of ephrins, we examined neural crest migration and ephrin expression in Delta-1 mutant mice. Using Sox-10 as a marker, we noted that neural crest cells moved through both rostral and caudal halves of the somites in mutants, consistent with the finding that ephrinB2 levels are significantly reduced in the caudal-half somites. Later, mutant embryos had aberrantly fused and/or reduced dorsal root and sympathetic ganglia, with a marked diminution in peripheral glia. These results show that Delta-1 is essential for proper migration and differentiation of neural crest cells. Interestingly, absence of Delta-1 leads to diminution of both neurons and glia in peripheral ganglia, suggesting a general depletion of the ganglion precursor pool in mutant mice.