The genital disc of Drosophila melanogaster

 The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development. Received: 4 February 1997 / Accepted: 22 May 1997     

Gradual acquisition of the developmental capacity to differentiate adult structures by the genital disc of Drosophila melanogaster

Summary Diplo-X flies homozygous for the transform-er-2 ^ts (tra-2 ^ts) mutation develop into females at 16° C, while they develop into males at 29° C (Belote and Baker 1982). By means of this conditional mutation, we have carried out a detailed analysis of the development of the genital disc. Temperature shifts between 16 and 29° C, in both directions, and temperature pulses at 29° C, have been applied during the larval growth of tra-2 ^ts homozygous diplo-X flies, and the external derivatives of the genital disc have been analysed. Genital discs shifted from 16 to 29° C rapidly lose their capacity to differentiate female genital structures, while they become able to differentiate male genital structures whose inventory is more complete the earlier in larval development the temperature shift is carried out; moreover, duplicated male genital structures were observed. In the shift from 29 to 16° C, the genital disc loses its capacity to differentiate male genital structures, while it becomes able to differentiate female genital structures. The inventory of male structures is smaller, and the inventory of the female structures is more complete, the earlier in larval development the temperature is shifted. No duplicated female or male genital structures were observed in the downshift experiment. With respect to the analia, the shift from 16 to 29° C resulted in the quick formation of pure male anal plates, while in the opposite shift the formation of pure female anal plates occurred gradually. Moreover, the time course for the dorsal and ventral anal plates to show normal female phenotype was different: when the dorsal anal plates were completely normal, it was still possible to find incomplete ventral anal plates. In the pulse experiment at 29° C, the genital disc is able to differentiate both female and male genital structures, although the inventory of the latter ones was not complete. In addition, the capacity of the genital disc to differentiate male genital structures depended on the duration of the temperature pulse. The anal plates were always female, although they showed a reduction in their size, the ventral female anal plate being more affected than the dorsal one. No male anal plates were observed. The results have revealed that the genital disc follows a sequence in its capacity to differentiate female or male adult structures. We suggest that this sequence reflects the sequence of determination events occurring in the genital disc during its larval growth. In addition, results shown here provide evidence for the existence in the female genital primordium of a set of cells capable of giving rise either to female genital structures (ventral vaginal plates) or to male genital structures (hypandrium and penis apparatus). We also present evidence supporting the previous idea of two primordia for the anal plates.     

Therapeutic effects of human STRO-3-selected mesenchymal precursor cells and their soluble factors in experimental myocardial ischemia

Stromal precursor antigen (STRO)-3 has previously been shown to identify a subset of adult human bone marrow (BM)-derived mesenchymal lineage precursors, which may have cardioprotective potential. We sought to characterize STRO-3-immunoselected and culture-expanded mesenchymal precursor cells (MPCs) with respect to their biology and therapeutic potential in myocardial ischemia. Immunoselection of STRO-3(+) MPCs enriched for fibroblastic colony forming units from unfractionated BM mononuclear cells (MNCs). Compared to mesenchymal stem cells conventionally isolated by plastic adherence, MPCs demonstrated increased proliferative capacity during culture expansion, expressed higher levels of early 'stem cell' markers and various pro-angiogenic and cardioprotective cytokines, and exhibited greater trilineage developmental efficiency. Intramyocardial injection of MPCs into a rat model of myocardial infarction (MI) promoted left ventricular recovery and inhibited left ventricular dilatation. These beneficial effects were associated with cardioprotective and pro-angiogenic effects at the tissue level, despite poor engraftment of cells. Treatment of MI rats with MPC-conditioned medium (CM) preserved left ventricular function and dimensions, reduced myocyte apoptosis and fibrosis, and augmented neovascularization, involving both resident vascular cells and circulating endothelial progenitor cells (EPCs). Profiling of CM revealed various cardioprotective and pro-angiogenic factors, which had biological activity in cultures of myocytes, tissue-resident vascular cells and EPCs. Prospective immunoselection of STRO-3(+) MPCs from BM MNCs conferred advantage in maintaining a population of immature MPCs during ex vivo expansion. Transplantation of culture-expanded MPCs into the post-MI heart resulted in therapeutic benefit, attributable at least in part to paracrine mechanisms of action. Thus, MPCs represent a promising therapy for myocardial ischemia.     

Spalt major controls the development of the notum and of wing hinge primordia of the Drosophila melanogaster wing imaginal disc

The Drosophila wing and the dorsal thorax develop from primordia within the wing imaginal disc. Here we show that spalt major (salm) is expressed within the presumptive dorsal body wall primordium early in wing disc development to specify notum and wing hinge tissue. Upon ectopic salm expression, dorsally located second leg disc cells develop notum and wing hinge tissue instead of sternopleural tissue. Similarly, by salm over-expression within the wing disc, wing blade formation is suppressed and a mirror-image duplication of the notum and wing hinge is formed. In large dorsal clones, which lack salm and its neighboring paralogue spalt related (salr), the cells of the notum primordium do not grow; these dorsal cells are not specified as notum, hence no notum outgrowth develops. These results suggest that the zinc finger factors encoded by the salm/salr complex play important roles in defining cells of the early wing disc as dorsal body wall cells, which develop into a large dorsal body wall territory and form mesonotum and some wing hinge tissue, and in delimiting the wing primordium. We also find that salm activity is down-regulated by its own product and by that of the Pax gene eyegone.     

Cell degeneration and elimination in the imaginal wing disc,caused by the mutationsvestigial andUltravestigial ofDrosophila melanogaster

Summary The mutationsvestigial (vg; recessive) andUltravestigial (vg ^U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg ^U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense (apoptosis). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg ^U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.     

The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages.     

Homeotic selector genes control the patterning of seven-up expressing cells in the Drosophila dorsal vessel

The linear cardiac tube of Drosophila, the dorsal vessel, is an important model organ for the study of cardiac specification and patterning in vertebrates. In Drosophila, the Hox segmentation gene abdominal-A (abd-A) is required for the specification of a functionally distinct heart region at the posterior of the dorsal vessel, from which blood is pumped anteriorly through a tube termed the aorta. Since we have previously shown that the posterior part of the aorta is specified during embryogenesis to form the adult heart during metamorphosis, we determined if the embryonic aorta is also patterned by the function of Hox segmentation genes. Using gain- and loss-of-function experiments, we demonstrate that the three Hox genes expressed in the posterior aorta and heart are sufficient to confer heart or posterior aorta fate throughout the dorsal vessel. Additionally, we demonstrate that Ultrabithorax and abd-A, but not Antennapedia, function to control cell number in the dorsal vessel. These studies add robustness to the model that homeotic selector genes pattern the Drosophila dorsal vessel, and further extend our understanding of how the cardiac tube is patterned in animal models.     

Isolation and characterization of mouse neural precursor cells in primary culture

Summary Primary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-d-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system. This study was entrusted to the Institute of Physical and Chemical Research (RIKEN) by the Science and Technology Agency (STA) and was financially supported by the Special Coordination Funds for Promoting Science and Technology. R. Shiurba is a recipient of a STA fellowship award from the Japanese Union of Scientists and Engineers.     

Discontinuous organization and specification of the lateral floor plate in zebrafish

The floor plate is a signaling center in the ventral neural tube of vertebrates with important functions during neural patterning and axon guidance. It is composed of a centrally located medial floor plate (MFP) and a bilaterally positioned lateral floor plate (LFP). While the role of the MFP as source of signaling molecules like, e.g., Sonic Hedgehog (Shh) is well understood, the exact organization and function of the LFP are currently unclear. Based on expression analyses, the one cell wide LFP in zebrafish has been postulated to be a homogenous structure. We instead show that the zebrafish trunk LFP is discontinuously arranged. Single LFP cells alternate with p3 neuronal precursor cells, which develop V3 interneurons along the anteroposterior (AP) axis. Our mutant analyses indicate that both, formation of LFP and p3 cells require Delta-Notch signaling. Importantly, however, the two cell types are differentially regulated by Hedgehog (HH) and Nkx2.2 activities. This implicates a novel mechanism of neural tube patterning, in which distinct cell populations within one domain of the ventral neural tube are differently specified along the AP axis. We conclude that different levels of HH and Nkx2.2 activities are responsible for the alternating appearance of LFP and p3 neuronal progenitor cells in the zebrafish ventral neural tube.     

Essential roles of myosin phosphatase in the maintenance of epithelial cell integrity of Drosophila imaginal disc cells

Reorganization of the actin cytoskeleton and contraction of actomyosin play pivotal roles in controlling cell shape changes and motility in epithelial morphogenesis. Dephosphorylation of the myosin regulatory light chain (MRLC) by myosin phosphatase is one of the key events involved. Allelic combinations producing intermediate strength mutants of the Drosophila myosin-binding subunit (DMBS) of myosin phosphatase showed imaginal discs with multilayered disrupted morphologies, and extremely mislocated cells, suggesting that DMBS is required to maintain proper epithelial organization. Clonal analyses revealed that DMBS null mutant cells appear to retract basally and localization of apical junction markers such as DE-cadherin is indetectable in most cells, whereas phosphorylated MRLC and F-actin become heavily concentrated apically, indicating misconfiguration of the apical cytoskeleton. In agreement with these findings, DMBS was found to concentrate at the apical domain suggesting its function is localized. Phenotypes similar to DMBS mutants including increased migration of cells were obtained by overexpressing the constitutive active form of MRLC or Rho-associated kinase signifying that the phenotypes are indeed caused through activation of Myosin II. The requirement of DMBS for the integrity of static epithelial cells in imaginal discs suggests that the regulation of Myosin II by DMBS has a role more general than its previously demonstrated functions in morphogenetic events.     

Oligodendrocyte precursor cells from different brain regions express divergent properties consistent with the differing time courses of myelination in these regions

Different CNS regions exhibit different temporal patterns of oligodendrocyte generation and myelinogenesis. Characterization of oligodendrocyte-type-2 astrocyte progenitor cells (here abbreviated as O-2A/OPCs) isolated from different regions indicates these developmental patterns are consistent with properties of the specific O-2A/OPCs resident in each region. Marked differences were seen in self-renewal and differentiation characteristics of O-2A/OPCs isolated from cortex, optic nerve and optic chiasm. In conditions where optic nerve-derived O-2A/OPCs generated oligodendrocytes within 2 days, oligodendrocytes arose from chiasm-derived cells after 5 days and from cortical O-2A/OPCs only after 7-10 days. These differences, which appear to be cell-intrinsic (and may be related to intracellular redox state), were manifested both in reduced percentages of clones producing oligodendrocytes and in a lesser representation of oligodendrocytes in individual clones. In addition, responsiveness of optic nerve-, chiasm- and cortex-derived O-2A/OPCs to thyroid hormone (TH) and ciliary neurotrophic factor (CNTF), well-characterized inducers of oligodendrocyte generation, was inversely related to the extent of self-renewal observed in basal division conditions. Our results demonstrate hitherto unrecognized complexities among the precursor cells thought to be the immediate ancestors of oligodendrocytes, and suggest that the properties of these different populations may contribute to the diverse time courses of myelination in different CNS regions.     

Plexin-A4 is expressed in oligodendrocyte precursor cells and acts as a mediator of semaphorin signals

Class 3 semaphorin acts as a guidance clue for both cell migration and nerve fiber projection. The signal of class 3 semaphorin travels via a receptor complex consisting of neuropilins and Plexin-A subfamily. Although it has been reported that class 3 semaphorin acts as a repellent for oligodendrocyte precursor cells (OPCs), which migrate actively during brain development, the expression of Plexin-A subfamily has not been reported in OPCs yet. Therefore, it is currently unclear how semaphorin signals can travel in OPCs. In the present study, the expression of Plexin-A4 (PlexA4) was first demonstrated in a newly established OPC line and OPCs in developing brain. In the OPC line, repulsion for process extension was caused by both Sema3A and Sema6A, and the effect of the semaphorins was diminished in cells expressing PlexA4 lacking the cytoplasmic domain. These results strongly suggest that PlexA4 expressed in OPCs acts as a mediator of semaphorin signals.     

N-cadherin-based adherens junction regulates the maintenance,proliferation, and differentiation of neural progenitor cells during development

This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin.     

Expression of gooseberry-proximal in the Drosophila developing nervous system responds to cues provided by segment polarity genes

Summary Segment polarity genes define the cell states that are required for proper organization of each metameric unit of the Drosophila embryo. Among these, the gooseberry locus has been shown to be composed of two closely related genes which are expressed in an overlapping single-segment periodicity. We have used specific antibodies raised against the protein product of the gooseberry proximal (gsb-p) gene to determine the spatial distribution of this antigen in wild type embryos, and to monitor the effects of segment polarity mutants on the pattern of the gsb-p protein distribution. We find that the gsb-p protein accumulates beneath each posterior axonal commissure in the progeny of neuroblasts deriving from the epidermal compartments of wingless (wg) and engrailed (en) expression. The results of this analysis support the idea that gsb-p has a specific role in the control of cell fates during neurogenesis, and indicate that en and wg provide critical positional cues to define the domain in which gsbp will be activated. Furthermore, these data suggest that, in order to be expressed in the embryonic CNS, gsb-p may preliminarily require activity of the gooseberry-distal gene in the epidermis. Offprint requests to: S. Côté     

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced--stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced--induced for 2 weeks before seeding into scaffolds; and (3) uninduced--cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.     

Identification and characterization of label-retaining cells in mouse pancreas

Pancreatic stem cells (PSCs) may play an important role in maintaining and repairing pancreatic tissues. However, both the existence and localization of PSCs in adult mammalian pancreas still remain elusive. In order to locate the potential pancreatic progenitor/stem cells, we used the tracing label-retaining cells (LRCs) method and identified slow-cycling cells in mouse pancreas. Characterization of the LRCs revealed that the differentiation marker-negative LRCs were located not only within and around the islets but also around the acini and ducts. About 30% of the LRCs around the acini and ducts expressed c-Met, which is a putative pancreatic progenitor/stem cell marker. Moreover, the LRCs around the acini could be activated to form duct-like structures in response to pancreatic damage, and the involvement of these LRCs in the neogenesis of islets and focal areas could also be observed in acini. Our data suggest that the LRCs located around the acini and ducts may represent potential pancreatic progenitor/stem cells, and characterization of these cells may aid in further identification of the specific markers of pancreatic progenitor/stem cells.