CPS49-induced neurotoxicity does not cause limb patterning anomalies in developing chicken embryos.

Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using ß-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide.

Repeated removal of developing limb buds permanently reduces appendage size in the highly-regenerative axolotl.

Matching appendage size to body size is fundamental to animal function. Generating an appropriately-sized appendage is a robust process executed during development which is also critical for regeneration. When challenged, larger animals are programmed to regenerate larger limbs than smaller animals within a single species. Understanding this process has important implications for regenerative medicine. To approach this complex question, models with altered appendage size:body size ratios are required. We hypothesized that repeatedly challenging axolotls to regrow limb buds would affect their developmental program resulting in altered target morphology. We discovered that after 10 months following this experimental procedure, limbs that developed were permanently miniaturized. This altered target morphology was preserved upon amputation and regeneration. Future experiments using this platform should provide critical information about how target limb size is encoded within limb progenitors.

First Neuromuscular Contact Correlates with Onset of Primary Myogenesis in Rat and Mouse Limb Muscles.

Skeletal muscle development has been the focus of intensive study for many decades. Recent advances in genetic manipulation of the mouse have increased our understanding of the cell signalling involved in the development of muscle progenitors which give rise to adult skeletal muscles and their stem cell populations. However, the influence of a vital tissue type - the peripheral nerve-has largely been ignored since its earliest descriptions. Here we carefully describe the timing in which myogenic progenitors expressing Pax3 and Pax7 (the earliest markers of myogenic cells) enter the limb buds of rat and mouse embryos, as well as the spatiotemporal relationship between these progenitors and the ingrowing peripheral nerve. We show that progenitors expressing Pax3 enter the limb bud one full day ahead of the first neurites and that Pax7-expressing progenitors (associated with secondary myogenesis in the limb) are first seen in the limb bud at the time of nerve entry and in close proximity to the nerve. The initial entry of the nerve also coincides with the first expression of myosin heavy chain showing that the first contact between nerves and myogenic cells correlates with the onset of myogenic differentiation. Furthermore, as the nerve grows into the limb, Pax3 expression is progressively replaced by Pax7 expression in myogenic progenitors. These findings indicate that the ingrowing nerve enters the limb presumptive muscle masses earlier than what was generally described and raises the possibility that nerve may influence the differentiation of muscle progenitors in rodent limbs.

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance.

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.

Phosphorylation of c-Jun in avian and mammalian motoneurons in vivo during programmed cell death: an early reversible event in the apoptotic cascade.

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo. We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.

Neurotrophin-independent attraction of growing sensory and motor axons towards developing Xenopus limb buds in vitro.

The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors. These findings indicate that developing limb buds in Xenopus release diffusible factors other than neurotrophins, able to attract growth of sensory and motor axons over long distances.

Neurotrophins are required for nerve growth during development.

Although the requirement of neurotrophins for the prevention of cell death in the peripheral nervous system is well established, their physiological involvement in nerve growth is still unclear. To address this question, we generated a mouse that expresses the green fluorescent protein in post-mitotic neurons, allowing the repeated visualization of all motor and sensory axons during development. We imaged the growth of these axons into the limb bud of day 10.5 embryos. Sensory axons, but rarely motor axons, were targeted to ectopically placed beads containing any of the neurotrophins NGF, BDNF, NT-3 or NT-4/5. Conversely, a combination of function-blocking monoclonal antibodies to NGF, BDNF and NT-3 dramatically inhibited elongation of both sensory and motor axons in the limb bud, indicating that the growth of mixed nerves is dependent upon neurotrophins during development.

Genetic and epigenetic mechanisms contribute to motor neuron pathfinding.

Many lines of evidence indicate that genetically distinct subtypes of motor neurons are specified during development, with each type having characteristic properties of axon guidance and cell-body migration. Motor neuron subtypes express unique combinations of LIM-type homeodomain factors that may act as intrinsic genetic regulators of the cytoskeletal events that mediate cell migration, axon navigation or both. Although experimentally displaced motor neurons can pioneer new routes to their targets, in many cases the axons of motor neurons in complete isolation from their normal territories passively follow stereotypical pathways dictated by the environment. To investigate the nonspecific versus genetically controlled regulation of motor connectivity we forced all motor neurons to express ectopically a LIM gene combination appropriate for the subgroup that innervates axial muscles. Here we show that this genetic alteration is sufficient to convert the cell body settling pattern, gene-expression profile and axonal projections of all motor neurons to that of the axial subclass. Nevertheless, elevated occupancy of the axial pathway can override their genetic program, causing some axons to project to alternative targets.

Targeting of the EphA4 tyrosine kinase receptor affects dorsal/ventral pathfinding of limb motor axons.

The Eph family of tyrosine kinase receptors has recently been implicated in various processes involving the detection of environmental cues such as axonal guidance, targeted cell migration and boundary formation. We have inactivated the mouse EphA4 gene to investigate its functions during development. Homozygous EphA4 mutant animals show peroneal muscular atrophy correlating with the absence of the peroneal nerve, the main dorsal nerve of the hindlimb. This phenotype is also observed, although with a lower penetrance, in heterozygotes. During normal hindlimb innervation, motor axons converge towards the sciatic plexus region at the base of the limb bud, where they must choose between dorsal and ventral trajectories within the limb. Among the axons emerging from the sciatic plexus, dorsal projections show higher levels of EphA4 protein than ventral axons. In EphA4 mutant mice, presumptive dorsal motor axons fail to enter the dorsal compartment of the limb and join the ventral nerve. Our data therefore suggest that the level of EphA4 protein in growing limb motor axons is involved in the selection of dorsal versus ventral trajectories, thus contributing to the topographic organisation of motor projections.

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated, nerve-dependent newt limb blastemas by rhGGF2.

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD-NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response.

Hepatocyte growth factor/scatter factor is a neurotrophic survival factor for lumbar but not for other somatic motoneurons in the chick embryo.

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed in the developing limb muscles of the chick embryo during the period of spinal motoneuron (MN) programmed cell death, and its receptor c-met is expressed in lumbar MNs during this same period. Although cultured motoneurons from brachial, thoracic, and lumbar segments are all rescued from cell death by chick embryo muscle extract (CMX) as well as by other specific trophic agents, HGF/SF only promotes the survival of lumbar MNs. Similarly, treatment of embryos in ovo with exogenous HGF/SF rescues lumbar but not other somatic MNs from cell death. Blocking antibodies to HGF/SF (anti-HGF) reduce the effects of CMX on MN survival in vitro and decrease the number of lumbar MNs in vivo. The expression of c-met on MNs in vivo is regulated by a limb-derived trophic signal distinct from HGF/SF. HGF/SF is a potent, select, and physiologically relevant survival factor for a subpopulation of developing spinal MNs in the lumbar segments of the chick embryo.

Stimulation of axon growth from the spinal cord by a regenerating limb blastema in newts.

The effects of limb blastemas of Pleurodeles waltl on axon growth from fragments of spinal cord were studied in vitro. Cultured in a defined medium, spinal cord fragments regenerated sparse, short axons. The culture of spinal fragments in the presence of blastemas greatly enhanced the length, number and survival of axons. Testing separately each of the two components of the blastema showed that only the mesenchyme exerts a neurotropic effect on the spinal fragments. Other tissues such as muscle or skin had a limited neurotrophic effect. Additionally, the neurotrophic activity of blastemas seems to be dependent of its proliferation status. Compared with blastemas of regenerating limbs from young animals, irradiated blastemas (devoid of mitotic activity) and blastemas of regenerating limbs from old animals or differentiated blastemas (both characterized by a low mitotic activity), exhibited a weaker neurotrophic influence. The blastema neurotrophic factor is not an attachment molecule but a soluble one and cannot be nerve growth factor (NGF) or fibroblast growth factor (FGF). It has a relatively low molecular weight (less than 15 kDa) and its protein nature was ascertained by its sensitivity to heating and proteases. As the production of this mesenchyme-derived neurotrophic factor depends upon mesenchymal cell proliferation of the blastema, we suggest that there is loop of positive regulation between spinal nerves and blastema. Blastema tissues may stimulate nerve regeneration allowing the stimulation of proliferation of blastema cells by regenerating nerve fibers. Alternatively, blastema cells may produce a neurotrophic factor whose secretion might be dependent on cell proliferation.

Developmental regulation of sensory neuron number and limb innervation in the mouse.

Although used widely in studies of naturally occurring cell death, systematic descriptions of the time course of changes in sensory neuron number and of limb innervation in the mouse are not available. The development of sensory innervation to the mouse forelimb was traced using the lipophilic carbocyanine dye, DiI, and correlated with neuron number in dorsal root ganglia contributing to the cervical enlargement. Axon invasion of the forelimb began at E10.5. Sensory axons reached the distal margin of the forelimb by E13.5. The difficulty of identifying immature neurons precluded estimating neuron numbers during the period of limb innervation. Neuron numbers in dorsal root ganglia (DRGs) C5-C8 increased from E14 to E16 and from E18 to P4. No evidence of a decline in neuron numbers was found during the developmental periods studied. Neuron number was compared in neonates and adults to determine if sensory neurons are added as body size increases as found in the frog [J. Comp. Neurol. 314 (1991) 106] and the rat [J. Comp. Neurol. 386 (1997) 8]. In contrast to previous findings, no difference was found in sensory neuron number between neonate and adult mice in either cervical or lumbar DRGs.

Muscle and tendon morphogenesis in the avian hind limb.

The proper development of the musculoskeletal system in the tetrapod limb requires the coordinated development of muscle, tendon and cartilage. This paper examines the morphogenesis of muscle and tendon in the developing avian hind limb. Based on a developmental series of embryos labeled with myosin and tenascin antibodies in whole mount, an integrative description of the temporal sequence and spatial pattern of muscle and tendon morphogenesis and their relationship to cartilage throughout the chick hind limb is presented for the first time. Anatomically distinct muscles arise by the progressive segregation of muscle: differentiated myotubes first appear as a pair of dorsal and ventral muscle masses; these masses subdivide into dorsal and ventral thigh, shank and foot muscle masses; and finally these six masses segregate into individual muscles. From their initial appearance, most myotubes are precisely oriented and their pattern presages the pattern of future, individual muscles. Anatomically distinct tendons emerge from three tendon primordia associated with the major joints of the limb. Contrary to previous reports, comparison of muscle and tendon reveals that much of their morphogenesis is temporally and spatially closely associated. To test whether reciprocal muscle-tendon interactions are necessary for correct muscle-tendon patterning or whether morphogenesis of each of these tissues is autonomous, two sets of experiments were conducted: (1) tendon development was examined in muscleless limbs produced by coelomic grafting of early limb buds and (2) muscle development was analyzed in limbs where tendon had been surgically altered. These experiments demonstrate that in the avian hind limb the initial morphogenetic events, formation of tendon primordia and initial differentiation of myogenic precursors, occur autonomously with respect to one another. However, later morphogenetic events, such as subdivision of muscle masses and segregation of tendon primordia into individual tendons, do require to various degrees reciprocal interactions between muscle and tendon. The dependence of these later morphogenetic events on tissue interactions differs between different proximodistal regions of the limb.

Transmembrane grasshopper Semaphorin I promotes axon outgrowth in vivo.

Members of the Semaphorin family of glycoproteins play an important role in axonal pathfinding by functioning as inhibitory guidance cues. Here we provide evidence that a transmembrane form of Semaphorin (Semaphorin I), which is expressed by bands of epithelial cells in the developing grasshopper limb bud, functions as an attractive/permissive cue for the growth cones of the subgenual organ. In addition, we demonstrate that Semaphorin I is needed for initial axonal outgrowth from the subgenual organ. These results are consistent with an alternative function for a transmembrane form of Semaphorin and may explain the previously reported arrest of the proximal extension of the subgenual organ growth cones in the absence of the Ti1 pioneer pathway.

Focal expression of glial cell line-derived neurotrophic factor in developing mouse limb bud.

Glial cell line-derived neurotrophic factor (GDNF) is known to support the survival of motoneurons in vitro and in vivo, as well as subpopulations of sensory neurons in vitro. To clarify the mechanisms by which GDNF supports these neurons, we examined the patterns of GDNF mRNA expression in relation to motor and sensory axons during early stages of mouse development. Between embryonic days (E) 10 and 12, a time when motor and sensory axons are entering the periphery, GDNF mRNA is expressed at high levels in a restricted region in proximal limb buds where axons converge and enter the limb. At later ages (E14-16), GDNF mRNA was detected in non-neuronal cells along peripheral nerve, in dermis, and in some muscles. To characterize cells that express GDNF in the proximal limb, GDNF expression in the forelimb was compared to expression patterns of two markers of muscle, Pax 3 and myogenin, as well as with the pan neurotrophin receptor (p75) which is expressed by Schwann cell precursors. We show that expression of GDNF in the proximal limb bud at E11-12 does not correlate with markers of muscle or Schwann cell precursors, which supports the idea that GDNF is expressed by mesenchymal cells in this region. Our results suggest that GDNF expression in proximal limb buds may function as a transient survival factor, particularly for motor neurons, before they reach their final targets. GDNF expression in muscle and dermis at later stages suggests that GDNF may have additional functions as motor and sensory neurons mature.

Abnormal cutaneous nerve outgrowth: a secondary effect of methyl triazene teratology in developing mouse limb buds.

The cutaneous branches originating from the superficial distal division of the ulnar nerve exhibit abnormal developmental features in 70.2% of the forelimb buds from embryos submitted 2 or 3 days earlier to methyl triazene administered to their pregnant mother. Similar abnormalities characterize the preaxial ventral cutaneous nerve of the thumb in 17.8% of forelimb primordia. The affected nerves undergo anticipated growth with respect to the normal schedule, follow abnormal pathways through areas of extensive cell death, and finally reach the apical ectoderm where they run in close contact with the basement membrane without forming a plexus. Histological observations gathered in pyronin-methyl green stained serial sections as well as in whole limb buds after cholinesterase method suggest that three factors probably contribute to modify nerve outgrowth: (1) a discrepancy between the rate of nerve progression and that of mesodermal growth in the prospective zeugopod territory which is preferentially affected by the teratogen; (2) facilitated nerve pathfinding into areas strongly hit by triazene-induced mesodermal cell death; and (3) alteration or abolition of some unknown ectodermal influence necessary to stimulate selective guidance of terminal sensory afferents and to maintain them transiently at some distance from the epidermis.

Expression and regulation of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in distinct avian motoneuron subsets.

We performed a detailed study of the expression of neurotrophin-3 and brain-derived neurotrophic factor transcripts in spinal motoneurons using in situ hybridization of serially sectioned chick embryos aged 3 to 8 days (E3 to E8). Neurotrophin-3 mRNA is detected in motoneuron subsets from E3.5 to E4 only in brachial segments of the neural tube and from E5 in both brachial and lumbar regions. Expression of brain-derived neurotrophic factor mRNA is first evident on E5 in a subset of brachial level motoneurons and from E6 also in motoneurons located in the rostral-most portion of the lateral motor column, as well as in the tail-innervating region of the spinal cord. Analysis along the rostrocaudal extent of the brachial lateral motor column reveals an overlap zone of expression of both neurotrophins of about two segments. In transverse sections of this region, it is observed that neurotrophin-3-positive motoneurons preferentially occupy the lateral part of the column, whereas brain-derived neurotrophic factor-producing motoneurons are localized in a more medial position. These results show that the two factors are synthesized at discrete axial levels of the spinal cord by distinct motoneuron subpopulations. Since brain-derived neurotrophic factor mRNA is expressed within the brachial but not the lumbar lateral motor column, we tested the possibility that brain-derived neurotrophic factor expression is regulated by the type of peripheral target, that is, the wing or the leg. Unilateral transplantation of a wing bud instead of a leg bud and vice versa, prior to the onset of peripheral innervation, failed to alter the original pattern of brain-derived neurotrophic factor mRNA observed in either level of the axis. Thus, the early synthesis of brain-derived neurotrophic factor by subsets of spinal motoneurons is independent of the type of peripheral target and may instead reflect intrinsic differences between motoneuron populations.

Pioneer growth cone migration in register with orthogonal epithelial domains in the grasshopper limb bud.

At the onset of neural development, pioneer growth cones can migrate over epithelia or neuroepithelia along stereotyped routes that establish the pattern of initial neural tracts. These migration routes may reflect the arrangement of distinct epithelial or neuroepithelial domains. In grasshopper limb buds, a pair of afferent pioneer neurons arise in the tibia and their growth cones migrate on a stereotyped path through the limb to the CNS. In the limb buds, circumferentially-oriented epithelial domains expressing semaphorin-I, annulin, or alkaline-phosphatase, and a longitudinal domain, expressing engrailed, have been described. Using multiple-labeling techniques, we describe the relationships of these domains to each other and to the pioneer neuron pathway. Taken together, these domains establish an orthogonal pattern of regionally specific epithelial molecular markers. During much of their migration across the limb epithelium, the pioneer growth cones are in register with the axes of circumferential or longitudinal epithelial domains.

Effects of nerve growth factor on sympathetic neuron development in normal and limbless chick embryos.

Wing bud removal in chick embryos has been shown to affect the generation of sympathetic neurons prior to the normal period of limb innervation [Saltis and Rush (1995) J. auton. nerv. Sys., 51, 117-127.]. Pyknotic activity occurred earlier within the peripherally deprived ganglion, suggesting that a precocious cell death of dividing sympathoblasts led to the reduced neuronal population. We have now sought to test whether the effect of limb bud extirpation can be overcome by the administration of nerve growth factor (NGF). Specifically, the peripherally deprived ganglion has been examined for mitotic activity and total neuronal numbers. In brachial ganglia from the operated side, neuron numbers decreased by 67% by embryonic day (E) 13, but by only 28% when NGF was administered from E9. Ganglia on the unoperated side were unaffected by the NGF treatment. In contrast, in embryos receiving NGF from E5 to E9, neuron numbers in the ganglia increased by more than 100%, on both the intact and operated side. This increase was accompanied by a greater proportion of 3H-thymidine-labelled neurons. We therefore conclude that NGF, in addition to its previously described role of preventing naturally occurring neuron death, can also affect the generation of sympathetic neurons. This ability of NGF to affect gangliogenesis is most likely achieved by increasing the survival of dividing neuroblasts, although a direct effect on mitosis has not been excluded.