Alteration of pectoral fin nerves following ablation of fin buds and by ectopic fin buds in the Japanese medaka fish

The role of the pectoral fin bud for outgrowth by fin axons was assessed by ablation of pectoral fin buds and by transplantation of fin buds to ectopic sites in the embryos of the Japanese medaka fish (Oryzias latipes). Normally nerves from segments 1-4 (S1-4) and less frequently the S5 nerve converged at the base of the fin bud by extending toward the fin bud on the ventral surface of the axial muscles (H. Okamoto and J. Y. Kuwada, 1991, Dev. Biol. 146). Following ablation of the fin bud before motor growth cones have begun to extend laterally, nerves in S1-5 followed a trajectory down the middle of each segment parallel to the borders of the metamerically arranged axial muscles rather than converging. This trajectory was similar to that of more posterior segmental nerves which do not converge toward the fin bud. When fin buds were transplanted to more posterior segments, nerves from S1-5 often changed their trajectories and extended to the base of ectopic buds. Furthermore, motor nerves from segments posterior to S5, which normally do not innervate the fin bud, also extended to the ectopic fin bud. When faced with both the host and ectopic fin bud, motor nerves extended to either fin bud or branched and extended to both fin buds. These results demonstrate that the early fin bud is necessary for correct outgrowth of fin nerves and suggest that the fin bud normally attracts fin nerves to its base. One possible mechanism for the attraction of motor growth cones by the fin bud is a long distance cue emitted by the fin bud.     

Fgf16 is essential for pectoral fin bud formation in zebrafish

Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in the zone of polarizing activity (ZPA) interact to induce and/or maintain each other's expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

Review of spino-occipital and spinal nerves in Tetraodontiformes,with special reference to pectoral and pelvic fin muscle innervation

The spino-occipital nerve (SO) and ventral rami of the spinal nerves (SV) in 10 tetraodontiform families and 5 outgroup taxa were examined, with special reference to pectoral and pelvic fin muscle innervation. Compared with the outgroup taxa, tetraodontiforms were characteristic in having SO3 + SV1 (SO3 in tetraodontids) that gave off several lateral subbranches to the pectoral fin base and SO participation in infracarinalis anterior innervation. SO and SV1 were connected with one another (6 patterns) before entering the pectoral fin muscles in most species, including the outgroup taxa, resulting in the participation of SV1 in the innervation of almost all of the pectoral fin muscles. SO3 + SV1 was present in all tetraodontiforms (except in 2 tetraodontids having only SO3) and the outgroup taxa, an upper dorsal branch uniformly extending dorsally into the pectoral fin base. The pectoral fin base also received a branch ventrally, but its identity differed (participation or nonparticipation of SV2). SV1 alone constituting the branch was a derived condition occurring in Aracanidae, Ostraciidae, Tetraodontidae, Diodontidae, and Molidae. No strong characters supporting a tetraodontiform sister group were recognized among the spino-occipital nerve and ventral rami of spinal nerves.     

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.     

Mechanism of pectoral fin outgrowth in zebrafish development

Fins and limbs, which are considered to be homologous paired vertebrate appendages, have obvious morphological differences that arise during development. One major difference in their development is that the AER (apical ectodermal ridge), which organizes fin/limb development, transitions into a different, elongated organizing structure in the fin bud, the AF (apical fold). Although the role of AER in limb development has been clarified in many studies, little is known about the role of AF in fin development. Here, we investigated AF-driven morphogenesis in the pectoral fin of zebrafish. After the AER-AF transition at ～36 hours post-fertilization, the AF was identifiable distal to the circumferential blood vessel of the fin bud. Moreover, the AF was divisible into two regions: the proximal AF (pAF) and the distal AF (dAF). Removing the AF caused the AER and a new AF to re-form. Interestingly, repeatedly removing the AF led to excessive elongation of the fin mesenchyme, suggesting that prolonged exposure to AER signals results in elongation of mesenchyme region for endoskeleton. Removal of the dAF affected outgrowth of the pAF region, suggesting that dAF signals act on the pAF. We also found that the elongation of the AF was caused by morphological changes in ectodermal cells. Our results suggest that the timing of the AER-AF transition mediates the differences between fins and limbs, and that the acquisition of a mechanism to maintain the AER was a crucial evolutionary step in the development of tetrapod limbs.     

Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo

Axon outgrowth between the spinal cord and the hindlimb of the chick embryo is constrained by three tissues that border axon pathways. Growth cones turn to avoid the posterior sclerotome, perinotochordal mesenchyme, and pelvic girdle precursor during normal development and after experimental manipulation. We wanted to know if these functionally similar barriers to axon advance also share a common molecular composition. Since the posterior sclerotome differentially binds peanut agglutinin (PNA) and since PNA binding is also typical of prechondrogenic differentiation, we examined the pattern of expression of PNA binding sites and cartilage proteoglycan epitopes in relation to axon outgrowth. We found that all three barrier tissues preferentially express both PNA binding sites and chondroitin-6-sulfate (C-6-S) immunoreactivity at the time when growth cones avoid these tissues. Moreover, both epitopes are expressed in the roof plate of the spinal cord and in the early limb bud, two additional putative barriers to axon advance. In contrast, neither epitope is detected in peripheral axon pathways. In the somites, this dichotomous pattern of expression clearly preceded the invasion of the anterior sclerotome by either motor growth cones or neural crest cells. However, in the limb, barrier markers disappeared from presumptive axon pathways in concert with the invasion of axons. Since this coordinate pattern suggested that the absence of barrier markers in these axon pathways requires an interaction with growth cones, we analyzed the pattern of barrier marker expression following unilateral neural tube deletions. We found that PNA-negative axon pathways developed normally even in the virtual absence of axon outgrowth. We conclude that the absence of staining with carbohydrate-specific barrier markers is an independent characteristic of the cells that comprise axon pathways. These results identify two molecular markers that characterize known functional barriers to axon advance and suggest that barrier tissues may impose patterns on peripheral nerve outgrowth by virtue of their distinct molecular composition.     

Involvement of HGF/MET signaling in appendicular muscle development in cartilaginous fish

In amniotes, limb muscle precursors de-epithelialize from the ventral dermomyotome and individually migrate into limb buds. In catsharks, Scyliorhinus, fin muscle precursors are also derived from the ventral dermomyotome, but shortly after de-epithelialization, they reaggregate within the pectoral fin bud and differentiate into fin muscles. Delamination of muscle precursors has been suggested to be controlled by hepatocyte growth factor (HGF) and its tyrosine kinase receptor (MET) in amniotes. Here, we explore the possibility that HGF/MET signaling regulates the delamination of appendicular muscle precursors in embryos of the catshark Scyliorhinus canicula. Our analysis reveals that Hgf is expressed in pectoral fin buds, whereas c-Met expression in fin muscle precursors is rapidly downregulated. We propose that alteration of the duration of c-Met expression in appendicular muscle precursors might underlie the evolution of individually migrating muscle precursors, which leads to the emergence of complex appendicular muscular systems in amniotes.     

Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Here we studied the expression of tenascin along developing peripheral nerves in chick embryos and tested its activity as a substrate for cultured neurons. Motor axons grow out through the tenascin-rich, anterior part of the sclerotome. Shortly after, tenascin surrounds axon fascicles of ventral roots. At the limb levels, outgrowing axons accumulate in the tenascin-containing girdle region forming a plexus. In the limb, tenascin first appears in bracket-like structures that surround the precartilage cell condensations of the femur and humerus, respectively. These regions coincide with the channels along which axons first grow in from the girdle plexus to form the limb nerves. Later, the major tenascin staining is associated with the cartilage and tendon primordia, and not with the limb nerves. We used tenascin as a substrate for cultured neural explants and single cells in order to test for its function in neurite outgrowth. Dissociated embryonic neurons of various types attached to mixed polylysine/tenascin substrates and sprouted rapidly after a lag of several hours. Outgrowth was inhibited and neurites were detached by anti-tenascin antibodies. On substrates coated with tenascin alone, neurite outgrowth was achieved from 3 day spinal cord explants. Whereas growth cones were well spread and rapidly moving, the neurites were poorly attached, straight and rarely branched. We speculate that in vivo tenascin allows axonal outgrowth, but inhibits branching and supports fasciculation of newly formed axons.     

The distribution of neural crest-derived Schwann cells from subsets of brachial spinal segments into the peripheral nerves innervating the chick forelimb.

Neural crest cells from brachial levels of the neural tube populate the ventral roots, spinal nerves, and peripheral nerves of the chick forelimb where they give rise to Schwann cells. The distribution of neural crest cells in the developing forelimb was examined using homotopic and heterotopic chick-quail chimeras to label neural crest cells from subsets of the brachial spinal segments. Neural crest cells from particular regions of the spinal cord populated ventral roots and spinal nerves adjacent to or immediately posterior to the graft. Crest cells also populated the brachial plexus in accord with their segmental origins. In the forelimb, neural crest cells populated muscle nerves with anterior brachial spinal segments populating nerves to anterior musculature of the forelimb and posterior brachial spinal segments populating nerves to posterior musculature. Similar patterns were seen following both homotopic and heterotopic transplantation. In both types of grafts, the distribution of neural crest cells largely matched the sensory and motor projection pattern from the same spinal segmental level. This suggests that neural crest-derived Schwann cells from a particular spinal segment may use sensory and motor fibers emerging from the same segmental level as substrates to guide their migration into the periphery.     

FGF7 and FGF10 directly induce the apical ectodermal ridge in chick embryos.

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.     

Cell death delineates axon pathways in the hindlimb and does so independently of neurite outgrowth

We wished to know whether the cell death and phagocytosis seen near the outgrowing nerve front in the hindlimb delineate axon pathways and, if so, whether the cells died only in the presence of growth cones. We unilaterally deleted the lumbosacral neural tube and reconstructed the patterns of neurite outgrowth and phagocytes during the stage when neurites first begin to colonize the thigh. In the control limbs, sensory and motor nerve pathways coincided with sites of phagocytosis, including those pathways that had yet to be colonized by growth cones. For instance, phagocytes were clustered at foci within the muscle masses where muscle nerves form a day later. However, they were not seen in adjacent, nonpathway regions such as posterior sclerotome or dorsal and ventral to the region of the plexus in which axons extend only posteriorly. Phagocytes were also seen in defined regions that are probably inaccessible to growth cones because they are too distant from pathways (i.e., subjacent to the apical ectodermal ridge) or express substances that are typical of precartilagenous tissues which may prohibit axon advance. In the experimental limbs, we conservatively estimated that neurite outgrowth was reduced to less than one-tenth (neurites were visible only with electron microscopy) or less than one-third of normal. Outgrowth extended less far distally and, in half the cases, motor innervation was completely abolished. Despite the extensive reduction in neurite outgrowth, the distribution of phagocytes was indistinguishable from that of the control side. Furthermore, the number of phagocytes did not differ significantly. We conclude that cell death delineates axon pathways remarkably well and does so without an interaction with growth cones; it is an independent characteristic of the axonal pathways and may be directly or indirectly important to axonal pathfinding. This is the first identification of a feature that characterizes prospective nerve pathways in the hindlimb.     

Growth pattern of pioneering chick spinal cord axons

The early growth pattern of axons in the embryonic chick spinal cord was studied by electron microscopy. Serial perisagittal thin sections were obtained from the lateral margins of spinal cords of stage 17 (S17) and S19 embryos. A simple stereotypic pattern of axonal growth was found. Axons originated from a dispersed population of presumptive interneurons located along the lateral spinal cord margin. They first grew ventrally in a nonfasciculative pattern and later turned at right angles and grew in a fasciculative manner longitudinally in the ventrolateral fasciculus. Growth along the circumferential pathway was analyzed in detail by reconstructing individual axons and growth cones from the S17 specimen. Most circumferential axons, regardless of their site of origin, grew in a parallel orientation, and each of their growth cones projected ventrally. This pattern suggested that circumferential growth cones were guided at many, if not all, points along their path. Study of the region in front of these seven growth cones, however, revealed no apparent structural basis for their guidance. Alternative guidance mechanisms are discussed. In conjunction with previous studies (e.g., Windle and Baxter, 1936; Lyser, 1966), these findings suggest that the circumferential-nonfasciculative and the longitudinal-fasciculative patterns of axonal growth are the two fundamental patterns followed by most early forming axons in the brain stem and spinal cord of all higher vertebrates.