Initial Stages Of Differentiation Research Articles

Caspase-1 activity is required for neuronal differentiation of PC12 cells: Cross-talk between the caspase and calpain systems

Previously, we have found that caspase-1 activity is increased during myoblast differentiation to myotubes. Here we show that caspase-1 activity is required for PC12 differentiation to neuronal-like cells. Caspase-1 is shown to be activated (by immunoblotting and by assessing activity in cell extracts) in the PC12 cells following the initial stage of differentiation. The inhibition of caspase-1 arrests PC12 cells at an intermediate stage of differentiation and prevents neurite outgrowth in these cells; the inhibition is reversed upon the removal of the inhibitor. Calpastatin (calpain endogenous specific inhibitor, and a known caspase substrate) is diminished at the later stages of PC12 cell differentiation, and diminution is prevented by caspase-1 inhibition. The degradation of fodrin (a known caspase and calpain substrate) is found in the advanced stage of differentiation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in two distinct differentiation processes (myoblast fusion and neuronal differentiation of PC12 cells) indicates a function for this caspase in differentiation processes, and suggests some common mechanisms underlying caspase roles in such processes.

146 EFFECT OF MATERNAL AGE AND PARITY ON THE PRESENCE AND DISTRIBUTION OF CADHERIN-E IN HAMSTER EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT AND TRANSPORT

Our knowledge of the factors that contribute to in vivo embryo development and transport are still incomplete. Although there is solid evidence that the hormonal state of the female regulates ovum transport in mammals, recently, because of significant differences between the rate of oviduct transport of fertilized and unfertilized eggs in several mammalian species, there have been some reports showing that the conceptus itself may auto regulates its own transport. During the initial phases of embryonic development the distribution of E-cadherin, a calcium dependent cell adhesion molecule, changes from a general distribution over the whole surface of the blastomeres to become almost exclusively localized over the points of contact between blastomeres. This change on E-cadherin localization has been related to the beginning of embryo compactation and is considered as the initial stage of differentiation. We have recently shown that pre-implantation embryo development and transport is highly synchronous in nuliparous young females (NYF), but not in adults. Nuliparous adult (8 months) females (NAF) and multiparous adult females (MAF) show significant asynchrony and retard on early embryo development. In NYF, all 8 cells embryos reached the uterus by 62 h after coitus. In adult females, both nuliparous and multiparous, a considerable number of 8 cells embryos fail to migrate into the uterus even at 67 h after coitus. E-cadherin distribution was studied by indirect immunofluorescence and confocal microscopy during preimplantation embryo development in three groups, NYF, MAF, and NAF, of regularly cycling golden hamster (Mesocricetus auratus) females. Females were mated with males of proven fertility. Only 15 minutes were allowing for mating. Time of ejaculation was registered. Females were sacrificed from 60 to 69 h after coitus. Corpora lutea were counted in both ovarian surfaces. Oviducts and uterine horns were flushed separately and embryo number, stage of development, and distribution were recorded. Cell numbers, cadherin distribution and the number of cadherin-dependent cell to cell contacts were established in at least 20 embryos per group and at each of the different hours of the study. Presence and distribution of E-cadherin on 4-, 6-, and 8-cell blastomeres embryos differs accordingly with the parity and age of the females. Differences are particularly important at the eight-cell stage 80 to 93% of the embryos from NYF showed normal cadherin dependent cell contacts with all neighboring cells. In the case of multiparous adult females (MAF) only 52 to 68% of the embryos showed normal cell to cell contacts. These results will be discussed in relation to the respective role that the female hamster and the conceptus itself play in early embryo development and transport. Our results also indicate that in spite of the significantly higher ovulation rate of old females, their reproductive efficiency were significantly lower than that of NYF.

Delineation of Signals Required for Thymocyte Positive Selection

Peptide/MHC complexes capable of inducing positive selection in mouse fetal thymic organ cultures fail to do so in suspension culture. Furthermore, this type of culture does not promote initial stages of differentiation, such as coreceptor down-modulation, unless peptides used for stimulation have (at least) weak agonist activity. We show in this study that signals provided in suspension culture by nonagonist peptide/MHC complexes on the surface of macrophages, even though apparently silent, are sufficient to promote complete phenotypic differentiation when CD4+CD8+ thymocytes are subsequently placed in a proper anatomical setting. Furthermore, the synergistic actions of suboptimal concentrations of phorbol esters and nonagonist peptide/MHC complexes can make the initial stages of positive selection visible, without converting maturation into negative selection. Thus, the correlation between efficiency of positive selection and the degree of coreceptor down-modulation on CD4+CD8+ thymocytes is not linear. Furthermore, these results suggest that the unique role of thymic stromal cells in positive selection is related not to presentation of self-peptide/MHC complexes, but most likely to another ligand.

Structural and ultrastructural aspects of ontogenesis and differentiation of resin secretory cavities in Hymenaea stigonocarpa (Fabaceae‐Caesalpinioideae) leaves

Cell lysis in the formation of secretory cavities in plants has been questioned by some authors and considered as result of technical artifacts. To describe the formation of secretory resin cavities in Hymenaea stigonocarpa leaves, leaflet samples at different stages of differentiation were collected, fixed, and processed for light and electron microscopy as per usual methods. The initial cells of secretory resin cavities are protodermal and grow towards the mesophyll ground meristem; these cells then divide producing cell groups that are distinguished by the shape and arrangement of cytoplasm, and density. At the initial stages of differentiation of the secretory cavities, some central cells in these groups show dark cytoplasm and condensed nuclear chromatin. Later, there is cell wall loosening, tonoplast and plasmalemma rupture resulting in cell death. These cells, however, maintain organelle integrity until lysis, when the cell wall degrades and the plasmalemma ruptures, releasing protoplast residues, marked characteristics of programmed cell death. The secretory epithelium remains active until complete leaf expansion when the cavity is filled with resin and the secretory activity ceases. There are no wall residues between central cells in adult cavities. Our results demonstrate lysigeny and the importance of ontogenetic studies in determining the origin of secretory cavities.

Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro

The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4 involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

Constitutively Active Mitogen-activated Protein Kinase Kinase 6 (MKK6) or Salicylate Induces Spontaneous 3T3-L1 Adipogenesis

Although much has been learned regarding the importance of p38 mitogen-activated protein kinase in inflammatory and stress responses, relatively little is known concerning its role in differentiation processes. Recently, we demonstrated that p38 mitogen-activated protein kinase activity is necessary for the differentiation of 3T3-L1 fibroblasts into adipocytes (Engelman, J. A., Lisanti, M. P., and Scherer, P. E. (1998) J. Biol. Chem. 273, 32111-32120). p38 activity is high during the initial stages of differentiation but decreases drastically as the fibroblasts undergo terminal differentiation into adipocytes. However, it remains unknown whether activation of p38 is sufficient to stimulate adipogenesis and whether the down-regulation of p38 activity in mature adipocytes is critical for maintaining adipocyte homeostasis. In this report, we have directly addressed these questions by analyzing 3T3-L1 cell lines harboring a specific upstream activator of p38 (a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) mutant, MKK6(Glu)) under the control of an inducible promoter. Induction of MKK6(Glu) in 3T3-L1 fibroblasts spurs adipocyte conversion in the absence of the hormonal mixture normally required for efficient differentiation of wild-type cells. However, activation of p38 in adipocytes leads to cell death. Furthermore, treatment of 3T3-L1 fibroblasts with salicylate, a potent stimulator of p38, produces adipocyte-specific changes consistent with those observed with induction of MKK6(Glu). Expression of MKK6(Glu) in NIH-3T3 fibroblasts (cells that do not differentiate into adipocytes under normal conditions) is capable of converting these fibroblasts into lipid-laden fat cells following hormonal stimulation. Thus, p38 activation has pro-adipogenic effects in multiple fibroblast cell lines.

Association of calpain (Ca2+-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts

Calpain isozymes (intracellular, Ca2+-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of μ-calpain and m-calpain (activated by μM and mM Ca2+, respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca2+-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca2+levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested. J. Cell. Biochem. 74:522–531, 1999. © 1999 Wiley-Liss, Inc.

Association of calpain (Ca(2+)-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts.

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.

The high‐pressure melting curve of Allende meteorite

Laser‐heated diamond‐cell experiments on Allende meteorite, a natural analog for undifferentiated terrestrial planetary mantles, yield a melting curve increasing from ∼2200 to 3200 K over the pressure range of 15–70 GPa, significantly below that of peridotite that is representative of the Earth's current upper mantle. Samples quenched from high temperatures and pressures reveal structures that may mimic the initial stages of differentiation between core and mantle materials. Micron‐scale chemical analyses support the idea that sulfur is enriched, and silicon, oxygen and aluminum are depleted, in the (potentially core‐forming) metal relative to the silicate phases.

The tip-top branching ureter

Organ rudiments with their epithelial bud and adjacent mesenchyme look much the same at their initial stage of differentiation. The subsequent branching of the epithelial anlagen determines the final pattern of the organs, but the mesenchyme provides essential signals for epithelial differentiation. Glial cell line derived neurotrophic factor (GDNF) has recently been shown to regulate ureteric branching morphogenesis and is thereby the first defined signalling molecule in the embryonic metanephric kidney. GDNF is expressed by the mesenchyme, binds to the tip of the ureteric bud and functions in both bud induction and bud orientation. The active receptor complex for GDNF includes the receptor tyrosine kinase Ret and a novel class of glycosylphosphatidylinositol-linked receptors, called GDNF family receptor αs.

COLOR‐PATTERN VARIATION IN LAKE ERIE WATER SNAKES: THE ROLE OF GENE FLOW

In an effort to clarify the evolutionary processes influencing color-pattern variation in Lake Erie island water snake (Nerodia sipedon) populations, rates of gene flow among island and mainland populations were estimated from patterns of allozymic variation detected using electrophoresis. Rates of gene flow were high with Nm, the number of migrants per generation, averaging 25.5 among island sites, 9.2 between the Ontario mainland and the islands, and 3.6 between the Ohio mainland and the islands. Based on estimates of current population size from mark-recapture work and of past population size extrapolated from the extent of shoreline habitat, values of m between island and mainland populations ranged from 0.0008-0.01. Synthesis of estimates of the rate of gene flow with information on inheritance of color pattern, the strength of natural selection, and population history supports the hypothesis that color-pattern variation in island populations results from a balance between gene flow and natural selection. However, depending on the mode of inheritance of color pattern, stochastic processes such as drift may have been important in the initial stages of differentiation between island and mainland populations.

Petrology of Calc-Alkaline Lavas at Volc n Ollag e and the Origin of Compositional Diversity at Central Andean Stratovolcanoes

Volcán Ollagüe (21°17'S) is a large stratovolcano that lies slightly east of the main axis of Quaternary Volcanoes in the Andean Central Volcanic Zone (CVZ). Euptive products range from basaltic andesite to dacite and define a high-K, calc-alkaline suite. This compositional range is similar to the collective compositional range of the other stratovolcanoes in the CVZ, and it provides a record of both early and late-stage differentiation processes operating at the stratovolcanoes. The volumetrically dominant andesitic and dacitic lavas are divided into four eruptive series on the basis of vent locations and petrography. In ascending stratigraphic order they are: the Vinta Loma, Chasca Orkho, post-collapse, and La Celosa series. Whole-rock compositions of the lavas are remarkably similar regardless of eruptive series. Variations in phenocryst assemblages and magmatic fo2 however, suggest differences in subliquidus volatile contents for magma chambers developed beneath the summit of the volcano versus those developed beneath the flanks. Basaltic andesite magmas are principally preserved as quenched inclusions within the andesitic and dacitie lava flows. Large ranges in isotopic ratios over a narrow compositional range indicate that the basaltic andesites were derived by crystal fractionation coupled with large amounts of crustal assimilation. Increasing Ce/Yb ratios with decreasing Yb contents further suggest that this initial stage of differentiation occurred at deep crustal levels where garnet was stable. Additional supporting evidence for differentiation in the deep crust includes isotopic and trace element compositions that indicate assimilation by the basaltic andesite magmas of a crust different from upper-crustal rocks exposed at present in the region. Whole-rock major and trace element trends of the dacitic lavas can be simulated largely by fractional crystallization of parental andesitic magma. The fractionating assemblages for the different eruptive series are consistent with the observed modes of the parent magmas. Small increases in Sr isotope ratios with increasing Rb contents indicate that the fractionating magmas also assimilated small amounts of wall rocks similar in composition to the upper-crustal basement to the volcano. Consideration of the chemical trends, mineral compositions, and eruptive history of Ollagüe rocks permits construction of a model for the evolution of shallow crustal magma chambers beneath the stratovolcanoes in the CVZ. At a relatively mature stage, the magma chambers may be compositionally, thermally, and density stratified. Temperatures estimated from Fe-Ti oxide and pyroxene thermometry for the chambers beneath Ollagüe range from 1000 to 790°C with increasing SiO2 from 59 to 67 wt.% in the upper reaches, and from 1150 to 1020°C with increasing SiO2 from 53 to 59 wt.% in the lower reaches. The occurrence of basaltic andesite magmatic inclusions within the intermediate lavas and the repeated eruption of monotonous composition andesitic magmas indicate that the shallow chambers are periodically replenished with parental basaltic andesite magmas. Ubiquitous, reversely zoned plagioclase and pyroxene phenocrysts in the lavas at Ollagüe suggest that convective cooling of the basaltic andesite releases buoyant derivative liquid that mixes with the overlying intermediate-composition body of the chambers. Further crystallization and differentiation of the intermediate magmas may take place in solidification zones at the boundaries of the magma chambers. If so, the return of residual liquid from the crystallizing margins and mixing with the interior are highly efficient such that magma differentiation can be modeled as a simple, homogeneous, fractional crystallization process.

Control of leaf and chloroplast development by the Arabidopsis gene pale cress.

Leaf plastids of the Arabidopsis pale cress (pac) mutant do not develop beyond the initial stages of differentiation from proplastids or etioplasts and contain only low levels of chlorophylls and carotenoids. Early in development, the epidermis and mesophyll of pac leaves resemble those of wild-type plants. In later stages, mutant leaves have enlarged intercellular spaces, and the palisade layer of the mesophyll can no longer be distinguished. To study the molecular basis of this phenotype, we cloned PAC and determined that this gene is regulated by light and has the capacity to encode an acidic, predominantly alpha-helical protein. The PAC gene appears to be a novel component of a light-induced regulatory network that controls the development of leaves and chloroplasts.

Identification of TGF-?-related genes in the early chick embryo

Transforming growth factor-β (TGF-β)-related genes have been postulated as possible mesoderm inducers in lower vertebrates. Activin, a member of the TGF-β family has been recently suggested to be directly involved in the induction of axial mesoderm in frogs and birds. Nodal, a novel TGF-β-like gene, seems to be essential for mesoderm formation in mice. In this work we have tried to characterize which growth factors of the TGF-β family are involved in the initial stages of differentiation of the chick embryo. Using degenerate primers to specific regions of the TGF-β super family in polymerase chain reactions (PCR), we have identified both TGF-β2 and TGF-β3 transcripts in early stages of chick development. In addition, we have identified a new gene which is transcribed during early chick development. We have called this gene Vgc1 since it is probably the chick homologue of the mouse Vg-related gene Vgrl. RNA coding for the Vgcl gene was detected by RNAse protection in early stage X blastoderms, gastrula and 3-day-old embryos. In situ hybridization experiments further indicate that Vgcl RNA is distributed uniformly throughout the various regions of the chick gastrula. The role of Vgcl in early chick development remains to be established.

ME1 and GE1: basic helix-loop-helix transcription factors expressed at high levels in the developing nervous system and in morphogenetically active regions.

Several class A basic helix-loop-helix (bHLH) transcription factors have been cloned from the developing mouse and chick nervous system. The cloned cDNAs (ME1, ME2, ME3, ME4, in the mouse and GE1, GE2 in the chick) have HLH coding regions highly homologous to other known class A bHLH genes. The genes corresponding to ME1 and GE1 are abundantly expressed during development of the central nervous system. ME1 and GE1 are expressed in proliferating neuroblasts and in cells at the initial stages of differentiation (for example in the external granule cell layer of the cerebellum and in the lateral region of the ventricular zone in the developing neural tube and cortex). They are also expressed at high levels in morphogenetically active regions such as limb buds, somites and mesonephric tubules. The expression of ME1 and GE1 decreases once cellular differentiation is over. Based on the expression of ME1 and GE1 in regions of active cellular proliferation and differentiation and on the known role of other bHLH factors in development, we suggest that ME1 and GE1 play important roles during development of the nervous system as well as in other organ systems.

Histone acetylation in Zea mays. II. Biological significance of post-translational histone acetylation during embryo germination.

Multiple forms of histone acetyltransferases and histone deacetylases, which have been separated and characterized in the accompanying manuscript (López-Rodas, G., Georgieva, E. I., Sendra, R., and Loidl, P. (1991) J. Biol. Chem. 266, 18745-18750), together with in vivo acetate incorporation, were studied during the germination of Zea mays embryos. Total histone acetyltransferase activity increases during germination with two maxima at 40 and 72 h after start of germination. This fluctuation is mainly due to the cytoplasmic B-enzyme which predominantly acetylates histone H4 up to the diacetylated form. The nuclear histone acetyltransferase A2, specific for H3, is low throughout germination, except at 24 h, when it transiently becomes the main activity. Both enzymes are also present in the dry embryo, whereas the second nuclear enzyme A1, specific for H3 and H4, is absent in the initial stage of differentiation. The two histone deacetylases, HD1 and HD2, exhibit entirely different patterns. Whereas HD1 activity is low in the dry embryo and increases during germination, HD2 is the predominant enzyme at the start of differentiation, but almost disappears at later stages. Analysis of the in vivo acetate incorporation reveals that H4 is present in up to tetraacetylated subspecies. The pattern of acetate incorporation into core histones closely resembles the fluctuations of histone acetyltransferase B. Based on the analysis of thymidine kinase activity a close correlation was established between histone acetyltransferase B and DNA replication, whereas the A2 enzyme is associated with transcriptional activity. Histone deacetylase HD1 obviously serves a specific function in the dry embryo and could be a prerequisite for DNA repair processes. The study confirms the idea of DNA repair processes. The study confirms the idea of multiple functions of histone acetylation and assigns distinct enzymes, involved in this modification, to certain nuclear processes.

In vitro differential developmental toxicity of vitamin A congeners.

Several forms of vitamin A were tested in the in vitro hydra assay for their developmental toxicity hazard potential and site of action on progressive ontogenesis. Retinol, retinyl acetate, retinaldehyde, all trans retinoic acid, and 13 cis retinoic acid were tested fully, and each was established clearly as being able to perturb development of artificial hydra "embryos" at, or near, adult toxic treatment levels. All forms of vitamin A tested interfered with differentiation, but, although the alcohol, acetate, and aldehyde forms (group I) prevented the initial stages of differentiation from occurring, the acid forms (group II) allowed the initial stages of differentiation to occur but not the final differentiation of tentacle buds. Group I compounds produced the developmental toxicity endpoint after as little as 24 h of transient exposure on the first day of development, but had no permanent effect on development at their minimal affective developmental concentration (D-MAC) when exposure began after the first day of development. In contrast, transient 24-h exposure to group II forms did not interfere with development. At, or even above, a concentration greater than the D-MAC, more continuous exposure to them was required to interfere with differentiation. Consistent with tests of other chemicals, the concentrations needed to produce effects in hydra bore no relation to those needed to produce effects in mammals.

Carbohydrate histochemistry of the epidermis of Natrix piscator during its sloughing cycle

Carbohydrate histochemistry of the scale and hinge epidermis of the chequered water snake, Nalrix piscator, throughout the sloughing cycle, has been described. The small amount of mucopolysaccharide present in the Oberhautchen, mesos layer, α‐layer and β‐Mayer (in its initial stage of differentiation) is comparable with that in amphibian epidermis and the epidermis of certain freshwater fish undergoing keratinization. Moderate amounts of mucopolysaccharide in the lacunar tissue and clear layer may protect against environmental pathogens and retain water to protect the epidermis from desiccation. Mucous cells could not be located in the epidermis throughout the sloughing cycle, contrary to some previous observations that they occur in the hinge region. The general absence of glycogen in the epidermis in most stages of the sloughing cycle suggests that the glycogen metabolized in the epidermis is utilized immediately, in view of the high energy requirements of proliferation and differentiation. Accumulation of glycogen granules in the presumptive α‐layer in stage 2 and in the clear layer, presumptive Oberhautchen and presumptive β‐Mayer in stage 3 is correlated with low energy requirements, indicating a slowing down of the process of keratinization of cells in these layers.

Vascular system in the developing wing bud of normal and talpid3 mutant chick embryos

The vascular limb mesoderm probably plays a prominent role in limb pattern formation in both normal and talpid 3 chick embryos. The differential vascularization, or its consequences, is a factor controlling the initial stages of differentiation in the developing bud. The axial artery runs from the subclavian artery to the distal region of the normal limb bud, whereas in the talpid 3 only secondary blood vessels develop. In the talpid 3, the gene permits the chondrogenic regions to grow and at the same time keeps the peripheral regions to a certain size. The mesenchyme tissue lies within the effective range of a metabolic gradient extending from either the ectodermal surface or the peripheral vessels to the limb axis.

Ultrastructural analysis of the in vitro differentiation of female rat preadipocytes.

In an attempt to characterize the preadipocytes of the adipose tissue of female rat, we studied by electron microscopy the differentiation of the cells into mature adipocytes in in vitro cultures. The preadipocytes arose from the stroma-vascular fraction of perirenal and perigenital adipose tissue. Culture of the preadipocytes in an enriched medium consisting of Dulbecco's medium supplemented with 10% fetal calf serum, antibiotics, rat triglycerides (0.5%), insulin (290 nM) and Tween 80 (0.1 mg/ml) induced their adipose conversion. The morphology of preadipocytes changed progressively. They accumulated fat granules, droplets and finally globules, which fused together. The cell organelles featured qualitative and quantitative modifications. The nucleus migrated with most mitochondria and a part of the Golgi system towards the cell periphery; the rough endoplasmic reticulum, dilated at the initial stage of differentiation became less and less conspicuous; the perinuclear Golgi system was dispersed between lipid droplets during fat accumulation; thick bundles of microfilaments, localized beneath the plasma membrane disappeared; large lipid droplets were surrounded by a network of microfilaments; many microvesicles and some "rosettes" typical of mature adipocytes could be observed. Nevertheless, the ultrastructural criteria did not allow to clearly discriminate the undifferentiated cells: early preadipocytes (without lipid droplets), adipoblasts and fibroblasts, all of these being probably present in the culture system.