Expression of 18-wheeler in the follicle cell epithelium affects cell migration and egg morphology in Drosophila.

The Drosophila ovary is a model system for examining the genetic control of epithelial morphogenesis. The somatic follicle cells form a polarized epithelium surrounding the 16-cell germ line cyst. The integrity of this epithelium is essential for the successful completion of oogenesis. Reciprocal signaling between germ line and somatic cells establishes embryonic and eggshell polarity. The follicle cells are responsible for shaping the egg and secreting the eggshell. Follicle cells at the boundary between the nurse cells and the oocyte migrate centripetally to cover the anterior end of the oocyte and secrete the operculum. Dorsal anterior main body follicle cells undergo elaborate patterning to produce the dorsal appendages. We have examined the expression of the Toll-like receptor, 18-wheeler (18w), in the ovary and find it to be restricted to subpopulations of follicle cells. Females carrying loss-of-function 18w mutant clones in their ovaries show delayed follicle cell migrations. The eggs laid by such females also show morphological defects in egg shape and dorsal appendage morphology. We propose that the 18W protein plays an adhesive or signaling role in regions of the epithelium engaged in cell migration.     

Shh maintains dermal papilla identity and hair morphogenesis via a Noggin-Shh regulatory loop

During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh(-/-) skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh-Noggin signaling loop to drive hair follicle morphogenesis.     

Cellular mechanisms of neural fold formation and morphogenesis in the chick embryo

The mechanisms underlying neural fold formation and morphogenesis are complex, and how these processes occur is not well understood. Although both intrinsic forces (i.e., generated by the neuroepithelium) and extrinsic forces (i.e., generated by non-neuroepithelial tissues) are known to be important in these processes, the series of events that occur at the neural ectoderm-epidermal ectoderm (NE-EE) transition zone, resulting in the formation of two epithelial layers from one, have not been fully elucidated. Moreover, the region-specific differences that exist in neural fold formation and morphogenesis along the rostrocaudal extent of the neuraxis have not been systematically characterized. In this study, we map the rostrocaudal movements of cells that contribute to the neural folds at three distinct brain and spinal cord levels by following groups of dye-labeled cells over time. In addition, we examine the morphology of the neural folds at the NE-EE transition zone at closely-spaced temporal intervals for comparable populations of neural-fold cells at each of the three levels. Finally, we track the lateral-to-medial displacements that occur in the epidermal ectoderm during neural groove closure. The results demonstrate that neural fold formation and morphogenesis consist of a series of processes comprising convergent-extension movements, as well as epithelial ridging, kinking, delamination, and apposition at the NE-EE transition zone. Regional differences along the length of the neuraxis in the respective roles of these processes are described.     

Differential adherence of epithelium overlying gut-associated lymphoid tissue. An ultrastructural study

The relative adherence of follicle associated and nonfollicle associated epithelial cells in intestinal lymphoid tissues was compared morphologically. Incubation of Peyer's patches and appendix with hyaluronidase resulted in detachment of M cells and other epithelial cells overlying lymphoid follicle surfaces but not of villus or colonic epithelial cells. Enzymatic treatment of intestinal lymphoid tissues produced superficial erosions of follicle epithelium which exposed a porous and fenestrated basal lamina. After enzymatic treatment, detached M cell-containing follicle epithelium was characterized by intracellular vacuoles, widening of intercellular spaces, cell rounding, disappearance of microvilli, and loss of M cell-lymphocyte associations. Enzymatic treatment was responsible for removal of follicle epithelium, since Peyer's patches and appendix tissues incubated with medium alone did not exhibit appreciable epithelial detachment. These results, showing that the adherence of epithelial cells overlying intestinal lymphoid follicles is more labile than that of villus and colonic epithelial cells, may be of pathophysiologic significance in follicle ulcerative processes.     

Relevance of apoptosis in the female reproductive system

Apoptosis is a genetically controlled form of cell suicide. Due to the cyclic nature of the female reproductive system, the ovary, the endometrium and the mammary gland sustain continuous cycles of cell growth and apoptosis in response to hormonal changes. Apoptotic cell death plays multiple roles during embryonic and organ development. It is involved in sculpturing tissues and serves to delete structures that are no longer required. It is clear that apoptosis plays an active and important role in ovarian physiological functions. Apoptosis plays a major role during folliculogenesis and dominant follicle selection and also plays part in corpus luteum regression. In addition, it has been shown that programmed cell death plays important roles in the mammary gland development and ductal morphogenesis. During puberty, lumen formation is associated with the selective apoptosis of centrally located cells. In turn, postlactational involution of the mammary gland is characterized by the secretory epithelial cells undergoing programmed cell death. Apoptosis has also been associated with physiological, as well as pathological, endometrial processes such as cancer and endometriosis. The delicate balance between apoptosis and cell proliferation is essential in controlling the cyclical growth of the reproductive tissues and plays an important role in the prevention of neoplastic transformation.     

Neuroglian stabilizes epithelial structure during Drosophila oogenesis.

The vertebrate L1 family of cell adhesion molecules (CAMs) and their fly homolog, Neuroglian, are members of the immunoglobulin (Ig) superfamily of CAMs. In general, Ig CAMs have been found to play critical roles in mediating axon guidance. One Ig CAM, NCAM, has also been implicated in maintaining epithelial integrity and suppressing metastatic dissemination of tumor cells. Other Ig CAMs, such as Nrg, are also expressed in epithelia. We thus tested the hypothesis that, like NCAM, Nrg might also be required for maintaining epithelial integrity and for inhibiting tumor invasion. We used the Drosophila follicular epithelium to determine the function of Nrg in vivo in maintaining epithelial structure, and in regulating the motility of migrating border cells and invasive tumorous follicle cells. Nrg(167) is expressed on the lateral membrane of follicle cells. Loss of Nrg(167) causes border cells to delay delamination and causes other follicle cells to delaminate inappropriately. The delaminated cells have aberrant epithelial polarity manifested as severe mislocalization of apical and basal membrane proteins, and uniform localization of lateral membrane proteins. Furthermore, loss of Nrg(167) dramatically enhances the invasive phenotype associated with loss of Discs Large, a neoplastic tumor suppressor. These results indicate that Nrg(167) stabilizes epithelial polarity by regulating junctional adhesion and function in normal and tumorous epithelia. Our data also suggest that Ig superfamily members have significant functional redundancy in maintaining epithelial polarity, with individual members playing subtle, unique roles during epithelial morphogenesis.     

A contribution to the study of the histogenesis of the bursal lymphoid follicles in Gallus domesticus

Numerous investigators have shown that the development of the bursal lymphoid follicle in chicken begins with the formation of epithelial gemmations called epithelial buds. The mesenchymal stem cells migrate into these epithelial buds and give rise to the lymphoid cells of the follicle medulla. However, research carried out in our laboratory, using the semi-thin serial section technique and the electron microscope, leads us to think that the early stages of the development of the bursal lymphoid follicle might take place in the following way: the stem cells migrate towards the proximal surface of the lining epithelium where they collect in an invagination. These cells gradually increase in number and a cellular mass is formed which, therefore, does not grow in the epithelial framework but under it. The epithelial invagination near the mesenchymal mass becomes more and more pronounced, making the epithelium progressively thinner until it is finally necrotized. At this point, the cells of the mesenchymal anlage protrude into the bursal lumen.     

Epithelial-mesenchymal interactions are linked to neovascularization

Lung morphogenesis is dependent on interactions between mesenchymal and epithelial cells. We have previously demonstrated that inhibition of neovascularization by endothelial monocyte-activating polypeptide (EMAP) II also attenuates fetal lung morphogenesis in vivo, and hypothesized that epithelial-mesenchymal interactions are regulated by vascular signals. To address this postulate, we evaluated the formation of epithelial cysts in vitro and assessed this complex interaction through: (i) identification of vascular formation in vitro; (ii) assessment of the effect of selective vascular inhibition on cell viability, proliferation, and cellular interactions as measured by epithelial cyst formation; and (iii) examination of whether there is an interdependent relationship between epithelial and mesenchymal cells and a vascular mediator's protein expression. Vascular networks in vitro formed in direct relationship to the presence of epithelial cysts. Disruption of the vasculature by delivery of a selective antiangiogenic protein EMAP II was associated with disruption of epithelial cyst formation. Lastly, control of the vascular formation regulatory protein EMAP II is a direct result of epithelial-mesenchymal cell interactions. These findings suggest that vascular formation modulates and is modulated by the normal cellular communication and interactions that direct lung morphology.     

Pathways and Fate of Migratory Cells During Late Tooth Organogenesis

Tissue recombination experiments and cell lineage analyses of the developing neural crest have documented the role and central pathways of migratory cells during early craniofacial development. In the present study, regional pathways of cells during late peripheral morphogenesis were investigated using the crown stage tooth organ as a model. Homing targets during tooth integument formation were analyzed to understand the fate of migratory cells involved in late tooth organogenesis and the developmental origin of periodontal tissues. After surgical removal of the oral mucosa, the oral aspect of the dental follicle of lower first mouse molar teeth was labeled using a fluorescent contact dye. Following sacrifice after 0, 2, 4, and 6 days, labeled cells were detected in the dental follicle, in the alveolar bone, and in the periodontal ligament adjacent to the molar root. The distribution of labeled tissues was reconstructed three-dimensionally via confocal microscopy. Using a tooth molar organ culture system, labeled cells within the dental follicle were documented traveling in the apical direction. Our results indicated that cell migration during tooth organogenesis was following specific pathways and that cells within the circumference of the dental follicle were migrating in the apical direction. We speculate that migratory cells passing through the dental follicle connective tissue may contribute to the formation of the periodontium. The present documentation visualizes pathways, role, and dynamics of extensive cell movements during late tooth organogenesis.     

Midkine expression correlating with growth activity and tooth morphogenesis in odontogenic tumors

Midkine (MK; a low molecular weight heparin-binding growth factor) is a multifunctional cytokine. MK plays a role in morphogenesis of many organs including teeth through epithelial-mesenchymal interactions. We immunohistochemically examined MK expression in various human odontogenic tumors. There was no difference in positive rate and intensity of MK between benign odontogenic tumors and their malignant counterparts. Ameloblastoma showed MK localization in the peripheral columnar cells in budding processes from the parenchyma, which frequently expressed proliferating cell nuclear antigen. MK was also preferentially expressed in keratinized cells in acanthomatous ameloblastoma and keratocystic odontogenic tumor. In odontogenic mixed tumors except for odontoma, intense immunoreactivity to MK was found in epithelial follicles, the surrounding odontogenic ectomesenchymal tissue, and the basement membrane between them. Intensity in the odontogenic ectomesenchyme decreased in relation to distance from the epithelial follicles. No expression was found in tumor cells associated with production of dental hard tissues in odontogenic mixed tumors including odontoma. These findings suggested that MK is involved in the reciprocal interaction between odontogenic epithelium and odontogenic ectomesenchymal tissue in areas without dental hard tissue formation in odontogenic mixed tumors. Coexpression of MK and proliferating cell nuclear antigen was also observed in epithelial follicles and highly cellular nodules in the ectomesenchyme of odontogenic mixed tumors. MK is considered to mediate growth activity of odontogenic tumors and cell differentiation of odontogenic mixed tumors through molecular mechanisms similar to those involved in morphogenesis of the tooth.     

Pathways and fate of migratory cells during late tooth organogenesis

Tissue recombination experiments and cell lineage analyses of the developing neural crest have documented the role and central pathways of migratory cells during early craniofacial development. In the present study, regional pathways of cells during late peripheral morphogenesis were investigated using the crown stage tooth organ as a model. Homing targets during tooth integument formation were analyzed to understand the fate of migratory cells involved in late tooth organogenesis and the developmental origin of periodontal tissues. After surgical removal of the oral mucosa, the oral aspect of the dental follicle of lower first mouse molar teeth was labeled using a fluorescent contact dye. Following sacrifice after 0, 2, 4, and 6 days, labeled cells were detected in the dental follicle, in the alveolar bone, and in the periodontal ligament adjacent to the molar root. The distribution of labeled tissues was reconstructed three-dimensionally via confocal microscopy. Using a tooth molar organ culture system, labeled cells within the dental follicle were documented traveling in the apical direction. Our results indicated that cell migration during tooth organogenesis was following specific pathways and that cells within the circumference of the dental follicle were migrating in the apical direction. We speculate that migratory cells passing through the dental follicle connective tissue may contribute to the formation of the periodontium. The present documentation visualizes pathways, role, and dynamics of extensive cell movements during late tooth organogenesis.     

Cell polarity and spindle orientation in the distal epithelium of embryonic lung

A proper balance between self‐renewal and differentiation of lung‐specific progenitors at the distal epithelial tips is absolutely required for normal lung morphogenesis. Cell polarity and mitotic spindle orientation play a critical role in the self‐renewal/differentiation of epithelial cells and can impact normal physiological processes, including epithelial tissue branching and differentiation. Therefore, understanding the behavior of lung distal epithelial progenitors could identify innovative solutions to restoring normal lung morphogenesis. Yet little is known about cell polarity, spindle orientation, and segregation of cell fate determinant in the embryonic lung epithelium, which contains progenitor cells. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized and highly mitotic with characteristic perpendicular cell divisions. Consistent with these findings, mInsc, LGN, and NuMA polarity proteins, which control spindle orientation, are asymmetrically localized in mitotic distal epithelial progenitors of embryonic lungs. Furthermore, the cell fate determinant Numb is asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. These findings provide evidence for polarity in distal epithelial progenitors of embryonic lungs and provide a framework for future translationally oriented studies in this area. Developmental Dynamics 240:441–445, 2011. © 2011 Wiley‐Liss, Inc.     

Cyclical changes in epithelial cells of the vaginal cul-de-sac of brushtail possums (Trichosurus vulpecula)

The aim of this study was to describe and quantify the changes that occur in cul-de-sac tissue, in particular to epithelial cells and their constituents, at specific stages of the estrous cycle in the brushtail possum. Stereological techniques were used to quantify changes in cul-de-sac epithelial cells collected at four stages of the estrous cycle; the time of removal of pouch young (RPY; n = 5), of initial follicle development (n = 5), of preovulatory follicle formation (n = 5), of midluteal stage (n = 4), and again at RPY (n = 5) after completion of the experiment to examine for any effects due to season or time. Tissue was weighed and processed for light microscopy, transmission electron microscopy, and stereological analysis. Cul-de-sac epithelial cell volume increased approximately 17-fold at the time of preovulatory follicle formation compared with that at the time of RPY, before declining (approximately four-fold greater than at RPY) during the midluteal phase. Epithelial cell volume enlargement was correlated strongly with the size of the preovulatory follicle present, and maximum size was coincidental with the formation of extracellular spaces and projection of cell processes between lateral cell membranes. Maximum cell volume was associated with an approximate 25-fold and six-fold increase in cytoplasmic and nuclear volume, respectively. Enlargement of the epithelial cells coincided with an increase in cytoplasmic organelle numbers, microvilli prominence, and accumulation of secretory vesicles. In the possum, the cul-de-sac epithelial cell undergoes phenomenal remodelling during the estrous cycle to accommodate an approximate 17-fold increase in volume. This increase in cell volume is coincident with morphological changes characteristic of secretory activity and appears to be under estrogen regulation.     

The cellular basis of the convergence and extension of the Xenopus neural plate.

There is great interest in the patterning and morphogenesis of the vertebrate nervous system, but the morphogenetic movements involved in early neural development and their underlying cellular mechanisms are poorly understood. This paper describes the cellular basis of the early neural morphogenesis of Xenopus laevis. The results have important implications for neural induction. Mapping the fate map of the midneurula (Eagleson and Harris: J. Neurobiol. 21:427-440, 1990) back to the early gastrula with time-lapse video recording demonstrates that the prospective hindbrain and spinal cord are initially very wide and very short, and thus at the beginning of gastrulation all their precursor cells lie within a few cell diameters of the inducing mesoderm. In the midgastrula, the prospective hindbrain and spinal cord undergo very strong convergence and extension movements in two phases: In the first phase they primarily undergo thinning in the radial direction and lengthening (extension) in the animal-vegetal direction, and the second phase is characterized primarily by mediolateral narrowing (convergence) and anterior-posterior lengthening (extension). These movements also occur in sandwich explants of the gastrula, thus demonstrating the local autonomy of the forces producing them. Tracing cell movements with fluorescein dextran-labeled cells in embryos or explants shows that the initial thinning and extension occurs by radial intercalation of deep cells to form fewer layers of greater area, all of which is expressed as increased length. The subsequent convergence and extension occurs by mediolateral intercalation of deep cells to form a longer, narrower array. These results establish that a similar if not identical sequence of radial and mediolateral cell intercalations underlie convergence and extension of the neural and the mesoderm tissues (Wilson and Keller: Development, 112:289-300, 1991). Moreover, these results establish that radial and mediolateral intercalation are the principal neural cell behaviors induced by the planar signals emanating from the dorsal involuting marginal zone (the Spemann organizer) in the early gastrula (Keller et al: Develop. Dynamics, 193: 218-234, 1992). Radial and mediolateral intercalation are induced among the 5 to 7 rows of cells comprising the prospective hindbrain and spinal cord, thus producing the massive convergence and extension movements that narrow and elongate these regions of the nervous system in the late gastrula. A more general significance of these results is that neural induction is best analyzed and understood in terms of the dynamics of the morphogenetic processes involved.     

Reappraisal of histogenesis in the bursal lymphoid follicle of the chicken

The development of the bursal follicle and the appearance of the follicle-associated epithelial (FAE) cell and the reticuloepithelial (REp) cell were studied. The stadied. The stages of development of the bursal follicle were observed by light and electron microscopy; an anticytokeratin monoclonal antibody was also used. At the beginning of folicle development, a mesenchymal cell cluster is observed in the tunica propria; the cluster becomes wedged in a niche of the surface epithelium, and gradually it is completely surounded by the epithelium itself, which closes under the clump of mesenchymal cells. The epithelial cells lying upon the mesenchymal clump become necrotic, and anumber of mesenchymal cells bulge out, forming the FAE cells. The epithelial cells that hav closed under the mesenchymal nodule become stratified and form the REp cells; they become star-shaped because the mdedullarylymphoid cells grow between them. Finally, the cortex in formed, possibly as a result of the migration of medullary cells before they peripheralize. it is concluded that FAE cells are not specialized It is concluded that FAE cells are not specialized epithelial cells, as they do not react to an anticytokeratin monoclonal antibody; on the contrary, they are formed by mesenchymal stemcells that bulge into the lumen and change their character after moving into the epithelium. The REp cells appear in the follicular primordium shortly after the bursal follicle begins to develop; the pronounced reactivity of the REp cells to an anticytokeratin monoclonal antibody supports the hypothesis of their epithelial origin.     

Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.