Topology of feather melanocyte progenitor niche allows complex pigment patterns to emerge.

Color patterns of bird plumage affect animal behavior and speciation. Diverse patterns are present in different species and within the individual. Here, we study the cellular and molecular basis of feather pigment pattern formation. Melanocyte progenitors are distributed as a horizontal ring in the proximal follicle, sending melanocytes vertically up into the epithelial cylinder, which gradually emerges as feathers grow. Different pigment patterns form by modulating the presence, arrangement, or differentiation of melanocytes. A layer of peripheral pulp further regulates pigmentation via patterned agouti expression. Lifetime feather cyclic regeneration resets pigment patterns for physiological needs. Thus, the evolution of stem cell niche topology allows complex pigment patterning through combinatorial co-option of simple regulatory mechanisms.

Granulocytosis and increased adhesion molecules after resistive loading of the diaphragm.

Upregulation of endothelial cell adhesion molecules, followed by an influx of granulocytes and macrophages, can contribute to exertion-induced skeletal muscle injury. The purpose of this study was to quantify circulating leukocyte subsets, diaphragm injury and infiltrating leukocyte subsets, and surface expression of vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1 in the diaphragm after inspiratory resistive loading (IRL). Eight New Zealand white rabbits underwent 1.5 h of IRL and seven control rabbits underwent a sham procedure. Blood samples, taken at baseline and 2, 6, 12, 24, 48 and 72 h after the onset of IRL or sham, showed that band cell counts had increased at 6 h post-IRL. Point counting of haematoxylin and eosin-stained cross-sections, sampled at 72 h post-IRL, showed greater injury in diaphragms from IRL rabbits compared with controls. Immunohistochemical processing showed increased expression of ICAM-1 and VCAM-1, and higher granulocyte and macrophage counts in IRL diaphragms than control diaphragms. Macrophages were the predominant inflammatory cells. Increased intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, and infiltration of granulocytes and macrophages may contribute to inspiratory resistive loading-induced diaphragm injury.

Free radical scavengers and diaphragm injury following inspiratory resistive loading.

Three groups of NZW rabbits were studied to examine the role of free radical scavengers in preventing diaphragm injury produced by inspiratory resistive load (IRL): control, IRL, and scavenger groups. An IRL (Pao: 45-55 cm H2O) was applied to the IRL and the scavenger groups on Day 1. Free radical scavengers (polyethylene glycol superoxide dismutase, N-acetylcysteine, and mannitol) were given (intravenously) to the scavenger group both before and after the IRL. All rabbits were killed on Day 3 to collect diaphragms. Point counting H&E-stained diaphragm x-sections indicated that abnormal diaphragm muscle in the IRL group was significantly greater than control (p < 0.01). However, it was significantly lower in the scavenger group than the IRL group (p < 0.05) and it did not differ from control. In vitro diaphragm physiological studies found that the twitch tension (p < 0.05) and maximal tension (p < 0.01) in the IRL group were significantly lower than control. The maximal tensions (p < 0.05) in the scavenger group were lower than control. After the fatigue protocol, diaphragmatic contractility in the scavenger group was similar to control and was better maintained compared with the IRL group. We conclude that free radical scavengers can prevent the development of diaphragm injury as evidenced by histology but the protection of diaphragm function is limited.

Conservation of early odontogenic signaling pathways in Aves.

Teeth have been missing from birds (Aves) for at least 60 million years. However, in the chick oral cavity a rudiment forms that resembles the lamina stage of the mammalian molar tooth germ. We have addressed the molecular basis for this secondary loss of tooth formation in Aves by analyzing in chick embryos the status of molecular pathways known to regulate mouse tooth development. Similar to the mouse dental lamina, expression of Fgf8, Pitx2, Barx1, and Pax9 defines a potential chick odontogenic region. However, the expression of three molecules involved in tooth initiation, Bmp4, Msx1, and Msx2, are absent from the presumptive chick dental lamina. In chick mandibles, exogenous bone morphogenetic protein (BMP) induces Msx expression and together with fibroblast growth factor promotes the development of Sonic hedgehog expressing epithelial structures. Distinct epithelial appendages also were induced when chick mandibular epithelium was recombined with a tissue source of BMPs and fibroblast growth factors, chick skin mesenchyme. These results show that, although latent, the early signaling pathways involved in odontogenesis remain inducible in Aves and suggest that loss of odontogenic Bmp4 expression may be responsible for the early arrest of tooth development in living birds.

beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin.

We explored the role of beta-catenin in chicken skin morphogenesis. Initially beta-catenin mRNA was expressed at homogeneous levels in the epithelia over a skin appendage tract field which became transformed into a periodic pattern corresponding to individual primordia. The importance of periodic patterning was shown in scaleless mutants, in which beta-catenin was initially expressed normally, but failed to make a punctuated pattern. To test beta-catenin function, a truncated armadillo fragment was expressed in developing chicken skin from the RCAS retrovirus. This produced a variety of phenotypic changes during epithelial appendage morphogenesis. In apteric and scale-producing regions, new feather buds with normal-appearing follicle sheaths, dermal papillae, and barb ridges were induced. In feather tracts, short, wide, and curled feather buds with abnormal morphology and random orientation formed. Epidermal invaginations and placode-like structures formed in the scale epidermis. PCNA staining and the distribution of molecular markers (SHH, NCAM, Tenascin-C) were characteristic of feather buds. These results suggest that the beta-catenin pathway is involved in modulating epithelial morphogenesis and that increased beta-catenin pathway activity can increase the activity of skin appendage phenotypes. Analogies between regulated and deregulated new growths are discussed.

Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia.

Periodic patterning is a fundamental organizing process in biology. Using a feather reconstitution assay, we traced back to the initial stage of the patterning process. Cells started from an equivalent state and self-organized into a periodic pattern without previous cues or sequential propagation. When different numbers of dissociated mesenchymal cells were confronted with a piece of same-sized epithelium, the size of feather primordia remained constant, not the number or interbud spacing, suggesting size determination is intrinsic to dissociated cells. Increasing bone morphogenetic protein (BMP) receptor expression in mesenchymal cells decreased the size of primordia while antagonizing the BMP pathway with Noggin increased the size of primordia. A threshold number of mesenchymal cells with a basal level of adhesion molecules such as NCAM were sufficient to trigger the patterning process. The process is best visualized by the progressive restriction of beta-catenin transcripts in the epidermis. Therefore, feather size, number and spacing are modulated through the available morphogen ligands and receptors in the system.

Epidermal dysplasia and abnormal hair follicles in transgenic mice overexpressing homeobox gene MSX-2.

The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin beta 1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages.

Successive formative stages of precartilaginous mesenchymal condensations in vitro: modulation of cell adhesion by Wnt-7A and BMP-2.

High-density chick limb bud cell culture is a useful model to study mesenchymal condensatifons and chondrogenesis. Most previous studies have focused on the effects of soluble reagents on terminal chondrogenic differentiation and have not defined the early cellular processes and signaling events. In this study, we defined five successive stages in the differentiation process: 1) dissociated cells, 2) small aggregates, 3) formation of cell clusters, 4) precartilaginous condensations, and 5) cartilage nodule. We used RCAS retrovirus-mediated Wnt-7a gene transduction to test the effect of Wnt-7a on the differentiation process. We found that Wnt-7a suppressed chondrogenic differentiation. Wnt-7a did not inhibit the initiation of condensation formation but blocked the progression of precartilaginous condensations to cartilage nodules. The Wnt-7a-transduced cultures showed characteristics of a less mature culture with persistent expression of NCAM, N-cadherin, wider distribution of integrin beta1 and fibronectin, and suppression of tenascin-C. BMP-2 is known to enhance chondrogenic differentiation in these cultures by promoting cell clusters to form continuous sheet-like precartilaginous condensations. However, cultures exposed to both BMP-2 and Wnt-7a showed inhibition of chondrogenic differentiation. Different signaling molecules such as Wnt-7a and BMP-2 may have antagonistic effects on cartilage differentiation and the gradient of the two molecules may be involved in defining the boundaries of the initial precartilaginous condensation. We propose that the shape of the precartilaginous condensations may be modulated by local concentrations of signaling molecules, such as Wnt-7a and BMP-2, which act to alter cell-substrate and cell-cell adhesions.

Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model.

How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity revealed a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiated all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression was induced later as a dot within the primordia. RCAS was used to overexpress Wnt-7a in reconstituted feather explants derived from stage 29 dorsal skin to further test its function in feather formation. Control skin formed normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression led to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also had inhibited elongation of the P-D axes. This was not due to a lack of cell proliferation, which actually was increased although randomly distributed. While morphogenesis was perturbed, differentiation proceeded as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 were diffusely expressed. The results showed that ectopic Wnt-7a expression enhanced properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds.

Msx-2 and the regulation of organ size: epidermal thickness and hair length.

During organogenesis, the issue of size regulation is as important as shape and differentiation. We propose that the regulation of the dimensions of the epithelium and its appendages (length, width, thickness) are based on regulation of cell numbers in specific sites, reflecting the input and output of cells in that region. This process is in turn regulated by the flow from the domain of proliferating cells to the domain of postmitotic differentiated cells. When the homeobox gene Msx-2 is over-expressed in transgenic mice under the control of the CMV promoter, the epidermis is thickened with hyperproliferation and hyperkeratosis. Hairs are shorter and the matrix region is shrunken. We suggest that Msx-2 may be one of the regulators involved in the control of organ size, and the above phenotypes are the manifestations of an increased cellular flow from proliferation domain to differentiation domain in the tissue.

Determinants of diaphragmatic injury.

Limb muscles can be injured during and after vigourous contractions. However, this injury is most evident under specific conditions. The strength and type of muscle contraction as well as the contractile status of the muscle are important determinants of injury. The initiating event leading to muscle injury is not clearly understood but there are several leading theories. The respiratory muscles are of obvious importance to survival, and fatigue or injury to them has been hypothesized to be prevented by various mechanisms. One such mechanism is reduced activation by the central nervous system. In this review information on the neural activation of the breathing muscles during inspiratory loading is discussed and reveals that neural activation to the diaphragm, the main inspiratory muscle, is high. Previous studies investigating the presence of muscle fatigue immediately after such inspiratory loading have shown little evidence of it. However, based on information from limb muscles, delayed or secondary muscle injury might occur and could produce deleterious effects on respiratory muscle function. Recent evidence shows that chronic low intensity inspiratory loading can produce diaphragmatic injury (Reid et al.) and secondary or delayed muscle injury can occur three days after an acute period of high intensity inspiratory loading. The results reviewed in this article suggest that the respiratory muscles, specifically the diaphragm, are not spared from injury or the results of muscle injury. Diaphragmatic function during the period of secondary muscle injury is markedly impaired and thus respiratory muscle injury is a phenomenon that warrants further investigation.

Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning; however, no molecular candidates have been identified. Here we show that the feather tract is initiated by a continuous stripe of Shh, Fgf-4, and Ptc expression in the epithelium, which then segregates into discrete feather primordia that are more strongly Shh and Fgf-4 positive. The primordia also become Bmp-2 and Bmp-4 positive. Bead-mediated delivery of BMPs inhibits local feather formation in contrast with the activators, SHH and FGF-4, which induce feather formation. Both FGF-4 and SHH induce local expression of Bmp-4, while BMP-4 suppresses local expression of both. FGF-4 also induces Shh. Based on these findings, we propose a model that involves (1) homogeneously distributed global activators that define the field, (2) a position-dependent activator of competence that propagates across the field, and (3) local activators and inhibitors triggered in sites of individual primordia that act in a reaction-diffusion mechanism. A computer simulation model for feather pattern formation is also presented.

Delayed diaphragm injury and diaphragm force production.

The present study was designed to examine the effect of delayed diaphragm injury produced by inspiratory resistive loading (IRL) on diaphragm force production. On Day 1, three groups of anesthetized and intubated NZW rabbits (n = 7 in each group) were subjected to moderate IRL (Pao approximately 30 cm H2O), high IRL (Pao approximately 45 cm H2O), or no load for 1.5 h. On Day 3, the baseline twitch transdiaphragmatic pressure (Pdi) and Pdi at 10 to 80 Hz were measured during bilateral phrenic stimulation and these measurements were repeated after another IRL (high level) in all three groups. Diaphragm injury was assessed by the point-counting technique. Marked diaphragm injury was observed in the high-IRL group (p < 0.01), but no significant diaphragm injury was observed in the moderate-IRL or control groups. The baseline twitch Pdi was maintained in both IRL groups, whereas the baseline Pdi-frequency values in the high-IRL group were significantly reduced at most frequencies (p < 0.05). The decreases in twitch and Pdi at different frequencies were more pronounced after the IRL on Day 3 in the high-IRL group compared with controls. Moderate IRL did not decrease diaphragm force either before or after the high IRL on Day 3. We conclude that the diaphragm injury induced by high IRL has a significant impact on diaphragm force production and the attendant force loss produced by IRL is dependent on the intensity of inspiratory loading.

Load dependence of secondary diaphragm inflammation and injury after acute inspiratory loading.

Chronic or prolonged low-intensity loading of the inspiratory muscles has recently been shown to produce diaphragm injury. The present study was designed to examine whether an acute episode of inspiratory resistive loading (IRL) could produce secondary diaphragm inflammation and injury. On Day 1, three groups of anesthetized and intubated New Zealand White rabbits were subjected to moderate IRL (Pao of approximately 30 cm H2O), high IRL (Pao of approximately 45 cm H2O), or no load for 1.5 h. On Day 3, costal and crural diaphragms, parasternals, and gastrocnemius muscles were taken to assess injury by point counting. Normal muscle, abnormal and inflamed muscle, and connective tissue on hematoxylin and eosin-stained cross-sections were expressed as percentage of the total points for that cross-section. For the costal diaphragm, both the abnormal muscle (7.3 +/- 0.6% versus 1.1 +/- 0.2%; p < 0.001) and connective tissue (8.0 +/- 0.6% versus 5.7 +/- 0.2%; p < 0.01) in the high IRL group were higher than control, whereas in the moderate IRL group they were not significantly different from control. Total calpain-like activity was increased in the moderate IRL group but not in the high IRL group. Injury was observed in the parasternal muscles but to a lesser extent. No injury was observed in the gastrocnemius muscle. We conclude that secondary diaphragm injury occurs after acute IRL but only when the IRL exceeds the fatigue threshold.

Reversible paralysis with status asthmaticus, steroids, and pancuronium: clinical electrophysiological correlates.

Prolonged neuromuscular weakness has been identified after neuromuscular blockade in intensive care unit patients on mechanical ventilation. Previously reported electromyographic studies in these patients documented both neurogenic features and features consistent with a myopathy. We recorded sequential electrophysiological parameters during recovery from neuromuscular blockade in 5 patients with clinical weakness. An evolving pattern was identified. The early features were in keeping with previous reports of neurogenic changes, and this evolved into features consistent with a primary myopathy. Several potential underlying mechanisms are discussed.

Asymmetric expression of Notch/Delta/Serrate is associated with the anterior-posterior axis of feather buds.

We studied the roles of Notch, Delta, and Serrate in vertebrate epithelial appendage morphogenesis using feather as a model and found the following. (1) C-Notch-1, C-Delta-1, and C-Serrate-1 are not expressed at the early placode stage and are therefore not involved in the determination of bud versus interbud compartments. (2) From symmetric short buds to asymmetric long buds, C-Delta-1 and C-Serrate-1 are expressed in the posterior bud mesenchyme in a nested fashion, while C-Notch-1 is expressed as a stripe perpendicular to the anterior-posterior (A-P) axis and positioned posterior to the midpoint. (3) Epithelial-mesenchymal recombination with rotation led to the disappearance of these genes followed by their reappearance with new positions appearing to predict their new morphological orientation. (4) Conditions leading to branched buds (e.g., recombination of later buds) show polarized staining patterns before branching occurs. (5) Conditions leading to symmetrical round buds (e.g., treated with the protein kinase A agonist forskolin) suppress expression of all three genes. These results lead us to hypothesize that Notch, Delta, and Serrate are involved in establishing the A-P asymmetry of feather buds.

Molecular histology in skin appendage morphogenesis.

Classical histological studies have demonstrated the cellular organization of skin appendages and helped us appreciate the intricate structures and function of skin appendages. At this juncture, questions can be directed to determine how these cellular organizations are achieved. How do cells rearrange themselves to form the complex cyto-architecture of skin appendages? What are the molecular bases of the morphogenesis and histogenesis of skin appendages? Recently, many new molecules expressed in a spatial and temporal specific manner during the formation of skin appendages were identified by molecular biological approaches. In this review, novel molecular techniques that are useful in skin appendage research are discussed. The distribution of exemplary molecules from different categories including growth factors, intracellular signaling molecules, homeobox genes, adhesion molecules, and extracellular matrix molecules are summarized in a diagram using feather and hair as models. We hope that these results will serve as the ground work for completing the molecular mapping of skin appendages which will refine and re-define our understanding of the developmental process beyond relying on morphological criteria. We also hope that the listed protocols will help those who are interested in this venture. This new molecular histology of skin appendages is the foundation for forming new hypotheses on how molecules are mechanistically involved in skin appendage development and for designing experiments to test them. This may also lead to the modulation of healing and regeneration processes in future treatment modalities.

FGF induces new feather buds from developing avian skin.

Induction of skin appendages involves a cascade of molecular events. The fibroblast growth factor (FGF) family of peptide growth factors is involved in cell proliferation and morphogenesis. We explored the role of the FGFs during skin appendage induction using developing chicken feather buds as a model. FGF-1, FGF-2, or FGF-4 was added directly to the culture medium or was released from pre-soaked Affigel blue beads. Near the midline, FGFs led to fusion of developing feather buds, representing FGFs' ability to expand feather bud domains in developing skin. In lateral regions of the explant where feather placodes have not formed, FGF treatment produces a zone of condensation and a region with an increased number of feather buds. In ventral epidermis that is normally apteric (without feathers), FGFs can also induce new feather buds. Like normal feather buds, the newly induced buds express Shh. The expression of Grb, Ras, Raf, and Erk, intracellular signaling molecules known to be downstream to tyrosine kinase receptors such as the FGF receptor, was enriched in feather bud domains. Genistein, an inhibitor of tyrosine kinase, suppressed feather bud formation and the effect of FGF. These results indicate that there are varied responses to FGFs depending on epithelial competence. All the phenotypic responses, however, show that FGFs facilitate the formation of skin appendage domains.

Effect of the beta-agonist clenbuterol on dexamethasone-induced diaphragm dysfunction in rabbits.

The present study was designed to examine whether clenbuterol (CLEN) could reduce dexamethasone (DEX)-induced diaphragm dysfunction. We studied four groups of New Zealand white (NZW) rabbits, each receiving one of the following daily injections subcutaneously for 2 wk: saline (control), DEX 3 mg/kg, DEX 3 mg/kg + CLEN 2 mg/kg, and CLEN 2 mg/kg. Diaphragm fiber cross-sectional areas (CSA) were measured. Twitch transdiaphragmatic pressure (Pdi) and tetanic Pdi were measured during bilateral phrenic stimulation both before and after 60 min of inspiratory resistive loading (IRL). DEX produced a marked atrophy of type IIa and type IIb diaphragm fibers. This diaphragm atrophy was prevented by CLEN in the DEX plus CLEN group. CLEN alone increased CSAs of all three types of diaphragm fibers. Significant reductions in twitch Pdi and tetanic Pdi at all stimulation frequencies both before and after IRL were observed similarly in the DEX group as well as in the DEX plus CLEN group compared with the control animals. We conclude that DEX produces significant diaphragm atrophy and decreases diaphragmatic contractility. CLEN produces hypertrophy of the diaphragm and minimizes diaphragm atrophy induced by DEX, but it has no demonstrable protective effect on DEX-induced diaphragm dysfunction.