Expression of cell-adhesion molecules in embryonic induction. II. Morphogenesis of adult feathers

The developmental appearance of cell-adhesion molecules (CAMs) was mapped during the morphogenesis of the adult chicken feather. Neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), as well as substrate molecules (laminin and fibronectin), were compared in newborn chicken skin by immunohistochemical means. N-CAM was found to be enriched in the dermal papilla, which was closely apposed to L-CAM-positive papillar ectoderm. The two CAMs were then co-expressed in cells of the collar epithelium. Subsequently generated barb epithelia expressed only L-CAM, but N-CAM reappeared periodically on cells between developing barbs and barbules. N-CAM first appeared on a single L-CAM-positive basilar cell located in each valley flanked by two adjacent barb ridges. Subsequently, the expression of N-CAM extended one cell after another to include the whole basilar layer. N-CAM also appeared in the L-CAM-positive axial-plate epithelia, beginning in a single cell located at the ridge base. The two collectives of N-CAM-positive epithelia constituting the marginal and axial plates then disintegrated, leaving interdigitating spaces between keratinized structures that had previously expressed L-CAM. The morphological transformation from an epithelial cylinder to a three-level branched feather pattern is thus achieved by coupling alternating CAM expression in linked cell collectives with specific differentiation events, such as keratinization. During all of these morphogenetic processes, laminin and fibronectin formed a continuous basement membrane separating pulp from feather epithelia, and were excluded from the sites involved in periodic appearances of N-CAM. The same staining pattern described for developing chickens persisted in the feather follicles of adult chicken tissue that have gone through several cycles of molting. Cyclic expression of the two different CAMs underlies each of the different morphological events that are generated epigenetically during feather morphogenesis.     

Pattern formation in epithelial development: the vertebrate limb and feather bud spacing.

The ectoderm of the vertebrate limb and feather bud are epithelia that provide good models for epithelial patterning in vertebrate development. At the tip of chick and mouse limb buds is a thickening, the apical ectodermal ridge, which is essential for limb bud outgrowth. The signal from the ridge to the underlying mesoderm involves fibroblast growth factors. The non-ridge ectoderm specifies the dorsoventral pattern of the bud and Wnt7a is a dorsalizing signal. The development of the ridge involves an interaction between dorsal cells that express radical fringe and those that do not. There are striking similarities between the signals and genes involved in patterning the limb ectoderm and the epithelia of the Drosophila imaginal disc that gives rise to the wing. The spacing of feather buds involves signals from the epidermis to the underlying mesenchyme, which again include Wnt7a and fibroblast growth factors.     

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.     

Expression of Hex during feather bud development

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.     

Dynamic Lunatic fringe expression is correlated with boundaries formation in developing mouse teeth

The formation of boundaries is a fundamental organizing principle during development. The Notch signalling pathway regulates this developmental patterning mechanism in many tissues. Recent data suggest that Notch receptors are involved in boundary determination during odontogenesis. It remains, however, uncertain if other components of the Notch pathway are also important for compartmental lineage restrictions in teeth. Here we report on the expression of the Lunatic fringe gene, which encodes a secreted signalling molecule regulating the Notch pathway, during the development of mouse teeth. Lunatic fringe is expressed in both epithelial and mesenchymal components of the developing molar. The expression pattern of Lunatic fringe in the epithelium is complementary to that of the Notch receptors. Lunatic fringe is asymmetrically expressed in the incisor epithelium during its antero-posterior rotation. This expression pattern defines the lingual comportment of the incisor epithelium whereas the labial comportment is defined by Notch2 expression.     

Involvement of Hex in the initiation of feather morphogenesis

In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.     

Lunatic fringe null female mice are infertile due to defects in meiotic maturation

We have demonstrated that Notch genes are expressed in developing mammalian ovarian follicles. Lunatic fringe is an important regulator of Notch signaling. In this study, data are presented that demonstrate that radical fringe and lunatic fringe are expressed in the granulosa cells of developing follicles. Lunatic fringe null female mice were found to be infertile. Histological analysis of the lunatic fringe-deficient ovary demonstrated aberrant folliculogenesis. Furthermore, oocytes from these mutants did not complete meiotic maturation. This is a novel observation because this is the first report describing a meiotic defect that results from mutations in genes that are expressed in the somatic granulosa cells and not the oocytes. This represents a new role for the Notch signaling pathway and lunatic fringe in mammalian folliculogenesis.     

Feather buds exert a polarizing activity when transplanted to chick limb buds

Homeoproteins have been shown to be expressed in a position-specific manner along the anterior-posterior axis in the developing chick feather bud, as seen also in the developing limb bud. These facts raise the possibility that there may be common mechanistic features in the establishment of the anterior-posterior polarity between both organs. In order to investigate this possibility, feather bud tissues were transplanted into the anterior region of limb buds to determine whether feather bud tissues possess properties such as the zone of polarizing activity of the limb bud. The manipulated limb bud formed a mirror image duplication of the skeletal elements, mainly (2)2234 digit pattern or sometimes 3(2)234. Both the anterior and posterior halves of feather bud tissue exhibited almost equal activity in inducing ectopic skeletal elements. Hox d-12 and Hox a-13 were expressed coordinately around the transplanted site of the operated limb bud. This secondary axis-inducing activity of the feather bud was enhanced when grafts were pretreated with trypsin. In contrast, the presumptive feather bud tissue and inter-feather bud tissue did not induce a secondary axis of the limb bud. These results suggest that the feather bud contains a region that exerts polarizing activity and that this region may play key roles in the formation of the anterior-posterior and, if it exists, proximal-distal axis of the feather bud, possibly via the regulation of region specific expression of Hox genes.     

Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.     

From buds to follicles: matrix metalloproteinases in developmental tissue remodeling during feather morphogenesis

Organogenesis involves a series of dynamic morphogenesis and remodeling processes. Since feathers exhibit complex forms, we have been using the feather as a model to analyze how molecular pathways and cellular events are used. While several major molecular pathways have been studied, the roles of matrix degrading proteases and inhibitors in feather morphogenesis are unknown. Here we addressed this knowledge gap by studying the temporal and spatial expression of proteases and inhibitors in developing feathers using mammalian antibodies that cross react with chicken proteins. We also investigated the effect of protease inhibitors on feather development employing an in vitro feather bud culture system. The results show that antibodies specific for mammalian MMP2 and TIMP2 stained positive in both feather epithelium and mesenchyme. The staining co-localized in structures of E10-E13 developing feathers. Interestingly, MMP2 and TIMP2 exhibited a complementary staining pattern in developing E15 and E20 feathers and in maturing feather filaments. Although they exhibited a slight delay in feather bud development, similar patterns of MMP2 and TIMP2 staining were observed in in vitro culture explants. The broad spectrum pharmacological inhibitors AG3340 and BB103 (MMP inhibitors) but not Aprotinin (a plasmin inhibitor) showed a reversible effect on epithelium invagination and feather bud elongation. TIMP2, a physiological inhibitor to MMPs, exhibited a similar effect. Markers of feather morphogenesis showed that MMP activity was required for both epithelium invagination and mesenchymal cell proliferation. Inhibition of MMP activity led to an overall delay in the expression of molecules that regulate either early feather bud growth and/or differentiation and thereby produced abnormal buds with incomplete follicle formation. This work demonstrates that MMPs and their inhibitors are not only important in injury repair, but also in development tissue remodeling as demonstrated here for the formation of feather follicles.     

beta-catenin signaling can initiate feather bud development.

Intercellular signaling by a subset of Wnts is mediated by stabilization of cytoplasmic beta-catenin and its translocation to the nucleus. Immunolocalization of beta-catenin in developing chick skin reveals that this signaling pathway is active in a dynamic pattern from the earliest stages of feather bud development. Forced activation of this pathway by expression of a stabilized beta-catenin in the ectoderm results in the ectopic formation of feather buds. This construct is sufficient to induce bud formation since it does so both within presumptive feather tracts and in normally featherless regions where tract-specific signals are absent. It is also insensitive to the lateral inhibition that mediates the normal spacing of buds and can induce ectopic buds in interfollicular skin. However, additional patterning signals cooperate with this pathway to regulate gene expression within domains of stabilized beta-catenin expression. Localized activation of this pathway within the bud as it develops is required for normal morphogenesis and ectopic activation of the pathway leads to abnormally oriented buds and growths on the feather filaments. These results suggest that activation of the beta-catenin pathway initiates follicle development in embryonic skin and plays important roles in the subsequent morphogenesis of the bud.     

Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin.

To understand cell interactions during induction of skin appendages, we studied the roles of adhesion molecules N-CAM, tenascin, integrin, and fibronectin during feather development. Tenascin appeared in a periodic pattern on epithelia and was so far the earliest molecule detected in placodes. Three placode domains were identified: the anterior was positive for tenascin, the distal positive for N-CAM, and the posterior lacking both. Integrin appeared in dermal-epidermal junctions of placodes. In feather buds, sagittal sections revealed a transient anterior-posterior asymmetry with tenascin and N-CAM enriched in the anterior mesoderm. Tangential sections revealed a lateral-medial asymmetry with tenascin distributed in a ring shape and N-CAM in an "X" shape. Integrin was diffusely distributed within buds. Later tenascin and N-CAM were enriched in dermal papilla, the inducer of skin appendages. Perturbation of embryonic skin explant cultures with antibodies showed that anti-integrin beta 1 and anti-fibronectin blocked epithelial-mesenchymal interaction, anti-N-CAM caused uneven segregation of mesenchymal condensation, and anti-tenascin inhibited feather bud elongation. Dose-response curves showed gradual effects by these antibodies. The results indicated that these adhesion molecules are independently regulated and each contributes in different phases during morphogenesis of skin appendages.     

Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (beta-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1alpha) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (alpha-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium.     

Gradients of homeoproteins in developing feather buds

Homeoproteins are functionally involved in pattern formation. Recently, homeoproteins have been shown to be distributed in a graded fashion in developing limb buds. Here we examine the expression of homeoproteins in chicken feather development by immunocytochemical localization. We find that XlHbox 1 antigen is present in cell nuclei and is distributed in a gradient in the mesoderm of developing feather buds, with strongest expression in the anterior-proximal region. The gradient is most obvious in feather buds from the mid-trunk level. Feather buds from the scapular level express very high levels of XlHbox 1 and feather buds from the caudal region express no XlHbox 1, suggesting that a broad gradient along the body axis is superimposed on a smaller gradient within each individual feather bud. Feather ectoderm also expresses XlHbox 1 antigen but without an obvious graded pattern. Another homeoprotein, Hox 5.2, is also expressed in developing feather buds in a graded way, and its distribution pattern is partially complementary to that of XlHbox 1. These observations suggest that homeoproteins may be involved in setting up the anteroposterior polarity of cell fields at different levels, first for the body axis, then for the limb axis and finally for the feather axis.     

Expression of multiple delta-protocadherins during feather bud formation

The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.     

山鸡椒雄花花芽发育形态解剖特征观察

采用体视显微镜、扫描电镜和石蜡切片技术对山鸡椒（Litsea cubeba（Lour.） Pers.）雄花花芽分化发育的外部形态和内部解剖结构进行了观察研究。结果显示:（1）山鸡椒雄花花芽分化发生可分为5个时期,即未分化期、花序原基分化期、苞片原基分化期、花原基分化期和花器官分化期,其中花器官分化期又可细分为花被原基分化期、雄蕊原基分化期和雌蕊原基分化期;各相邻分化时期存在一定重叠现象;花期从翌年1月上旬至3月下旬。（2）雄花成熟结构中具有独特的雄蕊蜜腺,蜜腺绿色且形态不规则,着生于内轮雄蕊基部,分布于花丝两侧,夹在内外轮雄蕊的花丝之间,与内轮花丝紧密相连。（3）雄蕊花药四室,花药壁发育属于基本型;腺质绒毡层;小孢子母细胞减数分裂过程中胞质分裂属于连续型;成熟花粉为2-细胞花粉粒;成熟花粉粒外壁刺突较多,刺突基部膨大,外壁露出部分粗糙,无薄壁区,有少数小穿孔。（4）山鸡椒雄花中绝大多数雌蕊发育至腹缝线卷合形成子房室时停止,柱头发育不良或者败育,花柱缩短或缺失,不能受精,直到开花结束,即发生退化。本研究明确了山鸡椒雄花花芽发育发生各个阶段时间、形态变化特点及外部形态变化特征,山鸡椒小孢子发生、雄配子体发育至散粉期变化特点和规律以及雄花中退化雌蕊发育的进程,可为山鸡椒优良品种选育、调控花期和提高结实率提供一定的参考。     

穗花牡荆花芽分化过程中形态和生理指标变化

以穗花牡荆为研究材料,通过探究其花芽分化进程和生理特性,为花期调控技术提供成花机理。采用物候期观察和石蜡切片相结合的方法并测定花芽分化过程中相关生理指标,研究花发育过程中的形态和生理变化。结果表明,穗花牡荆花芽分化为一年多次分化型,其进程可划分为七个时期：未分化期、总轴花序原基分化期、初级分轴花序原基分化期、次级分轴花序原基分化期、小花原基分化期、花器官分化前期和花器官分化后期。同一植株不同位置花芽及同一花序中不同单花分化的进程不同,第一季花期后各阶段的花芽分化形态常存在重叠。花芽分化过程中不同时期叶片和花芽的可溶性糖和可溶性蛋白质含量均有上升下降的变化,总体上叶片中营养物质含量高于花芽保证营养供应。花芽分化过程中,IAA、ABA、CTK和GA3整体水平上先升后降有利于花芽分化进行。研究认为,花芽中大量的可溶性糖和蛋白质积累及较高的碳氮比,有利于穗花牡荆花芽形态分化顺利完成。低水平的GA3/ABA和IAA/CTK有利于花序的形成,ABA/CTK和ABA/IAA比值升高促进小花原基和小花萼片原基的分化, GA3/CTK、GA3/ABA和GA3/IAA比值升高促进花瓣原基、雄雌蕊原基发育。