Persistent larval sensory neurones are required for the normal development of the adult sensory afferent projections in Drosophila

We have tested the hypothesis that larval neurones guide growth of adult sensory axons in Drosophila. We show that ablation of larval sensory neurones causes defects in the central projections of adult sensory neurones. Spiralling axons and ectopic projections indicate failure in axon growth guidance. We show that larval sensory neurones are required for peripheral pathfinding, entry into the CNS and growth guidance within the CNS. Ablation of subsets of neurones shows that larval sensory neurones serve specific guidance roles. Dorsal neurones are required for axon guidance across the midline, whereas lateral neurones are required for posterior growth. We conclude that larval sensory neurones pioneer the assembly of sensory arrays in adults.     

Establishment of neuronal connectivity during development of the Drosophila larval visual system

We used confocal microscopy in conjunction with specific antibodies and enhancer trap strains to investigate the development of specific neuronal connections in a simple model system, the larval visual system of Drosophila. We find that the establishment of axonal projections from the larval photoreceptor neurons to their central nervous system targets involves a series of discrete steps. During embryogenesis, the larval optic nerve contacts several different cell types, including optic lobe pioneer (OLP) neurons and a number of glial cells. We demonstrate that OLP neurons are present and project normally in glass (gl) mutant embryos in which the larval optic nerve fails to develop, suggesting that they do not depend on interactions with the larval optic nerve for differentiation and proper axonal projection. The OLPs fail to differentiate properly in disconnected (disco) mutant embryos, where appropriate connections between the larval optic nerve and its targets in the brain are not formed. The disco gene is expressed in the OLPs and may therefore act autonomously to direct the differentiation of these cells. Taken together, our results suggest that the OLPs act as an intermediate target required for the establishment of normal optic nerve projection and connectivity. © 1995 John Wiley & Sons, Inc.     

Regulation of adult mammalian intrinsic axonal regeneration by NF-κB/STAT3 signaling cascade

The inflammatory response is a critical regulator for the regeneration of axon following nervous system injury. Nuclear factor-kappa B (NF-κB) is characteristically known for its ubiquitous role in the inflammatory response. However, its functional role in adult mammalian axon growth remains elusive. Here, we found that the NF-κB signaling pathway is activated in adult sensory neurons through peripheral axotomy. Furthermore, inhibition of NF-κB in peripheral sensory neurons attenuated their axon growth in vitro and in vivo. Our results also showed that NF-κB modulated axon growth by repressing the phosphorylation of STAT3. Furthermore, activation of STAT3 significantly promoted adult optic nerve regeneration. Taken together, the findings of our study indicated that NF-κB/STAT3 cascade is a critical regulator of intrinsic axon growth capability in the adult nervous system.     

Experimental analysis of character coupling across a complex life cycle: Pigment pattern metamorphosis in the tiger salamander,Ambystoma tigrinum tigrinum

Developmental relationships among characters are expected to bias patterns of morphological variation at the population level. Studies of character development thus can provide insights into processes of adaptation and the evolutionary diversification of morphologies. Here I use experimental manipulations to test whether larval and adult pigment patterns are coupled across metamorphosis in the tiger salamander, Ambystoma tigrinum tigrinum (Ambystomatidae). Previous investigations showed that the early larval pigment pattern depends on interactions between pigment cells and the lateral line sensory system. In contrast, the results of this study demonstrate that the major features of the adult pigment pattern develop largely independently of both the early larval pattern and the lateral lines. These results suggest that ontogenetic changes that occur across metamorphosis decouple larval and adult pigment patterns and could thereby facilitate independent evolutionary modifications to the patterns during different stages of the life cycle. J. Morphol. 237:53–67, 1998. © 1998 Wiley-Liss, Inc.     

The fate of specific motoneurons and sensory neurons of the pregenital abdominal segments inTenebrio molitor (Insecta : Coleoptera) during metamorphosis

The anatomy and innervation of the lateral external muscle and sensory cells located in the ventral region of pregenital abdominal segments were examined at the larval and adult stages ofTenebrio molitor (Coleoptera). All seven muscles located in this region degenerate during the pupal stage, whilst only the lateral external median (lem) appears in the adult. Backfillings of the motor nerve innervating this muscle reveal that, at both larval and adult stages, it is innervated by ten neurons. Intracellular records from the muscle fibres show that two neurons are inhibitory, and at least five are excitatory. There are also two unpaired neurons. A variety of sensory organs are located in the ventral region of the larvae, whilst only campaniform sensilla are found in the adult. At both stages, the innervation pattern of the sensory nerve branches is very similar. Also, the central projections of the sensory cells occupy similar neuropilar areas. Finally, prolonged intracellular records from the lem muscle revealed that, at the larval stage, it participates only in segmental or intersegmental reflexes, whilst in the adult it has a primary expiratory role in ventilation. The results show that extensive changes occur in the number of muscles located in the ventral region of the pregenital abdominal segments, as well as in the arrangement and number of sensory neurons, in the structure of the exoskeleton, and even in the central nervous system. In contrast, only minor changes are observed in the sensory and motor nerve branches, in the sensory projections, and in the number and the location of the motoneurons innervating the lateral external median muscle. Correspondence to: G. Theophilidis     

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. We present data in this study showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point — the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance.     

Development of wing sensory axons in the central nervous system of Drosophila during metamorphosis

The development of new, adult-specific axonal pathways in the central nervous system (CNS) of insects during metamorphosis is still largely uncharacterized. Here we used axonal labeling with DiI to describe the timing and pattern of growth of sensory axons originating in the wing of Drosophila as they establish their adult projection pattern in the CNS during pupal life. The wing of Drosophila carries a small number of readily identifiable sensory organs (sensilla) whose neurons are located in the periphery and whose axons travel along specific routes within the adult CNS. The neurons are born and undergo axonogenesis in a characteristic order. The order of axon arrival in the CNS appears to be the same as that of their development in the periphery. Within the CNS, the formation of four prominent axon bundles leading to distant termination sites is followed by the formation of a compact axon termination site near the point of wing nerve entry into the CNS. This sensillum-specific pattern persists into adulthood without discernible modification. We also find a small number of axons filled with DiI prior to the formation of the four permanent bundles. We have only been able to fill them for a few hours in early pupal life and therefore consider them to be transient. The bundles of wing sensory axons travel within tracts that contain other axons as well. Using immunocytochemistry, the tracts start to be histologically identifiable at around 12 h after pupariation (AP), and grow substantially as metamorphosis proceeds. Wing sensory neurons are found in the tracts by 18–20 h AP and the full adult pattern is established by 48 h AP. When sensory axons first enter the CNS, they fan out in the region where their appropriate tracts are located, but they do not wander extensively. They quickly form bundles that become increasingly compact over time. Calculations show that the rate of axon extension within the CNS varies from bundle to bundle and is equal to or greater than that of the same axons growing through wing tissue. © 1995 John Wiley & Sons, Inc.     

Enhanced axon outgrowth and improved long‐distance axon regeneration in sprouty2 deficient mice

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal‐regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2^+/? neuron cultures, whereas homozygous Spry2^?/? neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2^+/? mice recovered faster in motor but not sensory testing paradigms (Spry2^?/? mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP‐43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2^?/? mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4‐positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long‐distance axon regeneration in injured peripheral nerves. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 217–231, 2015     

Nervous network in larvae of the ascidian Ciona intestinalis

 With the use of the monoclonal antibody UA301, which specifically recognizes the nervous system in ascidian larvae, the neuronal connections of the peripheral and central nervous systems in the ascidian Ciona intestinalis were observed. Three types of peripheral nervous system neurons were found: two located in the larval trunk and the other in the larval tail. These neurons were epidermal and their axons extended to the central nervous system and connected with the visceral ganglion directly or indirectly. The most rostral system (rostral trunk epidermal neurons, RTEN) was distributed bilateral-symmetrically. In addition, presumptive papillar neurons in palps were found which might be related to the RTEN. Another neuron group (apical trunk epidermal neurons, ATEN) was located in the apical part of the trunk. The caudal peripheral nervous system (caudal epidermal neurons, CEN) was located at the dorsal and ventral midline of the caudal epidermis. In the larval central nervous system, two major axon bundles were observed: one was of a photoreceptor complex and the other was connected with RTEN. These axon bundles joined in the posterior sensory vesicle, ran posteriorly through the visceral ganglion and branched into two caudal nerves which ran along the lateral walls of the caudal nerve tube. In addition, some immunopositive cells existed in the most proximal part of the caudal nerve tube and may be motoneurons. Received: 8 September 1997 / Accepted: 14 December 1997     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Neurometamorphosis of the ear in the gypsy moth,Lymantria dispar,and its homologue in the earless forest tent caterpillar moth,Malacosoma disstria

The adult gypsy moth, Lymantria dispar (Lymantriidae: Noctuoidea) has a pair of metathoracic tympanic ears that each contain a two-celled auditory chordotonal organ (CO). The earless forest tent caterpillar moth, Malacosoma disstria (Lasiocampidae: Bombycoidea), has a homologous pair of three-celled, nonauditory hindwing COs in their place. The purpose of our study was to determine whether the adult CO in both species arises from a preexisting larval organ or if it develops as a novel structure during metamorphosis. We describe the larval metathoracic nervous system of L. dispar and M. distria, and identify a three-celled chordotonal organ in the anatomically homologous site as the adult CO. If the larval CO is severed from the homologue of the adult auditory nerve (IIIN1b1) in L. dispar prior to metamorphosis, the adult develops an ear lacking an auditory organ. Axonal backfills of the larval IIIN1b1 nerve in both species reveal three chordotonal sensory neurons and one nonchordotonal multipolar cell. The axons of these cells project into tracts of the central nervous system putatively homologous with those of the auditory pathways in adult L. dispar. Following metamorphosis, M. disstria moths retain all four cells (three CO and one multipolar) while L. dispar adults possess two cells that service the auditory CO and one nonauditory, multipolar cell. We conclude that the larval IIIN1b1 CO is the precursor of both the auditory organ in L. dispar and the putative proprioceptor CO in M. disstria and represents the premetamorphic condition of these insects. The implications of our results in understanding the evolution of the ear in the Lepidoptera and insects in general are discussed. © 1996 John Wiley & Sons, Inc.     

Morphology of the larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L

Cell population and neuropile morphology of larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L., are compared. The larval brain is in continuous transition, the processes of adult brain development being underway from the earliest larval stages. It is characterized by a less diverse population of cells and more homogenous fiber areas than those of the adult. Neuroblasts, which divide to form the neurones of the adult brain, occur either in discrete proliferation centers or scattered among the larval ganglion cells. The larval brain contains, in addition to small homogeneous antennal centers and a distinct larval optic center, rapidly developing adult optic centers, corpora pedunculata, and protocerebral bridge. The larval brain lacks a central body. Major differences between larval and adult brains are clearly related to the increased dependence of the adult upon sensory input from the eyes and antennae.     

Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867)

Jungblut, L.D., Pozzi, A.G. and Paz, D.A. 2010. Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867). — Acta Zoologica (Stockholm) 92 : 305–315. The olfactory and the vomeronasal system are the two major chemosensory systems found in terrestrial vertebrates. Among tetrapods, amphibians are unique in having an aquatic larval stage, followed by metamorphosis to a terrestrial adult. In the present work, we studied the histological development of the olfactory and vomeronasal organ and associated multicellular glands of the toad Rhinella (Bufo) arenarum, from early poshatching larva to postmetamorphic toadlets. As in other bufonids, the olfactory epithelium of R. arenarum in larvae is divided into dorsal and ventral branches in the rostral and mid‐nasal regions. At metamorphic climax, the larval pattern changes drastically and the adult olfactory configuration develops. Bowman’s glands appear in the olfactory epithelium of R. arenarum at the onset of metamorphic climax. The vomeronasal epithelium develops early in larval development in R. arenarum, around the time of operculum development. Interestingly, a novel sensory epithelium develops in the floor of the principal chamber of R. arenarum at metamorphic climax. This novel sensory epithelium resembles larval sensory epithelium lacking Bowman’s glands, and suggests that these animals would be able to sense not only air‐borne, but also water‐borne odors during their adult terrestrial life.     

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda)

 Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered. Received: 23 June 1998 / Accepted: 25 November 1998     

The proprotein convertase amontillado (amon) is required during Drosophila pupal development

Peptide hormones governing many developmental processes are generated via endoproteolysis of inactive precursor molecules by a family of subtilisin-like proprotein convertases (SPCs). We previously identified mutations in the Drosophila amontillado (amon) gene, a homolog of the vertebrate neuroendocrine-specific Prohormone Convertase 2 (PC2) gene, and showed that amon is required during embryogenesis, early larval development, and larval molting. Here, we define amon requirements during later developmental stages using a conditional rescue system and find that amon is required during pupal development for head eversion, leg and wing disc extension, and abdominal differentiation. Immuno-localization experiments show that amon protein is expressed in a subset of central nervous system cells but does not co-localize with peptide hormones known to elicit molting behavior, suggesting the involvement of novel regulatory peptides in this process. The amon protein is expressed in neuronal cells that innervate the corpus allatum and corpora cardiaca of the ring gland, an endocrine organ which is the release site for many key hormonal signals. Expression of amon in a subset of these cell types using the GAL4/UAS system in an amon mutant background partially rescues larval molting and growth. Our results show that amon is required for pupal development and identify a subset of neuronal cell types in which amon function is sufficient to rescue developmental progression and growth defects shown by amon mutants. The results are consistent with a model that the amon protein acts to proteolytically process a diverse suite of peptide hormones that coordinate larval and pupal growth and development.     

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens

 A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration. Received: 6 November 1998 / Accepted: 12 December 1998     

Peripheral synapses at identifiable mechanosensory neurons in the spider Cupiennius salei : synapsin-like immunoreactivity

Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7–8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5–6.8 μm long and located 0.5–6.6 μm apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results.Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity. Received: 20 April 1998 / Accepted: 18 August 1998     

Metamorphic changes in the central projects of hair sensilla inTenebrio molitor L. (Insecta: Coleoptera)

Summary This paper describes the afferent projections of hair sensilla of the pro- and mesothoracic legs and the lateral thoracic sclerites of larval and adultTenebrio molitor and the corresponding set of pupal hair sensilla. The sensory neurons that innervate the hair sensilla of larval or adult insects project somatotopically into the thoracic neuropil. Different types of sensilla on the same region of the body surface project to the same zone of the ipsilateral thoracic ventral neuropil but exhibit different arborization patterns. Although there is a profound reorganization of body surface sensilla, the basic somatotopic layout of the larva is maintained in the adult. The sensory neurons that innervate the pupal hair sensilla possess central projections similar to those of the corresponding adult sensory neurons. The central projections of pupal sensory neurons are somatotopically oriented. Their projection pattern is serially homologous in the thoracic and the abdominal ganglia. The central projection pattern of the described pupal sensory neurons is constant throughout pupation. MAb 22C10 immunoreactivity allows an estimate of the timing of the early differentiation of the imaginal sensory neurons originating during pupation. Ablation experiments indicate that pupal sensory neurons influence the central projection pattern of the differentiating imaginal sensory neurons.     

Guidance of regenerating motor axons in vivo by gradients of diffusible peripheral nerve‐derived factors

During development of the central nervous system, neurons rely on target‐derived factors to guide their outgrowing processes. Several CNS target‐derived chemoattractive and repellant factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target‐derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve‐released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve‐released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target‐derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 212–219, 2000