Chick Hindlimb Research Articles

Normal skeletal pattern formation in chick limb bud with a mesenchymal hole is mediated by adjustment of cellular properties along the anterior–posterior axis in the limb bud

The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 ​h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 ​h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.

Cover Image

Seven-day old chick hindlimbs (wild-type on the left, talpid2 on the right), stained for nuclei in blue (DAPI) and osteochondroprogenitors in yellow (Sox9). The increase in the number and identity of the digits in the talpid2 mutant, which were among Dr. Fallon's favorite subject of study, provides an important window into understanding how pattern and form arise in development and in evolution. From: Atavisms in the avian hindlimb and early developmental polarity of the limb; Bonatto Paese CL, Hawkins MB, Brugmann SA, Harris MP; DevDyn 250:9:1358–1367; https://doi.org/10.1002/dvdy.318

Differential effect of frequency and duration of mechanical loading on fetal chick cartilage and bone development.

Developmental engineering strategies aim to recapitulate aspects of development in vitro as a means of forming functional engineered tissues, including cartilage and bone, for tissue repair and regeneration. Biophysical stimuli arising from fetal movements are critical for guiding skeletogenesis, but there have been few investigations of the biomechanical parameters which optimally promote cartilage and bone development events in in vitro explants. The effect of applied flexion-extension movement frequencies (0.33 and 0.67 Hz) and durations (2 h periods, 1, 2 or 3 × per day) on knee (stifle) joint cartilage shape, chondrogenesis and diaphyseal mineralisation of fetal chick hindlimbs, cultured in a mechanostimulation bioreactor, were assessed both quantitatively and qualitatively. It was hypothesised that increasing frequency and duration of movements would synergistically promote cartilage and bone formation in a dose-dependent manner. Increasing loading duration promoted cartilage growth, shape development and mineralisation of the femoral condyles and tibiotarsus. While increasing frequency had a significant positive effect on mineralisation, hyaline cartilage growth and joint shape were unaffected by frequency change within the ranges assessed, and there were limited statistical interactions between the effects of movement frequency and duration on cartilage or bone formation. Increased glycosaminoglycan deposition and cell proliferation may have contributed to the accelerated cartilage growth and shape change under increasing loading duration. The results demonstrated that frequencies and durations of applied biomechanical stimulation differentially promoted cartilage and bone formation, with implications for developmentally inspired tissue engineering strategies aiming to modulate tissue construct properties.

Blocking mechanosensitive ion channels eliminates the effects of applied mechanical loading on chick joint morphogenesis.

Abnormalities in joint shape are increasingly considered a critical risk factor for developing osteoarthritis in life. It has been shown that mechanical forces during prenatal development, particularly those due to fetal movements, play a fundamental role in joint morphogenesis. However, how mechanical stimuli are sensed or transduced in developing joint tissues is unclear. Stretch-activated and voltage-gated calcium ion channels have been shown to be involved in the mechanoregulation of chondrocytes in vitro. In this study, we analyse, for the first time, how blocking these ion channels influences the effects of mechanical loading on chick joint morphogenesis. Using in vitro culture of embryonic chick hindlimb explants in a mechanostimulation bioreactor, we block stretch-activated and voltage-gated ion channels using, respectively, gadolinium chloride and nifedipine. We find that the administration of high doses of either drug largely removed the effects of mechanical stimulation on growth and shape development in vitro, while neither drug had any effect in static cultures. This study demonstrates that, during joint morphogenesis, mechanical cues are transduced—at least in part—through mechanosensitive calcium ion channels, advancing our understanding of cartilage development and mechanotransduction.This article is part of the Theo Murphy meeting issue ‘Mechanics of development’.

Characterising the effects of in vitro mechanical stimulation on morphogenesis of developing limb explants

Mechanical forces due to fetal movements play an important role in joint shape morphogenesis, and abnormalities of the joints relating to abnormal fetal movements can have long-term health implications. While mechanical stimulation during development has been shown to be important for joint shape, the relationship between the quantity of mechanical stimulation and the growth and shape change of developing cartilage has not been quantified. In this study, we culture embryonic chick limb explants in vitro in order to reveal how the magnitude of applied movement affects key aspects of the developing joint shape. We hypothesise that joint shape is affected by movement magnitude in a dose-dependent manner, and that a movement regime most representative of physiological fetal movements will promote characteristics of normal shape development. Chick hindlimbs harvested at seven days of incubation were cultured for six days, under either static conditions or one of three different dynamic movement regimes, then assessed for joint shape, cell survival and proliferation. We demonstrate that a physiological magnitude of movement in vitro promotes the most normal progression of joint morphogenesis, and that either under-stimulation or over-stimulation has detrimental effects. Providing insight into the optimal level of mechanical stimulation for cartilage growth and morphogenesis is pertinent to gaining a greater understanding of the etiology of conditions such as developmental dysplasia of the hip, and is also valuable for cartilage tissue engineering.

AP-2β is a transcriptional regulator for determination of digit length in tetrapods

The species-specific morphology of digits in the tetrapod limb, including the length and number of metacarpal, metatarsal, and phalangeal bones, suggests that a common developmental mechanism for digit formation is modified in a species-specific manner. Here, we examined the function of the AP-2β transcription factor in regulating digit length in the chicken autopod. Mutations in the gene encoding AP-2β are associated with Char syndrome, a human autosomal dominant disorder. Char syndrome patients exhibit autopod skeletal defects, including loss of phalanges and shortened fingers, suggestive of a function for AP-2β in normal digit development. The ectopic expression of two different dominant-negative forms of chick AP-2β, equivalent to mutant forms associated with human Char syndrome, in the developing chick hindlimb bud resulted in defective digit formation, including reductions in the number and length of phalanges and metatarsals. A detailed analysis of the AP-2β expression pattern in the limb bud indicated a correlation between the pattern/duration of AP-2β expression in the limb mesenchyme and digit length in three amniote species, the chicken, mouse and gecko. In addition, we found that AP-2β expression was downstream of Fgf signals from the apical ectodermal ridge, which is crucial in digit morphogenesis, and that excessive AP-2β function resulted in dysregulated digit length. Taken together, these results suggest that AP-2β functions as a novel transcriptional regulator for digit morphogenesis.

Expression and Methylation of FGF2, TGF-β and Their Downstream Mediators during Different Developmental Stages of Leg Muscles in Chicken

A number of growth factors determine the proliferation of myoblasts and therefore the number of ultimate myofibers. The members of transforming growth factor-beta (TGF-β) family and the fibroblast growth factor 2 (FGF2) have profound effects on skeletal myoblasts proliferation in various animal systems. To investigate their involvement in different stages of avian skeletal muscle development in vivo, we detected the mRNA expression and DNA methylation profiles of TGF-β2, TGF-β3, FGF2 and their downstream mediators in leg muscles at embryonic day 10, day of hatch and day 45 posthatch, using both Arbor Acres meat-type and White Leghorn egg-type chickens. By real-time PCR, we found that the expression levels of TGF-β2, TGF-β3, Smad3 and FGF2 were significantly (P≤0.01) higher at embryonic day 10, a developmental window of abundant fetal myoblasts expansion, by comparison to day of hatch and day 45 posthatch. The methylation status of the 5′ end region of these four genes was examined subsequently. A section of a CpG island in the 5′ end region of FGF2 was significantly hypomethylated (P≤0.01) at embryonic day 10, compared with neonatal and postnatal stages in both stocks. Our results suggested that TGF-β2, TGF-β3, Smad3 and FGF2 may play important roles in fetal myoblasts proliferation in chicken hindlimb, and the transcription of FGF2 in this wave of myogenesis could be affected by DNA methylation in 5′ flanking region. These outcomes contribute to our knowledge of the growth factors in avian myogenesis. Further investigation is needed to confirm and fully understand their functions in fetal limb myogenesis in birds.

Irx1 and Irx2 Are Coordinately Expressed and Regulated by Retinoic Acid, TGFβ and FGF Signaling during Chick Hindlimb Development

The Iroquois homeobox (Irx) genes play a crucial role in the regionalization and patterning of tissues and organs during metazoan development. The Irx1 and Irx2 gene expression pattern during hindlimb development has been investigated in different species, but its regulation during hindlimb morphogenesis has not been explored yet. The aim of this study was to evaluate the gene expression pattern of Irx1 and Irx2 as well as their regulation by important regulators of hindlimb development such as retinoic acid (RA), transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling during chick hindlimb development. Irx1 and Irx2 were coordinately expressed in the interdigital tissue, digital primordia, joints and in the boundary between cartilage and non-cartilage tissue. Down-regulation of Irx1 and Irx2 expression at the interdigital tissue coincided with the onset of cell death. RA was found to down-regulate their expression by a bone morphogenetic protein-independent mechanism before any evidence of cell death. Furthermore, TGFβ protein regulated Irx1 and Irx2 in a stage-dependent manner at the interdigital tissue, it inhibited their expression when it was administered to the interdigital tissue at developing stages before their normal down-regulation. TGFβ administered to the interdigital tissue at developing stages after normal down-regulation of Irx1 and Irx2 evidenced that expression of these genes marked the boundary between cartilage tissue and non-cartilage tissue. It was also found that at early stages of hindlimb development FGF signaling inhibited the expression of Irx2. In conclusion, the present study demonstrates that Irx1 and Irx2 are coordinately expressed and regulated during chick embryo hindlimb development as occurs in other species of vertebrates supporting the notion that the genomic architecture of Irx clusters is conserved in vertebrates.

Heterochronically early decline of Hox expression prior to cartilage formation in the avian hindlimb zeugopod

The fibula, a zeugopod bone in the hindlimb, exhibits various morphologies in tetrapod species. The fibula in some species has a similar length with the other zeugopod element, the tibia, while other species have obvious differences in the sizes of the two elements. In the avian hindlimb, for example, the fibula is extremely short, thin, and truncated. Basic morphology of the fibula is established during development, and cartilage primordium of the bone emerges in a certain region defined by a distinct combination of expression of Hox genes (Hox code). In order to elucidate how the different morphologies are produced from a region that is defined as the fixed Hox code, we examined spatial and temporal patterns of Hoxd11/Hoxd12 expression in the developing limb bud, which defines the region from which the fibula emerges, in comparison with the sites of precartilaginous mesenchymal condensations representing regions for cartilage formation among chick, mouse, and gecko embryos. We found that in the chick hindlimb, expression of Hoxd11/Hoxd12 decreased and disappeared from the presumptive zeugopod region before cartilage formation. This heterochronically early decline of expression of Hox genes is strongly correlated with the peculiar trait of the fibula in the avian hindlimb, since in the other species examined, expression of those genes continued after the onset of cartilage formation. This is morphological phenotype-related because the early disappearance was not seen in the chick forelimb. Our results suggest that temporal change of the Hox code governs diversification in morphology of homologous structures among related species.

Genetic Evidence for a Contribution of EphA:EphrinA Reverse Signaling to Motor Axon Guidance

Repulsive Eph forward signaling from limb-derived ephrins guides the axons of lateral motor column (LMC) motor neurons. LMC axons also express ephrinAs, while their EphA receptors are expressed in the limb mesenchyme. In vitro studies have suggested that reverse signaling from limb-derived EphA4 to axonal ephrinAs might result in attraction of LMC axons. However, genetic evidence for this function is lacking. Here we use the Dunn chamber turning assay to show that EphA proteins are chemoattractants and elicit fast turning responses in LMC neurons in vitro. Moreover, ectopic expression of EphA4 in chick hindlimb changes the limb trajectory of LMC axons. Nervous system-specific deletion of EphA4 in mice resulted in fewer LMC axon projection errors than the ubiquitous deletion of EphA4. Additionally, a signaling-incompetent EphA4 mutant partially rescued guidance errors in the hindlimb, suggesting that limb-derived EphA4 contributes to the establishment of LMC projections. In summary, we provide evidence for a role of EphA:ephrinA attractive reverse signaling in motor axon guidance and in vivo evidence of in-parallel forward Eph and reverse ephrin signaling function in the same neuronal population.

Knockdown of ephrin-A5 expression by 40% does not affect motor axon growth or migration into the chick hindlimb.

Bidirectional signaling between Eph receptor tyrosine kinases and their cell-surface protein signals, the ephrins, comprises one mechanism for guiding motor axons to their proper targets. During projection of motor axons from the lateral motor column (LMC) motor neurons of the spinal cord to the hindlimb muscles in chick embryos, ephrin-A5 has been shown to be expressed in the LMC motor axons until they reach the base of the limb bud and initiate sorting into their presumptive dorsal and ventral nerve trunks, at which point expression is extinguished. We tested the hypothesis that this dynamic pattern of ephrin-A5 expression in LMC motor axons is important for the growth and guidance of the axons to, and into, the hindlimb by knocking down endogenous ephrin-A5 expression in the motor neurons and their axons. No perturbation of LMC motor axon projections was observed in response to this treatment, suggesting that ephrin-A5 is not needed for LMC motor axon growth or guidance.

Growth of limb muscle is dependent on skeletal-derived Indian hedgehog

During embryogenesis, muscle and bone develop in close temporal and spatial proximity. We show that Indian Hedgehog, a bone-derived signaling molecule, participates in growth of skeletal muscle. In Ihh −/− embryos, skeletal muscle development appears abnormal at embryonic day 14.5 and at later ages through embryonic day 20.5, dramatic losses of hindlimb muscle occur. To further examine the role of Ihh in myogenesis, we manipulated Ihh expression in the developing chick hindlimb. Reduction of Ihh in chicken embryo hindlimbs reduced skeletal muscle mass similar to that seen in Ihh −/− mouse embryos. The reduction in muscle mass appears to be a direct effect of Ihh since ectopic expression of Ihh by RCAS retroviral infection of chicken embryo hindlimbs restores muscle mass. These effects are independent of bone length, and occur when Shh is not expressed, suggesting Ihh acts directly on fetal myoblasts to regulate secondary myogenesis. Loss of muscle mass in Ihh null mouse embryos is accompanied by a dramatic increase in myoblast apoptosis by a loss of p21 protein. Our data suggest that Ihh promotes fetal myoblast survival during their differentiation into secondary myofibers by maintaining p21 protein levels.

Versican V0 and V1 Direct the Growth of Peripheral Axons in the Developing Chick Hindlimb

Peanut agglutinin-binding disaccharides and chondroitin sulfate mark transient mesenchymal barriers to advancing motor and sensory axons innervating the hindlimbs during chick development. Here we show that the vast majority of these carbohydrates are at the critical stage and location attached to the versican splice variants V0 and V1. We reveal that the isolated isoforms of this extracellular matrix proteoglycan suppress axon extension at low concentrations and induce growth cone collapse and rapid retraction at higher levels. Moreover, we demonstrate that versican V0 and/or V1, recombinantly expressed in collagen-I gels or ectopically deposited in the hindlimbs of chicken embryos in ovo, cause untimely defasciculation and axon stalling. Consequently, severe disturbances of nerve patterning are observed in the versican-treated embryos. Our experiments emphasize the inhibitory capacity of versicans V0 and V1 in axonal growth and evidence for their function as basic guidance cues during development of the peripheral nervous system.

Retinoid signaling is involved in governing the waiting period for axons in chick hindlimb

During embryonic development in chick, axons pause in a plexus region for approximately 1 day prior to invading the limb. We have previously shown that this “waiting period” is governed by maturational changes in the limb. Here we provide a detailed description of the spatiotemporal pattern of Raldh2 expression in lumbosacral motoneurons and in the limb, and show that retinoid signaling in the limb contributes significantly to terminating the waiting period. Raldh2, indicative of retinoid signaling, first appears in hindlimb mesenchyme near the end of the waiting period. Transcripts are more abundant in connective tissue associated with predominantly fast muscles than predominantly slow muscles, but are not expressed in muscle cells themselves. The tips of ingrowing axons are always found in association with domains of Raldh2, but development of Raldh2 expression is not regulated by the axons. Instead, retinoid signaling appears to regulate axon entry into the limb. Supplying exogenous retinoic acid to proximal limb during the waiting period caused both motor and sensory axons to invade the limb prematurely and altered the normal stereotyped pattern of axon ingrowth without obvious effects on limb morphogenesis or motoneuron specification. Conversely, locally decreasing retinoid synthesis reduced axon growth into the limb. Retinoic acid significantly enhanced motor axon growth in vitro, suggesting that retinoic acid may directly promote axon growth into the limb in vivo. In addition, retinoid signaling may indirectly affect the waiting period by regulating the maturation of other gate keeping or guidance molecules in the limb. Together these findings reveal a novel function of retinoid signaling in governing the timing and patterning of axon growth into the limb.

A dynamic pattern of mechanical stimulation promotes ossification in avian embryonic long bones

We have performed a set of finite element analyses of embryonic chick hindlimb skeletal rudiments at several time points during development, around the time of initial bone formation. Using optical projection tomography, we created anatomically accurate rudiment and muscle morphologies for each stage. The changes in pattern and magnitude of biophysical stimuli (such as stress, strain, hydrostatic pressure and fluid flow) were computed, and were found to transform as bone formation proceeded in the rudiment. For each biophysical stimulus, a single concentration of the stimulus was predicted at the mid-diaphysis some time before initial bone formation begins. Then, several hours before ossification, two concentrations of the stimuli were predicted distal and proximal to the prospective bone collar. Once bone formation had begun, high concentrations of the stimuli were maintained proximal and distal to the bone collar. We propose the hypothesis that patterns of biophysical stimuli resulting from mechanical loading due to muscle contractions initiate and propagate ossification in avian embryonic long bones, whereby a region of the perichondrium experiences a period of time under high cyclic stimulus levels, promoting chondrocyte hypertrophy at the core and prompting bone collar formation some hours later.

Development of neuromuscular connections: guidance of motoneuron axons to muscles in the embryonic chick hindlimb.

A striking feature of the outgrowth of motor axons from the embryonic chick spinal cord is the accuracy with which they grow to correct target muscles in the limb. Studies involving the surgical manipulation of the early neural tube or limb bud have led to the conclusion that motoneurons are specified or acquire distinctive properties early in development that enable them to detect and respond to specific cues in the periphery. We have recently carried out studies to identify the cells or tissues responsible for producing these cues. Major contributors to the outgrowing axon's environment are the lumbosacral (LS) somites which give rise to limb muscle cells and the LS somatopleural mesoderm which gives rise to limb connective tissues. To determine whether the LS somites contain specific guidance information at stages when motoneurons are known to be specified, motor projections were assessed in chick embryos after early somitic tissue reversals. The finding of normal projection patterns suggests that the LS somites do not contain such information at early stages. To assess the role of the LS somatopleural mesoderm in specific axon guidance, we examined motor projections in embryos where the LS somatopleural mesoderm had been shifted anteriorly before limb bud formation. In these embryos the limb developed in an anteriorly shifted position. LS motoneuron axons entered the posterior part of the limb but then extended anteriorly along aberrant paths to innervate appropriate muscles. These observations suggest that it is the precursor of limb connective tissues that is the source of specific axon guidance cues.

Viewpoint 4

Viewpoint 4

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.

The developmental evolution of avian digit homology: an update.

The identity of avian digits has been unresolved since the beginning of evolutionary morphology in the mid-19th century, i.e. as soon as questions of phylogenetic homology have been raised. The main source of concern is the persistent discrepancy between anatomical/paleontological and embryological evidence over the identity of avian digits. In this paper, recent evidence pertaining to the question of avian digit homology is reviewed and the various ideas of how to resolve the disagreement among developmental and phylogenetic evidence are evaluated. Paleontological evidence unequivocally supports the hypothesis that the fully formed digits of maniraptoran theropods are digits DI, DII, and DIII, because the phylogenetic position of Herrerasaurus is resolved, even when hand characters are excluded from the analysis. Regarding the developmental origin of the three digits of the avian hand the discovery of an anterior digit condensation in the limb bud of chickens and ostriches conclusively shows that these three digits are developing from condensations CII, CIII, and CIV. The existence of this additional anterior condensation has been confirmed in four different labs, using four different methods: Alcian blue staining, PNA affinity histochemistry, micro-capillary regression and Sox9 expression. Finally, recent evidence shows that the digit developing from condensation CII has a Hox gene expression pattern that is found in digit DI of mice forelimb and chick hind limbs. The sum of these data supports the idea that digit identity has shifted relative to the location of condensations, known as Frame Shift Hypothesis, such that condensation CII develops into digit DI and condensation CIII develops into digit DII, etc. A review of the literature on the digit identity of the Italian Three-toed Skink or Luscengola (Chalcides chalcides), shows that digit identity frame shifts may not be limited to the bird hand but may be characteristic of "adaptive" digit reduction in amniotes (sensu Steiner, H., Anders, G., 1946. Zur Frage der Entstehung von Rudimenten. Die Reduktion der Gliedmassen von Chalcides tridactylus Laur. Rev. Suisse Zool. 53, 537-546) in general. In this mode of evolution two digits are lost, in the course of the adaptation of the three anterior digits to a function that does not require the two posterior digits. This evidence suggests that the evolution of digits in tetrapods can proceed at least on two distinct levels of integration, the level of digit condensations and that of adult digits.

The contributions of BMP4, positive guidance cues, and repulsive molecules to cutaneous nerve formation in the chick hindlimb

Our previous surgical manipulations have shown that the target ectoderm is necessary for the initial formation of one of the major cutaneous nerves in the embryonic chick limb (Honig, M.G., Camilli, S.J., Xue, Q.S., 2004. Ectoderm removal prevents cutaneous nerve formation and perturbs sensory axon growth in the chick hindlimb. Dev. Biol. 266, 27–42.). Moreover, the target ectoderm is required during a critical time period, at ∼St. 24, when those axons are about to diverge from the hindlimb plexus. To elucidate the underlying mechanisms, here we examined the effects of removing the ectoderm at St. 24 on a variety of molecules expressed within the limb. We find that, while ectoderm removal is accompanied by changes in the expression of Lmx1, fibronectin, EphA7, cDermo-1, and in the complement of muscle cells, these changes do not account for the cutaneous nerve deficit. In contrast, an upregulation of PNA-binding sites and a downregulation of Bmp4 appear to be associated with this nerve deficit. Exogenous BMP4 reversed the effect of ectoderm removal on cutaneous nerve formation, but did not act as a chemoattractant. Our results suggest that BMP4, together with permissive and repulsive molecules that growing cutaneous axons encounter in the local environment and with signaling molecules, originating from and/or dependent on the ectoderm, work in concert to ensure proper cutaneous nerve formation.