Hoxc13在毛囊发育中的作用

Hoxc13属于Hox(Homobox)基因家族Abd-B类成员之一, 与毛囊形成和毛发生长密切相关。毛发结构蛋白KP(角蛋白)和KAP(角蛋白关联蛋白)的表达都受Hoxc13的严格调控, Hoxc13表达水平会直接影响毛发的特性, 对维持毛囊的正常形态也至关重要。文章就Hoxc13的表达水平对毛囊发育和毛发生长的影响及Hoxc13与相关基因的调控进行了综述。     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.     

Cellular contribution to supernumerary limbs resulting from the interaction between developing and regenerating tissues in the axolotl

The relationship between limb development and limb regeneration is considered with regard to the mechanisms by which pattern is established during limb outgrowth. In a previous paper (Muneoka, K. and Bryant, S. V. 1982 Nature (London) 298, 369-371) the interaction between cells from the developing limb bud and the regenerating limb blastema was found to result in the production of organized supernumerary limb structures. In this paper the relative cellular contribution from developing and regenerating cells to supernumerary limbs resulting from contralateral grafts between limb buds and blastemas has been analyzed using the triploid cell marker in the axolotl. Results show that there is substantial participation from both developing and regenerating limb cells to all supernumerary limbs analyzed. These data lend further support to the hypothesis that developing and regenerating limbs utilize the same patterning mechanisms during limb outgrowth. This conclusion is discussed in terms of patterning models for developing and regenerating limbs and it is proposed that the rules of the polar coordinate model can best explain the behavior of cells during limb development as well as limb regeneration.     

Expression analysis of KAP9.2 and Hoxc13 genes during different cashmere growth stages by qRT-PCR method

Keratin-associated protein 9.2 (KAP9.2) and Homeobox C13 (Hoxc13) genes were chosen to study because of their biological functions involving hair formation. KAP9.2 gene belongs to the ultra high sulfur KAPs, which is important for hair formation and may have association with cashmere. Hoxc13 takes part in the formation of cashmere keratin and maintaining the normal structure of follicle. It has been reported that Hoxc13 gene exists binding site of KP and KAP genes at its promoter regions in mouse. So the expression of KAP9.2 and Hoxc13 genes was detected at anagen stage vs telogen stage by qRT-PCR. The data showed that KAP9.2 and Hoxc13 gene had similar expression trend at different stages, which indicated that there was interaction between them. KAP9.2 and Hoxc13 gene had lower expression level in anagen than that of in telogen of cashmere growth. In anagen, KAP9.2 and Hoxc13 expressed lower in high cashmere yield individuals than that of in low cashmere yield ones. In telogen, the result was reverse. The study would provide the evidence of involvement of KAP9.2 and Hoxc13 in hair periodic growth.     

Hoxc12 expression pattern in developing and cycling murine hair follicles

We examine the Hoxc12 RNA expression pattern during both hair follicle morphogenesis and cycling in direct comparison to its only upstream neighbor, Hoxc13. Expression of both genes is restricted to the epidermal part of the follicle excluding the outer root sheath and interfollicular epidermis in a distinct stage-dependent and cyclical manner. During the progressive growth phase (anagen) of developing and cycling follicles, the distinct proximo-distal expression domain of Hoxc12 overlaps only proximally, at the upper-most region of the bulb, with the more proximally restricted Hoxc13 domain. This arrangement of the expression domains of the two genes along the proximal-toward-distal axis of increasing follicular differentiation correlates with the sequential expression of first Hoxc13 and then Hoxc12. This indicates a reversal of the typical temporal colinearity of Hox gene activation otherwise observed along the anterior-posterior morphogenetic axis of the embryo (review: Cell 78 (1994) 191).     

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.     

Cyclopamine induces digit loss in regenerating axolotl limbs

Axolotls, with their extensive ability to regenerate as adults, provide a useful model for studying the mechanisms of regeneration in a vertebrate, in hopes of understanding why other vertebrates cannot regenerate. Although the expression of many genes has been described in regeneration, techniques for gain and loss of function analyses have been limited. We demonstrated in a previous study that gain of function for secreted proteins was possible in the axolotl using the vaccinia virus to drive expression of the transgene. In this study, we used a pharmacological approach made possible by the existence of a naturally occurring compound that specifically blocks shh signaling, cyclopamine. The treatment of axolotls with cyclopamine during the process of limb regeneration caused a loss of digits similar to that described for the shh knockout mouse. Our results further demonstrate that shh signaling and function are conserved during limb regeneration in urodeles as in limb development in other vertebrates.     

Expression ofHoxDGenes in Developing and Regenerating Axolotl Limbs

Hoxgenes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior–posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns ofHoxDgenes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary ofHoxd-11expression is conserved with respect to morphological landmarks, but there is no anterior–distal expansion of the posterior domain ofHoxd-11expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5′HoxDgenes are reexpressed during limb regeneration. The change in the expression pattern ofHoxd-11during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. BothHoxd-8andHoxd-10are expressed in non-regenerating wounds, butHoxd-11is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.     

Proteomic analysis of blastema formation in regenerating axolotl limbs

Background

For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.

Results

Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.

Conclusion

This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.     

Myoblasts are aligned with collagen fibrils in regenerating frog tadpole tails

Myoblasts in the regenerating frog tadpole tail differentiate from mesen chymal cells that lie next to the basement membrane of the epidermis of the tail. As these cells elongate and form myotubes, they orientate uniformly in the longitudinal axis of the tail. The collagen fibrils of the basement membrane adjacent to the myogenic cells are also orientated in the tail axis just prior to and during the time when the myogenic cells are elongating. This has been demonstrated by transmission electron microscopy of thin sections, by differential interference contrast microscopy of isolated basement membranes, and by scanning electron microscopy of the inner surface of the basement membrane. Since elongating myoblasts are in contact with the longitudinally orientated fibrils, the latter could provide directional cues to the elongating myoblasts. This proposition is supported by the finding that isolated basement membranes readily orientate cells that are cultured upon their inner surfaces.     

Expression of neurotrophins and Trk receptors in the developing,adult, and regenerating avian cochlea

We studied the expression of neurotrophins and their Trk receptors in the chicken cochlea. Based on in situ hybridization, brain-derived neurotrophic factor (BDNF) is the major neurotrophin there, in contrast to the mammalian cochlea, where neurotrophin-3 (NT-3) predominates. NT-3 mRNA labeling was weak and found only during a short time period in the early cochles. During embryogenesis, BDNF mRNA was first seen in early differentiating hair cells. Afferent cochlear neurons expressed trkB mRNA from the early stages of gangliogenesis onward. In accordance, in vitro, BDNF promoted survival of dissociated neurons and stimulated neuritogenesis from ganglionic explants. High levels of BDNF mRNA in hair cells and trkB mRNA in cochlear neurons persisted in the mature cochlea. In addition, mRNA for the truncated TrkB receptor was expressed in nonneuronal cells, specifically in supporting cells, located adjacent to the site of BDNF synthesis and nerve endings. Following acoustic trauma, regenerated hair cells acquired BDNF mRNA expression at early stages of differentiation. Truncated trkB mRNA was lost from supporting cells that regenerated into hair cells. High levels of BDNF mRNA persisted in surviving hair cells and trkB mRNA in cochlear neurons after noise exposure. These results suggest that in the avian cochlea, peripheral target-derived BDNF contributes to the onset and maintenance of hearing function by supporting neuronal survival and regulating the (re)innervation process. Truncated TrkB receptors may regulate the BDNF concentration available to neurites, and they might have an important role during reinnervation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 1019–1033, 1997     

Hoxd13 expression in the developing limbs of the short-tailed fruit bat, Carollia perspicillata

Bat forelimbs are highly specialized for sustained flight, providing a unique model to explore the genetic programs that regulate vertebrate limb diversity. Hoxd9-13 genes are important regulators of stylopodium, zeugopodium, and autopodium development and thus evolutionary changes in their expression profiles and biochemical activities may contribute to divergent limb morphologies in vertebrates. We have isolated the genomic region that includes Hoxd12 and Hoxd13 from Carollia perspicillata, the short-tailed fruit bat. The bat Hoxd13 gene encodes a protein that shares 95% identity with human and mouse HOXD13. The expression pattern of bat Hoxd13 mRNA during limb development was compared with that of mouse. In bat and mouse hindlimbs, the expression patterns of Hoxd13 are relatively similar. However, although the forelimb Hoxd13 expression patterns in both organisms during early limb bud stages are similar, at later stages they diverge; the anterior expression boundary of bat Hoxd13 is posterior-shifted relative to the mouse. These findings, compared with the Hoxd13 expression profiles of other vertebrates, suggest that divergent Hoxd13 expression patterns may contribute to limb morphological variation.     

Retinoic-acid-induced pattern completion in regenerating double anterior limbs of urodeles

The effects of retinoic acid on the regeneration of double anterior lower arms in the adult newt, Notophthalmus viridescens, were investigated. Normally, double anterior lower arms regenerate a hypomorphic, symmetrical pattern of structures, which are distally complete; and double anterior upper arms regenerate a hypomorphic, symmetrical but distally incomplete pattern of structures. In limbs with a normal anteroposterior axis, the major effect of retinoic acid is to alter the proximodistal (PD) positional value of cells local at the amputation level to a much more proximal value, thereby creating duplications in the regenerate of structures proximal to the amputation plane (Thoms and Stocum, '84). Therefore, we predicted that double anterior lower arms treated with retinoic acid would regenerate like double anterior upper arms. However, in a substantial number of cases, each half of these double anterior lower arms regenerated a limb that was complete in the anteroposterior (AP) axis, with asymmetry corresponding to the half of origin. In addition, these regenerates were serially duplicated in the PD axis. These results indicate that retinoic acid can posteriorize the positional value of midline cells, leading to restoration of normal AP pattern, when the set of posterior-half positional values is removed from the cross section of the limb.