Showing papers in "Development in 1989"

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

Abstract: An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.

An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo.

Abstract: Embryonic stem cells (ES) cells were injected into host blastocysts either in groups of 10–15 cells or as single cells in order to test their developmental potential in the developing embryo. The analysis of midgestation chimaeras, by electrophoretic separation of glucose phosphate isomerase (GPI) isozymes, showed that ES cells were capable of colonizing trophectoderm and primitive endoderm derivatives at a low frequency, as well as producing a high rate of chimaerism in tissues of the fetus and extraembryonic mesoderm.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Abstract: The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

Mitotic domains reveal early commitment of cells in Drosophila embryos.

Abstract: The importance of cell–cell interactions in embryonic development was first described by Driesch (1891), who showed that any of the blastomeres of the 2-cell or 4-cell sea-urchin embryo is capable of forming a complete embryo if cultured in isolation; this implied that in normal development each blastomere is aware of the other and will only form a half- or quarter-embryo, as appropriate. And it was only ten years later that Spemann (1901) discovered the phenomenon of embryonic induction, recently reviewed by Gurdon (1987) and defined as an interaction in which the differentiation of one group of cells is affected by a signal from an adjacent group. Thus the significance of cell signalling during development has been appreciated for almost a century, but, as has frequently been remarked, progress in the molecular analysis of the phenomenon has been slow compared with that in the younger disciplines of, for example, immunology and molecular biology.

Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study

Abstract: In mouse embryos, acquisition of the nuclear lamin polypeptides A/C varies according to developmental stage and tissue type. In order to determine the precise time points and cell types in which lamin A/C are first observed, we have used two monoclonal antibodies in immunofluorescence studies of different tissues of developing mouse embryos and of young mice. One antibody (mAB346) is specific for lamins A and C, while the other (PKB8) detects lamins A, B and C. Dividing uterine development into three phases--germ layer formation, organogenesis and tissue differentiation--our results show that lamin A/C expression in the embryo proper is not observed until the third phase of development. Lamin A/C first appears at embryonic day 12 in muscle cells of the trunk, head and the appendages. Three days later it is also seen in cells of the epidermis where its appearance coincides with the time of stratification. In the simple epithelial of lung, liver, kidney and intestine, as well as in heart and brain, lamins A/C do not appear until well after birth. Embryonal carcinoma (EC) cells express lamin B but not lamin A/C. Lamin A/C expression is noted in some EC cells after they are induced to differentiate and in several differentiated teratocarcinoma cell lines. Our results suggest that commitment of a cell to a particular pathway of differentiation (assayed by cell-type-specific expression of intermediate filament proteins) usually occurs prior to the time that lamin A/C can be detected. Thus lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events.

Stem cells: the generation and maintenance of cellular diversity

Abstract: Introduction Stem cells in adult self-renewing tissues Haemopoiesis Epidermis Gastrointestinal epithelium General characteristics of stem cells Asymmetric divisions Predetermined asymmetry Yeast C. elegans What are the mechanisms of predetermined asymmetric divisions? Asymmetry without lineage involvement Stochastic events Stem cell heterogeneity Environmental regulation Conclusions What is the difference between two daughter cells that result from an asymmetric division? Unlimited self renewal versus finite transit amplifying divisions The differentiation process is unidirectional Conclusions and prospects

Identification of an adult-specific glial progenitor cell.

Abstract: We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundamentally different from their counterparts in perinatal animals. In our studies on bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, we have seen that O-2Aadult progenitor cells can be distinguished from O-2Aperinatal progenitors by their morphology and antigenic phenotype, their much longer cell cycle time (65 h versus 18 h), slower rate of migration rate (4 microns h-1 versus 21 microns h-1), and their time course of differentiation into oligodendrocytes or type-2 astrocytes in vitro (less than or equal to 3 days versus greater than 5 days). At least some of the differences between O-2Aadult and O-2Aperinatal progenitor cells appear to be clearly related to the differing cellular requirements of the adult and perinatal central nervous system (CNS). The properties of the O-2Aadult progenitor cells may make these cells ideally suited for the needs of the adult CNS, where rapid exponential increases in the number of oligodendrocytes and O-2A progenitor cells would be inappropriate. However, the properties of the O-2Aadult progenitor cells are such that they may not be able to replace oligodendrocytes in sufficient numbers to repair extensive or recurrent damage in the adult brain, such as in patients suffering from the human demyelinating disease multiple sclerosis. Moreover, available information about other tissues suggests that the transition from perinatal to adult progenitor cell types may represent a developmental mechanism of general importance.

Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny.

Abstract: Quail-chick intracoelomic grafts of organ rudiments were used to study the origin of endothelia and haemopoietic cells during avian organogenesis in conjunction with the monoclonal antibody QH1 which recognizes the quail haemangioblastic lineage. Results differed according to the germ-layer constitution of the grafted rudiments. In the case of the limb buds, endothelial cells from the host invaded the graft through an angiogenic process. Haemopoietic progenitors from the host also colonized the grafted bone marrow. In contrast, rudiments of internal organs provided their own contingent of endothelial precursors, a process termed vasculogenesis. Nevertheless, haemopoietic cells in these organs were all derived from the host. In the lung, this extrinsic cell population appeared regularly scattered around the parabronchi and had a macrophage-like phenotype. In the pancreas, the granulocytes which differentiate as dense aggregates located in the wall of the largest vessels were extrinsic. Similarly in the spleen, a mesodermal primordium that develops in close association with the pancreatic endoderm, endothelial cells were intrinsic and haemopoietic cells host-derived. This study demonstrates that, in ontogeny, vascularization obeys different rules depending on which germ layer the mesoderm is associated with: in mesodermal/ectodermal rudiments angiogenesis is the rule; in mesodermal/endodermal rudiments, vasculogenesis occurs. However, in these internal organs undergoing vasculogenesis, endothelial and haemopoietic cells have separate origins. We put forward the hypothesis that the endoderm induces the emergence of endothelial cells in the associated mesoderm. Formation of blood stem cells may also involve interactions between endoderm and mesoderm, but in this case the responding capacity of the mesoderm appears restricted to the paraaortic region.

A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus

Abstract: We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 × 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 × 10(3) Mr found in A6 cells. The 57 × 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anterior somites and on progressively more posterior somites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anterior somites and later appear below more posterior somites. The dorsal fin mesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.

Cortical rotation of the Xenopus egg: consequences for the anteroposterior pattern of embryonic dorsal development.

Abstract: We first review cortical-cytoplasmic rotation, a microtubule-mediated process by which the Xenopus egg, like other amphibian eggs, transforms its polarized cylindrical symmetry into bilateral symmetry within the first cell cycle after fertilization. This transformation, the earliest of many steps leading to dorsal development, involves the displacement of the egg's cortex relative to its cytoplasmic core by 30 degrees in an animal-vegetal direction. As rotation is progressively reduced by microtubule-depolymerizing agents, embryos develop with body axes progressively deleted for dorsal structures at the anterior end. With no rotation, ventralized embryos are formed. In an effort to comprehend this progressive effect on embryonic organization, we go on to review subsequent developmental process depending on rotation, and we propose, with evidence, that reduced rotation leads to a reduced number of vegetal dorsalizing cells, which induce during the blastula stage a Spemann organizer region of smaller than normal size. The reduced organizer then promotes a reduced amount of cell rearrangement (morphogenesis) at gastrulation. Reduced morphogenesis seems the proximate cause of the incompleteness of axial pattern, as shown further by the fact that embryos that are normal until the gastrula stage, if exposed to inhibitors of morphogenesis, develop body axes that are progressively less complete in their anterior dorsal organization the earlier their gastrulation had been blocked. We discuss why axial pattern might depend systematically on morphogenesis.

Development of adult sensilla on the wing and notum of Drosophila melanogaster.

Abstract: We have investigated the temporal pattern of appearance, cell lineage, and cytodifferentiation of selected sensory organs (sensilla) of adult Drosophila. This analysis was facilitated by the discovery that the monoclonal antibody 22C10 labels not only the neuron of the developing sensillum organ, but the accessory cells as well. The precursors of the macrochaetes and the recurved (chemosensory) bristles of the wing margin divide around and shortly after puparium formation, while those of the microchaetes and the stout and slender (mechanosensory) bristles of the wing margin divide between 9 h and 18 h after puparium formation (apf). The onset of sensillum differentiation follows the terminal precursor division within a few hours. Four of the cells in an individual microchaete organ are clonally related: A single first-order precursor cell divides to produce two second-order precursors; one of these divides into the neuron and thecogen cell, the other into the trichogen cell and tormogen cell. Along the anterior wing margin, two rounds of division generate the cells of the mechanosensory sensilla; here, no strict clonal relationship seems to exist between the cells of an individual sensillum. At the time of sensillum precursor division, many other, non-sensillum-producing cells within the notum and wing proliferate as well. This mitotic activity follows a spatially non-random pattern.

Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear.

Abstract: The stria vascularis of the mammalian cochlea is composed primarily of three types of cells. Marginal cells line the lumen of the cochlear duct and are of epithelial origin. Basal cells also form a continuous layer and they may be mesodermal or derived from the neural crest. Intermediate cells are melanocyte-like cells, presumably derived from the neural crest, and are scattered between the marginal and basal cell layers. The marginal cells form extensive interdigitations with the basal and intermediate cells in the normal adult stria. The stria also contains a rich supply of blood vessels. We investigated the role of melanocytes in the stria vascularis by studying its development in a mouse mutant, viable dominant spotting, which is known to have a primary neural crest defect leading to an absence of recognisable melanocytes in the skin. Melanocytes were not found in the stria of most of the mutants examined, and from about 6 days of age onwards a reduced amount of interdigitation amongst the cells of the stria was observed. These ultrastructural anomalies were associated with strial dysfunction. In the normal adult mammal, the stria produces an endocochlear potential (EP), a resting dc potential in the endolymph in the cochlear duct, which in mice is normally about +100 mV. In our control mice, EP rose to adult levels between 6 and 16 days after birth. In most of the mutants we studied, EP was close to zero at all ages from 6 to 20 days. Melanocyte-like cells appear to be vital for normal stria vascularis development and function. They may be necessary to facilitate the normal process of interdigitation between marginal and basal cell processes at a particular stage during development, and the lack of adequate interdigitation in the mutants may be the cause of their strial dysfunction. Alternatively, melanocytes may have some direct, essential role in the production of an EP by the stria. Melanocytes may be important both for normal strial development and for the production of the EP. We believe this is the clearest demonstration yet of a role for migratory melanocytes other than their role in pigmentation.

A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration

Abstract: To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.

Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice

Abstract: Transgenic mice have been generated that express the E. coli beta-galactosidase gene under the control of the promoter from the mouse heat-shock gene, hsp68. Sequences from -664 to +113 relative to the start of transcription of the hsp68 gene were sufficient to direct stress-induced expression of the beta-galactosidase gene in adult tail tissue and various tissues of fetal stages of development. Expression was detected in situ by staining with the chromogenic substrate, X-gal. The hybrid gene was refractory to induction in preimplantation embryos until the blastocyst stage of development, as reported for the endogenous hsp68 gene. No constitutive expression was observed by in situ staining or Northern analysis at any stage of development, even in tissues that constitutively express the endogenous hsp68 gene. We conclude that the hsp68 promoter region included in the construct contains sufficient sequence information for heat and arsenite inducibility, but it does not contain sequences controlling tissue-specific expression during development. This tightly regulated inducible promoter may provide a useful tool for short-term inducible gene expression in transgenic mice.

Positional information revisited

Abstract: Positional information has been suggested to play a central role in pattern formation during development. The strong version of positional information states that there is a cell parameter, positional value, which is related to position as in a coordinate system and which determines cell differentiation. A weaker version merely emphasises position as a key determinant in cell development and differentiation. There is evidence for boundaries and orthogonal axes playing an important role in positional systems. A positional signal is distinguished from an inductive interaction because the former specifies multiple states, confers polarity, and can act over a long range. A gradient in a diffusible morphogen is just one way of specifying position. There is now good evidence in several systems for substances which may be the morphogen for positional signalling. The product of the bicoid gene in early Drosophila development is the best prospect. Retinoic acid is unique in its ability to alter positional value and may also be a morphogen. The best evidence for positional value, a concept fundamental to positional information, remains a biological assay based on grafting. The idea of positional value uncouples differentiation and position, and allows considerable freedom for patterning. It is not clear whether positional value or differentiation involves a combinatorial mechanism. Interpretation of positional information remains a central problem. There is good evidence that cells can respond differentially to less than a two-fold change in concentration of a chemical signal. It may be that interpretation involves listing the sites at which a particular class of cell differentiation will occur. The problem is made less severe when blocks of cells are specified together as in mechanisms based on an isomorphic prepattern. Isomorphic prepatterns could establish repeated structures which are equivalent and which are then made non-equivalent by positional information. This would enable local differences to develop. The combination of these two mechanisms may be wide-spread. There is evidence that positional signals within a single animal and in related animals are conserved. It is not clear just how wide this conservation is, but it is at phylotypic stages, rather than in eggs, that similarity might be expected. It is nevertheless impressive that the polar coordinate model can be applied to regulation in systems as diverse as insects, vertebrates and protozoa. The molecular basis of positional signalling is just becoming accessible; the molecular basis of positional value is still awaited. A brief personal history of positional information is provided in an appendix.

Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration.

Abstract: We reported previously that epithelial cells in explants from neonatal rat lenses, when cultured in the presence of fibroblast growth factor (FGF), showed increased proliferation, cell migration and fibre differentiation; moreover, fibre differentiation in response to the basic form of FGF (bFGF) was virtually completely blocked by an anti-bFGF antibody. In the present study, we report a detailed analysis of the effects of bFGF on cells in the central region of lens epithelial explants. Proliferation in explants was assessed by measuring [3H]thymidine incorporation. Cell migration was measured by labelling cells in explants with fluorescein isothiocyanate (FITC)-dextran and monitoring them by UV fluorescence microscopy. Fibre differentiation in explants was assessed on the basis of beta-crystallin accumulation. This study showed that half maximal activities for the three responses, proliferation, migration and fibre differentiation, were achieved at different concentrations of bFGF, namely, 0.15, 3 and 40 ng ml-1, respectively. Thus, the response of lens epithelial cells to bFGF varied qualitatively, as well as quantitatively, as the concentration increased. Monitoring FITC-dextran injection cells for up to 5 days after exposure to bFGF allowed analysis of the interrelation between various responses to bFGF in individual cells. As expected some cells divided in response to FGF, mainly within the first three days. However, whether or not they divided, all labelled cells responded to FGF by migrating and elongating. Maximal migration occurred during the first day of culture and maximal elongation was achieved by day 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte.

Abstract: The anterior region of the Drosophila embryonic pattern is determined by a gradient of the bicoid (bcd) protein. The correct formation of this gradient requires the localization of bcd RNA to the anterior pole of the egg. Here we use a wholemount in situ technique to examine the process of bcd RNA localization during oogenesis and embryogenesis. While bcd protein becomes distributed in a gradient that extends throughout the anterior two thirds of the early embryo, bcd RNA remains restricted to a much smaller region at the anterior pole. The difference between these distributions indicates that the shape of the protein gradient must depend to some extent on the posterior movement of the protein after it has been synthesized. Four distinct phases of bcd RNA localization can be distinguished during oogenesis. Between stages 6 and 9 of oogenesis, the RNA accumulates in a ring at the anterior end of the oocyte. During the second phase, in stage 9–10a follicles, the RNA also localizes to the apical regions of the nurse cells, demonstrating that the nurse cells possess an intrinsic polarity. As the nurse cells contract during stages 10b–ll, all of the bcd RNA becomes localized to the cortex at the anterior end of the oocyte. During a final phase that must occur between stage 12 of oogenesis and egg deposition, the RNA becomes localized to a spherical region that occupies a slightly dorsal position at the anterior pole. Mutations in the maternal-effect genes, exuperantia (exu) and swallow (sww) , lead to an almost uniform distribution of bcd RNA in the early embryo, while staufen (stau) mutations produce a gradient of RNA at the anterior pole, exu mutations disrupt the second stage of bcd RNA localization during oogenesis, sww mutations disrupt the third, and stau mutations affect the fourth phase.

Expression pattern of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal development.

Abstract: The FGF-related proto-oncogene int-2 is implicated in mouse embryogenesis, since it is expressed in specific tissues during gastrulation and neurulation (Wilkinson et. al. 1988). Here, we describe the expression of this gene during subsequent fetal development, int-2 transcripts are restricted to Purkinje cells in the cerebellum and to regions of the developing retina containing early-stage differentiating cells. This high level expression is not detected in the mature cerebellum or retina. In addition, int-2 RNA is detected in the mesenchyme of the developing teeth and in sensory regions of the inner ear. This complex and dynamic pattern suggests multiple roles of this proto-oncogene during fetal development of the mouse.

Expression of engrailed during segmentation in grasshopper and crayfish

Abstract: We have used a monoclonal antibody that recognizes engrailed proteins to compare the process of segmentation in grasshopper, crayfish, and Drosophila. Drosophila embryos rapidly generate metameres during an embryonic stage characterized by the absence of cell division. In contrast, many other arthropod embryos, such as those of more primitive insects and crustaceans, generate metameres gradually and sequentially, as cell proliferation causes caudal elongation. In all three organisms, the pattern of engrailed expression at the segmented germ band stage is similar, and the parasegments are the first metameres to form. Nevertheless, the way in which the engrailed pattern is generated differs and reflects the differences in how these organisms generate their metameres. These differences call into question what role homologues of the Drosophila pair-rule segmentation genes might play in other arthropods that generate metameres sequentially.

Regeneration and pattern formation in planarians. III. that neoblasts are totipotent stem cells and the cells

Abstract: In most regenerating systems, blastema cells arise by dedifferentiation of functional tissue cells. In is still debatable whether dedifferentiated cells or a undifferentiated cells, the neoblasts, are the main cells. Moreover, it is unclear whether in the intact neoblasts are quiescent cells ‘reserved’ for serve as functional stem cells of all differentiated uncertainties partly stem from the failure to conventional labelling methods neoblasts from Here we describe a new approach to these problems regenerative and stem cell capabilities of purified differentiated cells when introduced, separately, into hosts. Introduction of neoblasts led to resumed blastema formation, and extended or complete survival differentiated cells, in contrast, never did so. neoblasts can be qualified as totipotent stem cells of blastema cells, while dedifferentiation does not either in intact or regenerating organisms. In strengthen the idea that different types of formation, linked to the tissular complexity of the present in the animal kingdom.

Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos

Abstract: We have used a monoclonal antibody directed against the C-terminus of the Drosophila invected homeodomain to detect a nuclear protein in brain cells of Xenopus laevis embryos. We refer to this antigen as the Xenopus EN protein. The EN protein is localized at midneurula stage to a band of cells in the anterior portion of the neural plate, on each side of the neural groove. Later in development, the expression coincides with the boundary of the midbrain and hindbrain, and persists at least to the swimming tadpole stage. These properties make the EN protein an excellent molecular marker for anterior neural structures. In embryos where inductive interactions between mesodermal and ectodermal tissues have been perturbed, the expression of the EN protein is altered; in embryos that have been anterodorsalized by LiCl treatment, the region that expresses the EN protein is expanded, but still well organized. In ventralized UV-irradiated embryos, the absence of the protein is correlated with the absence of anterior neural structures. In extreme exogastrulae, where the contacts between head mesoderm and prospective neurectoderm are lost, the EN protein is not expressed.

Mesoderm induction and mesoderm-inducing factors in early amphibian development.

Abstract: Introduction Experimental embryology and mesoderm induction The three-signal model of mesoderm formation Mesoderm-inducing factors Mesoderm-inducing factors as growth factors Xenopus embryos contain TGF-fi-like molecules Xenopus embryos contain FGF Elimination of inducing factors from Xenopus embryos Mesoderm-inducing factors and pattern formation Thresholds Mesoderm induction, gastrulation and developmental clocks Modulation of the effects of mesoderm-inducing factors: inhibitors and further cell-cell interactions Conclusions

Lineage-specific gene expression and the regulative capacities of the sea urchin embryo: a proposed mechanism

Abstract: Three aspects of early sea urchin development are reviewed, and conclusions derived that lead to a unified concept of how the initial specifications of differential gene activity may occur in this embryo. i. The embryo has an invariant cell lineage, and the lineage founder cells can be considered as regulatory spatial domains. That is, from each of these cells descend clones of progeny the members of which express the same set of lineage-specific genes. ii. From the extensive classical literature on blastomere plasticity, and some key modern experiments, are derived a system of inductive blastomere interactions, which accounts for the conditionality of lineage founder cell specification. That is, the fates of many of the lineage founder cells can apparently be altered if the normal spatial interrelationships within the embryo are perturbed. iii. Recent studies have been carried out by gene transfer, and are supported by in vitro analyses of DNA-protein interactions in the regulatory regions of two genes that are expressed in a lineage- specific manner. Expression of both of these markers of cell fate specification is controlled by diffusible DNA-binding factors (i.e. within each nucleus). A molecular mechanism is proposed, based on inductive effects on gene regulatory factors, which in principle provides a specific explanation of the regulative capacities for which this embryo is famous.

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

Abstract: It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells—oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.

Oligodendrocyte progenitors isolated directly from developing telencephalon at a specific phenotypic stage: Myelinogenic potential in a defined environment

Abstract: Oligodendroglia differentiate asynchronously in the developing central nervous system, passing through a series of stages identified by the sequential expression of specific differentiation antigens, culminating in the formation of the myelin sheath. In the work presented here, oligodendrocyte progenitors at a temporally narrow and well-defined phenotypic stage of development have been isolated in high purity and yield directly from postnatal rat telencephalon. This stage is identified by the expression of the O4 antigen, the earliest recognized surface marker specific for the oligodendroglial lineage, but the absence of the differentiation marker galactosylcerebroside (GalC). These O4+ GalC- progenitors first appear at birth (10(5)/telencephalon), 2-3 days before O4+ GalC+ oligodendrocytes. The work presented here demonstrates that a major subpopulation of O4+ GalC- progenitors (80%), which we have termed 'proligodendrocytes', is fully committed to terminal oligodendrocyte differentiation. A relatively small, maximal set of nutritional supplements are sufficient for proligodendrocytes to carry out the myelinogenic cascade of differentiated gene expression in a temporally normal manner, in quantitatively significant amounts, in normal ratios of myelin protein isoforms, and in a regulated relationship to the inclusion of myelin-specific products into myelin-like membrane sheets. An important corollary is that this step of myelinogenesis does not require contact with other cell types, in particular neurones and astrocytes, nor does it require unknown growth factors unique to these cell types. Additionally under these conditions, there exists a developmentally quiescent subpopulation (20%) of O4+ GalC- cells that may have significance for understanding the progenitors previously described in adult brain and suggested to be instrumental in remyelination under pathological conditions.

Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo

Abstract: The primary gene transcript for the adhesive extracellular matrix glycoprotein fibronectin (FN) is alternatively spliced in three regions (EIIIA, EIIIB and V). At least one of these regions (V) has been shown to encode cell-binding sites, suggesting that splicing represents a mechanism to create functionally different forms of FN at different times and places. In order to test this hypothesis, we have examined the extent of alternative splicing of fibronectin during embryonic development. The distribution of the different spliced forms of FN mRNA in developing chicken embryos was determined using probes specific for the spliced regions in ribonuclease protection and in situ hybridization experiments. At embryonic day 2-4 (E2-4), all three spliced regions were included wherever FN mRNA was detected. At E16, however, we found spatially distinct splicing differences within the embryo, with cell-type-specific splicing excluding EIIIA and/or EIIIB in some tissues. In contrast, we did not detect exclusion of the V region. In a more detailed developmental study of the simplest of these tissues, the chorioallantoic membrane, we found that EIIIB was preferentially excluded after the completion of growth. These results suggest that FN splicing is used during development as a mechanism to create different forms of FN within the extracellular matrix by the inclusion or exclusion of specific segments. The data are consistent with an essential role for one of these segments, EIIIB, in the migration and/or proliferation of embryonic cells prior to their terminal differentiation and also suggest possible roles for the EIIIA segment.

Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development.

Abstract: The role in early development of proteins secreted by oviduct epithelial cells has been investigated. Secreted proteins devoid of serum contamination have been produced by the surgical removal and immediate incubation of oviduct cells in [35S]methionine-containing medium. After electrophoretic separation, secreted polypeptides could be divided into those that were secreted uniformly throughout the oestrous cycle and a second class that showed a cyclical pattern of secretion. The first class of proteins represented a small proportion of total output whilst the predominant second class was composed mainly of polypeptides of Mr 92 and 46 x 10(3), respectively. Both of these polypeptide species, referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), are detected only during the first 4 to 5 days after oestrus when the embryos are located in the oviduct. Oviduct cells collected at oestrus and maintained thereafter in culture secrete the same pattern of proteins and follow the same time course as their counterparts in vivo. The interaction between the oviduct proteins and the developing embryo was studied firstly by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination studies showed that two polypeptides of Mr 92 and 46 x 10(3), respectively, were bound to the zona pellucida of eggs removed from the oviduct but were absent from eggs that had not had contact with the oviduct epithelium. That these newly acquired proteins represent SOP 92 and 46 is suggested by their electrophoretic mobility and their ability to bind to the zona of follicular eggs when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. The collection of unlabelled secreted proteins enabled us to produce a monoclonal antibody, which was used in the second series of experiments on oviduct-embryo interactions. The results confirmed that SOP 92 binds to the zona pellucida and moreover showed that this protein crosses the zona and becomes associated with the individual blastomeres of the developing embryo. These findings provide evidence that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by its epithelium.

Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis.

Abstract: Expression and the role of E- and P-cadherin in the histogenesis of the surface epidermis and hair follicles were examined using the upper lip skin of the mouse. P-cadherin is expressed exclusively in the proliferating region of these tissues, that is in the germinative layer of the surface epidermis, the outer root sheath and the hair matrix. E-cadherin is coexpressed in these layers but this molecule was also detected in non-proliferating regions such as the intermediate layer of the surface epidermis and the immature regions of the inner root sheath. Neither P- nor E-cadherin was detected in fully keratinized layers such as the horny layer of the surface epidermis, the outermost layer of the outer root sheath and the mature hair fibres. These two cadherins were not detected in dermal cells. We cultured pieces of the upper lip skin in vitro in the absence or presence of a monoclonal antibody to E-cadherin (ECCD-1) or to P-cadherin (PCD-1). In control cultures, skin morphogenesis normally occurred in a pattern whereby the hair follicles grew and dermal cells were condensed to form the dermal sheath. A mixture of ECCD-1 and PCD-1, however, induced abnormal morphogenesis in the skin in several respects. (1) The cuboidal or columnar arrangement of basal epithelial cells was distorted. (2) Hair follicles were deformed. (3) Condensation of dermal cells was suppressed, causing a homogeneous distribution of these cells. These results suggest that cadherins present in epidermal cells are involved not only in maintaining the arrangement of these cells but also in inducing dermal condensation.

Expression of transforming growth factor beta 2 RNA during murine embryogenesis

Abstract: We have studied the temporal and spatial expression of transforming growth factor beta 2 (TGF beta 2) RNA in mouse embryos from 10.5 days post coitum (p.c.) to 3 days post partum (p.p.) by in situ hybridization analysis. TGF beta 2 RNA is expressed in a variety of tissues including bone, cartilage, tendon, gut, blood vessels, skin and fetal placenta, and is in general found in the mesenchymal component of these tissues. The expression of TGF beta 2 RNA changes during development in a manner consistent with a role for the gene product in mediating mesenchymal-epithelial interactions.

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Abstract: Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.