Antibodies recognizing 20-hydroxyecdysone-dependent cell surface antigens during morphogenesis in Drosophila

Summary Polyclonal antibodies (anti-P116 and anti-P93) specific for two different hormone-dependent cell surface glycoproteins (P116 and P93) from Drosophila S3 cells have been produced. Anti-P116 and anti-P93 each immunoprecipitate substantially more of P116 and P93, respectively, from extracts of iodinated hormone-treated S3 cells compared to controls. Both antigens are present in control and 20-hydroxyecdysone treated imaginal discs, although apparent increases in antigen content are associated with hormone treatment. Immunofluorescent staining of whole discs with anti-P116 and anti-P93 reveals increased amounts of both antigens at the surface of hormone-treated discs compared to controls. Both antibodies were used to characterize the expression of their respective antigens during embryonic development, and both antibodies were found to recognize in embryos a third developmentally-regulated antigen with a relative mobility of approximately 220000. Our results indicate, at least in the case of P116 and P93, that 20-hydroxyecdysone-dependent cell surface antigens in imaginal discs may be regulated both by increasing the amounts of constitutively present proteins, and possibly through biochemical modifications, altering the localization of these proteins from a cytoplasmic to a cell surface domain.This research was supported by a grant from the National Science Foundation (PCM-84089255).     

Telocytes play a key role in prostate tissue organisation during the gland morphogenesis

Telocytes are CD34‐positive interstitial cells, known to exert several functions, one of which is a role in tissue organisation, previously demonstrated by telocytes in the myocardium. The existence of telocytes in the prostate has recently been reported, however, there is a lack of information regarding the function of these cells in prostate tissue, and information regarding the possible role of these cells in prostatic development. This study used immunofluorescence techniques in prostate tissue and prostatic telocytes in culture to determine the relationship between telocytes and prostate morphogenesis. Furthermore, immunofluorescent labelling of telocytes was performed on prostate tissue at different stages of early postnatal development. Initially, CD34‐positive cells are found at the periphery of the developing alveoli, later in the same region, c‐kit‐positive cells and cells positive for both factors are verified and CD34‐positive cells were predominantly observed in the interalveolar stroma and the region surrounding the periductal smooth muscle. Fluorescence assays also demonstrated that telocytes secrete TGF‐β1 and are ER‐Beta (ERβ) positive. The results suggest that telocytes play a changing role during development, initially supporting the differentiation of periductal and perialveolar smooth muscle, and later, producing dense networks that separate alveoli groups and form a barrier between the interalveolar region and periurethral smooth muscle. We conclude that telocytes play a relevant role in prostate tissue organisation during postnatal development.     

Overexpression of Sonic Hedgehog suppresses embryonic hair follicle morphogenesis

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development, we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype.     

Keratan sulfate expression during avian craniofacial morphogenesis

Summary Using the monoclonal antibody MZ15 in immunocytochemical and ultrastructural studies we have been able to determine the spatiotemporal pattern of keratan sulfate (KS) distribution during quail craniofacial morphogenesis. KS-containing proteoglycans are found associated with invaginating placodes (olfactory, lens and otic), in developing pronephric tubules, notochord, pharynx and endocardium, and display developmental regulation. The appearance of such proteoglycans (PGs) during placode morphogenesis is particularly striking and we suggest that they may be an important component of the extracellular matrix which has been previously implicated in mediating the morphogenetic interactions and cell movements occurring at these sites. The otic vesicle during stage 18–22 displays a notable asymmetric distribution of KS-containing PGs. The role that these molecules may play and the reasons for this regionalization are, as yet, unclear but it is conceivable that the distribution of proteoglycans at this stage reflects subsequent differentiative events during otocyst development. Furthermore, our ultrastructural observations indicate that over the developmental period studied (H & H stages 8–22) keratan sulfate exists in at least two proteoglycan forms. Some spatiotemporal correlation has been found to exist between the distributions of KS-containing PGs and type II collagen as previously reported by Thorogood et al. (1986). We suggest that the proteoglycan detected at such sites is cartilage-specific proteoglycan and that it plays an important role, together with type II collagen, in the “signalling” mechanism which specifies the subsequent pattern of the chondrocranium. It is proposed that this interaction at epithelio-mesenchymal interfaces in the developing head parallels the matrix-mediated tissue interaction between notochord and somites which results in the formation of the cartilaginous primordia of the vertebrae from the sclerotomes as reported by Lash and Vasan (1978).     

Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophila development

Background  

The Rho-kinases (ROCKs) are major effector targets of the activated Rho GTPase that have been implicated in many of the Rho-mediated effects on cell shape and movement via their ability to affect acto-myosin contractility. The role of ROCKs in cell shape change and motility suggests a potentially important role for Rho-ROCK signaling in tissue morphogenesis during development. Indeed, in Drosophila, a single ROCK ortholog, DRok, has been identified and has been found to be required for establishing planar cell polarity.     

Ephrin-Eph signaling in embryonic tissue separation

The physical separation of the embryonic regions that give rise to the tissues and organs of multicellular organisms is a fundamental aspect of morphogenesis. Pioneer experiments by Holtfreter had shown that embryonic cells can sort based on “tissue affinities,” which have long been considered to rely on differences in cell-cell adhesion. However, vertebrate embryonic tissues also express a variety of cell surface cues, in particular ephrins and Eph receptors, and there is now firm evidence that these molecules are systematically used to induce local repulsion at contacts between different cell types, efficiently preventing mixing of adjacent cell populations.     

Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB

Vertebrate neural tube formation involves two distinct morphogenetic events — convergent extension (CE) driven by mediolateral cell intercalation, and bending of the neural plate driven largely by cellular apical constriction. However, the cellular and molecular biomechanics of these processes are not understood. Here, using tissue-targeting techniques, we show that the myosin IIB motor protein complex is essential for both these processes, as well as for conferring resistance to deformation to the neural plate tissue. We show that myosin IIB is required for actin-cytoskeletal organization in both superficial and deep layers of the Xenopus neural plate. In the superficial layer, myosin IIB is needed for apical actin accumulation, which underlies constriction of the neuroepithelial cells, and that ultimately drive neural plate bending, whereas in the deep neural cells myosin IIB organizes a cortical actin cytoskeleton, which we describe for the first time, and that is necessary for both normal neural cell cortical tension and shape and for autonomous CE of the neural tissue. We also show that myosin IIB is required for resistance to deformation (“stiffness”) in the neural plate, indicating that the cytoskeleton-organizing roles of this protein translate in regulation of the biomechanical properties of the neural plate at the tissue-level.     

CYFIP dependent actin remodeling controls specific aspects of Drosophila eye morphogenesis

Cell rearrangements shape organs and organisms using molecular pathways and cellular processes that are still poorly understood. Here we investigate the role of the Actin cytoskeleton in the formation of the Drosophila compound eye, which requires extensive remodeling and coordination between different cell types. We show that CYFIP/Sra-1, a member of the WAVE/SCAR complex and regulator of Actin remodeling, controls specific aspects of eye architecture: rhabdomere extension, rhabdomere terminal web organization, adherens junctions, retina depth and basement membrane integrity. We demonstrate that some phenotypes manifest independently, due to defects in different cell types. Mutations in WAVE/SCAR and in ARP2/3 complex subunits but not in WASP, another major regulator of Actin nucleation, phenocopy CYFIP defects. Thus, the CYFIP-SCAR-ARP2/3 pathway orchestrates specific tissue remodeling processes.     

The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis

Ventral enclosure in Caenorhabditis elegans involves migration of epidermal cells over a neuroblast substrate and subsequent adhesion at the ventral midline. Organisation of the neuroblast layer by ephrins and their receptors is essential for this migration. We show that bus-8, which encodes a predicted glycosyltransferase, is essential for embryonic enclosure and acts in or with ephrin signalling to mediate neuroblast organisation and to permit epidermal migration. BUS-8 acts non-cell-autonomously in this process, and likely modifies an extracellular regulator of ephrin signalling and cell organisation. Weak and cold-sensitive alleles of bus-8 show that the gene has a separate and distinct post-embryonic role, being essential for epidermal integrity and production of the cuticle surface. This disorganisation of the epidermis and cuticle layers causes increased drug sensitivity, which could aid the growing use of C. elegans in drug screening and chemical genomics. The viable mutants are also resistant to infection by the pathogen Microbacterium nematophilum, due to failure of the bacterium to bind to the host surface. The two separate essential roles of BUS-8 in epidermal morphogenesis add to our growing understanding of the widespread importance of glycobiology in development.     

Appendage morphogenesis in Drosophila: a developmental study of the rotund (rn) gene

Summary The phenotype of rotund (rn) null alleles is described, and compared to wild type. The mutants are expressed zygotically and cause position specific defects in certain imaginal discs (antenna, legs, wing, haltere and proboscis) and their corresponding adult derivatives. In the discs, specific folds are absent in rn mutants compared to wild type. Clonal analysis shows that the rn ⁺ gene is partially autonomous in its expression in cells destined to form certain distal parts of the adult appendages. The results are consistent with the idea that the rn ⁺ gene is required for normal morphogenesis of specific distal parts of the adult appendages.     

The atypical Rho GTPase,RhoU, regulates cell-adhesion molecules during cardiac morphogenesis

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.     

Extracellular matrix control of mammary gland morphogenesis and tumorigenesis: insights from imaging

The extracellular matrix (ECM), once thought to solely provide physical support to a tissue, is a key component of a cell’s microenvironment responsible for directing cell fate and maintaining tissue specificity. It stands to reason, then, that changes in the ECM itself or in how signals from the ECM are presented to or interpreted by cells can disrupt tissue organization; the latter is a necessary step for malignant progression. In this review, we elaborate on this concept using the mammary gland as an example. We describe how the ECM directs mammary gland formation and function, and discuss how a cell’s inability to interpret these signals—whether as a result of genetic insults or physicochemical alterations in the ECM—disorganizes the gland and promotes malignancy. By restoring context and forcing cells to properly interpret these native signals, aberrant behavior can be quelled and organization re-established. Traditional imaging approaches have been a key complement to the standard biochemical, molecular, and cell biology approaches used in these studies. Utilizing imaging modalities with enhanced spatial resolution in live tissues may uncover additional means by which the ECM regulates tissue structure, on different length scales, through its pericellular organization (short-scale) and by biasing morphogenic and morphostatic gradients (long-scale). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.     

Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs

The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. We have reexamined the evagination of both the leg and wing discs and find that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreased as the tissue extended during evagination and we observed cell rearrangement to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. Our observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes.     

Endocytic and recycling endosomes modulate cell shape changes and tissue behaviour during morphogenesis in Drosophila

During development tissue deformations are essential for the generation of organs and to provide the final form of an organism. These deformations rely on the coordination of individual cell behaviours which have their origin in the modulation of subcellular activities. Here we explore the role endocytosis and recycling on tissue deformations that occur during dorsal closure of the Drosophila embryo. During this process the AS contracts and the epidermis elongates in a coordinated fashion, leading to the closure of a discontinuity in the dorsal epidermis of the Drosophila embryo. We used dominant negative forms of Rab5 and Rab11 to monitor the impact on tissue morphogenesis of altering endocytosis and recycling at the level of single cells. We found different requirements for endocytosis (Rab5) and recycling (Rab11) in dorsal closure, furthermore we found that the two processes are differentially used in the two tissues. Endocytosis is required in the AS to remove membrane during apical constriction, but is not essential in the epidermis. Recycling is required in the AS at early stages and in the epidermis for cell elongation, suggesting a role in membrane addition during these processes. We propose that the modulation of the balance between endocytosis and recycling can regulate cellular morphology and tissue deformations during morphogenesis.     

The role of the peripodial membrane in the morphogenesis of the eye-antennal disc ofDrosophila melanogaster

Summary The early morphogenesis of the eye-antennal disc ofDrosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial membrane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.