bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo
Authors: Hardy Rebecca , Reynolds Richard
Published: April 2021

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LBi, which binds to the surface ganglioside GD3 expressed on 0-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5’bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC+-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.
Origins and patterning of avian outflow tract endocardium
Authors: Noden Drew M.
Published: April 2021

Outflow tract endocardium links the atrioventricular lining, which develops from cardiogenic plate mesoderm, with aortic arches, whose lining forms collectively from splanchnopleuric endothelial channels, local endothelial vesicles, and invasive angioblasts. At two discrete sites, outflow tract endocardial cells participate in morphogenetic events not within the repertoire of neighboring endocardium: they form mesenchymal precursors of endocardial cushions.

The objectives of this research were to document the history of outflow tract endocardium in the avian embryo immediately prior to development of the heart, and to ascertain which, if any, aspects of this history are necessary to acquire cushion-forming potential. Paraxial and lateral mesodermal tissues from between somitomere 3 (midbrain level) and somite 5 were grafted from quail into chick embryos at 3–10 somite stages and, after 2–5 days incubation, survivors were fixed and sectioned. Tissues were stained with the Feulgen reaction to visualize the quail nuclear marker or with antibodies (monoclonal QH1 or polyclonals) that recognize quail but not chick cells. Many quail endothelial cells lose the characteristic nuclear heterochromatin marker, but they retain the species-specific epitope recognized by these antibodies.

Precursors of outflow tract but not atrioventricular endocardium are present in cephalic paraxial and lateral mesoderm, with their greatest concentration at the level of the otic placode. Furthermore, the ventral movement of individual angiogenic cells is a normal antecedent to outflow tract formation. Cardiac myocytes were never derived from grafted head mesoderm. Thus, unlike the atrioventricular regions of the heart, outflow tract endocardial and myocardial precursors do not share a congruent embryonic history.

The results of heterotopic transplantation, in which trunk paraxial or lateral mesoderm was grafted into the head, were identical, including the formation of cushion mesenchyme. This means that cushion positioning and inductive influences must operate locally within the developing heart tubes.
The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers
Authors: Alberga Audrey , Boulay Jean-Louis , Kempe Elisabeth , Dennefeld Christine , Haenlin Marc
Published: April 2021

The zygotic effect gene snail (sna) encodes a zinc-finger protein required for mesodenn formation in Drosophila embryos. By in situ analysis, sna transcripts are first detected at syncytial blastoderm and persist until very late stages of embryogenesis. Expression of sna is transient and is observed in tissues derived from all three germ layers. Prior to germband elongation, sna RNA accumulation is consistent with its genetically deter mined role in mesoderm formation. Starting at germ band elongation, a second phase of sna expression appears to be initiated, characterized by a highly dynamic accumulation of transcripts in the developing central and peripheral nervous systems. Translation of sna RNA is apparently delayed as the sna protein is not detected before the onset of gastrulation. Its regional distribution generally correlates with that of sna transcripts. The complex pattern of sna expression strongly suggests that the function of the gene is not restricted to mesoderm formation.
Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments
Authors: Mortillo Steven , Wassarman Paul M.
Published: April 2021

Egg zona pellucida glycoproteins mZP3 and mZP2 serve as primary and secondary sperm receptors, respectively, during initial stages of fertilization in mice [Wassarman (1988) A. Rev. Biochem. 57, 415–442]. These receptors interact with complementary egg-binding proteins (EBPs) located on the sperm surface to support speciesspecific gamete adhesion. Results of whole-mount autoradiographic experiments suggest that purified egg mZP3 and mZP2 bind preferentially to acrosome-intact (Al) and acrosome-reacted (AR) sperm heads, respectively [Bleil and Wassarman (1986)J. Cell Biol. 102, 1363–1371]. Here, we used purified egg mZP2, egg mZP3 and fetuin, which were coupled directly to colloidal gold (‘gold-probes’), to examine binding of these glycoproteins to membrane compartments of Al and AR sperm by transmission electron microscopy. mZP3 gold-probes were found associated primarily with plasma membrane overlying the acrosomal and post- acrosomal regions of Al sperm heads. They were also found associated with plasma membrane overlying the post-acrosomal region of AR sperm heads. mZP2 gold- probes were found associated primarily with inner acrosomal membrane of AR sperm heads, although some gold was associated with outer acrosomal membrane of Al sperm that had holes in plasma membrane overlying the acrosome. Fetuin gold-probes, used to assess background levels of binding, were bound at relatively low levels to plasma membrane and inner acrosomal membrane of Al and AR sperm, respectively. None of the gold-probes exhibited significant binding to sperm tails, or to red blood cells and residual bodies present in sperm preparations. These results provide further evidence that mZP2 and mZP3 bind preferentially to heads of AR and Al sperm, respectively. Furthermore, they demonstrate that mZP2 and mZP3 bind preferentially to different sperm membrane compartments and permit quantitative assessment of the distribution and abundance of EBPs on sperm.
Spatiotemporal expression of three gap junction gene products involved in fetomaternai communication during rat pregnancy
Authors: Risek Boris , Gilula Norton B.
Published: April 2021

The expression of three different members of the gap junction multigene family, ax (Cx43), ft (Cx32), and p2 (Cx26), was analysed in the rat implantation chamber (a structural unit containing fetal, extraembryonic and maternal components within the pregnant uterus) during mid-and late stages of gestation as well as in the delivering, post-partum and non-pregnant uterus. A differential, spatiotemporal and cell-type-specific regulation of gap junctional coexpression was observed for Pi and p2 • ″ a ″ epithelia examined (visceral, luminal and glandular), as well as for a, and ft in decidual cells and keratinocytes of the fetal epidermis, aj antigen was detected in the mesometrial stroma, mesometrial myometrium, connective tissue, mesothelia of the amnion and visceral yolk sac and in the allantoic mesodermal layer throughout gestation. In addition, expression of at in the placental basal zone and trophoblast giant cells coincided with the differentiation of these cells, fi expression was observed prominently in the chorionic villi of the placental labyrinth. The presence of Pi and P2 in the visceral epithelium (visceral yolk sac=the primary route for embryonic nourishment prior to the formation of the chorioallantoic placenta) and p2 in the chorionic villi (placental barrier=the major fetomaternai exchange route) suggests that gap junctions have an important role in fetomaternai communication.
Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo
Authors: Sutherland A. E. , Sanderson R. D. , Mayes M. , Seibert M. , Calarco P. G. , Damsky C. H. , Bernfield M.
Published: April 2021

Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast growth factor (bFGF) thus promoting bFGF association with its high- affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell – cell contact. At the blastocyst stage, syndecan is first detected at cell – cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectopiacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69×l03Mr), does not change during periimplantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.

Abbreviations: DGD, diethylene glycol distearate; bFGF, basic fibroblast growth factor; GAG, glycosaminoglycan; mAb, monoclonal antibody; PEG, polyethylene glycol.
Clonal analysis of astrocyte diversity in neonatal rat spinal cord cultures
Authors: Miller Robert H. , Szigeti Vilma
Published: April 2021

Within the mammalian CNS, astrocytes appear to be a heterogeneous class of cells. To assay the number of distinct types of astrocytes in the rat spinal cord, cell lineage and phenotypic analyses were carried out on cultures from newborn rat spinal cord and five distinct types of astrocytes were observed. Proliferating precur- sors for each class of astrocyte were isolated by low density culture and shown to give rise to 5 distinct and morphologically homogeneous clusters of GFAP+ astro- cytes. Immunocytochemical analysis with antibodies A2B5 and Ran-2, which identify different glial lineages in optic nerve cultures, demonstrated that many clusters included both A2B5+ and A2B5− cells. Similarly, many clusters also possessed a mixture of Ran-2 + and Ran-2− cells, suggesting that in spinal cord cultures, in contrast to optic nerve cultures, expression of these antigens is regulated by individual cells rather than by cell lineage.

Single-cell cloning studies, revealed that the abun- dance and proliferative capacity of individual astrocyte precursors differed depending on the type of astrocyte. To assay the effects of a complex cellular environment on the composition of astrocyte clones, lineage analysis was performed in complete spinal cord cultures using a replication deficient retrovirus. Although similar mor- phologically homogeneous clones of cells to those seen with single-cell clones were observed, the proliferative capacity and relative abundance of the distinct astrocyte precursors differed from that seen in single-cell cloning studies. Together these observations suggest that in spinal cord, gliogenesis is considerably more complex than in the optic nerve and that cultures of newborn rat spinal cord contain multiple, distinct populations of astrocytes.
Development of the indirect flight muscles of Drosophila
Authors: Fernandes Joyce , Bate Michael , Vijayraghavan K.
Published: April 2021

We have followed the pupal development of the indirect flight muscles (IFMs) of Drosophila melanogaster. At the onset of metamorphosis larval muscles start to histolyze, with the exception of a specific set of thoracic muscles. Myoblasts surround these persisting larval muscles and begin the formation of one group of adult indirect flight muscles, the dorsal longitudinal muscles. We show that the other group of indirect flight muscles, the dorsoventral muscles, develops simultaneously but without the use of larval templates. By morphological criteria and by patterns of specific gene expression, our experiments define events in IFM development.
Elements of the fruitless locus regulate development of the muscle of Lawrence, a male-specific structure in the abdomen of Drosophila melanogaster adults
Authors: Gailey Donald A. , Taylor Barbara J. , Hall Jeffrey C.
Published: April 2021

A genetically defined element of the fruitless (fru) locus in Drosophila melanogaster regulates the development of a male-specific muscle spanning the fifth abdominal segment in adult males, the ‘muscle of Lawrence’ (MOL). The region is defined by two cytological deletions, each with a breakpoint that co-maps with previously described mutant courtship phenotypes at cytogenetic interval 91B on the third chromosome. Flies that carry both of these deletions are viable, and males express abnormalities of courtship similar to those caused by the fru inversion breakpoint at 91B. In addition, these double-deletion males show the complete absence of the MOL, suggesting that they have little or no gene expression of a postulated MOL determinant; the musculature in the fifth abdominal segment of these mutants to indistinguishable from that of a normal female. Other mutant combinations that produce fruitless courtship phenotypes -including deletion and inversion breakpoints, and a marked transposon inserted at 91B -produce intermediate forms of the MOL. A new genetic variant, induced by imprecise excision of the marked transposon, is homozygous lethal and disrupts fru functions related to courtship and the MOL. The MOL is shown to be dispensable for fertility and is therefore not the causative factor of fru-induced behavioral sterility. These genetic variants and their phenotypic results are discussed with regard to a model for the organization of the fru locus.
Developmental changes in the subcellular distribution of the 43K (v1) polypeptides in Torpedo marmorata electrocyte: support for a role in acetylcholine receptor stabilization
Authors: Nghiêm Hoàng-Oanh , Hill Joseph , Changeux Jean-Pierre
Published: April 2021

Analysis of the relative amounts of the acetylcholine receptors (AChR) and of the 43K protein present in the membrane of developing electrocyte shows that massive accumulation of 43K protein is not required for induction of early AChR clustering. Furthermore, we demonstrate the existence of cytosol- and membrane-associated 43K polypeptide pools in Torpedo electrocyte. Epitope analysis shows that both pools of 43K protein are related to the long mRNA transcript and share similar antigenic determinants distributed throughout the protein sequence. Their partition between the cytosol and membrane fractions abruptly increases in favor of the membrane during the postsynaptic maturation phase of development, supporting a role for 43K protein in the stabilization and maintenance of the postsynaptic domain.
Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins
Authors: Lallier Thomas , Bronner-Fraser Marianne
Published: April 2021

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick β1, subunit of integrin, suggesting that β1integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (>1 μgml−1; Low-LM), but not at high coating concentration of laminin (10 μgml−1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required >0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165 000 Mr which is also found in immunoprecipitates using antibodies against the β1subunit of integrin and is likely to be an integrin a subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of β1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin.
The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo
Authors: Poirier Francoise , Chan C.-T. J. , Timmons Paula M. , Robertson Elizabeth J. , Evans Martin J. , Rigby Peter W. J.
Published: April 2021

The differentiation in vitro of murine embryonic stem cells to embryoid bodies mimicks events that occur in vivo shortly before and after embryonic implantation. We have used this system, together with differential cDNA cloning, to identify genes the expression of which is regulated during early embryogenesis. Here we describe the isolation of several such cDNA clones, one of which corresponds to the gene H19. This gene is activated in extraembryonic cell types at the time of implantation, suggesting that it may play a role at this stage of development, and is subsequently expressed in all of the cells of the mid-gestation embryo with the striking exception of most of those of the developing central and peripheral nervous systems. After birth, expression of this gene ceases or is dramatically reduced in all tissues.
A target-derived chemoattractant controls the development of the corticopontine projection by a novel mechanism of axon targeting
Authors: O’leary D. D. M. , Heffner C. D. , Kutka L. , López-Mascaraque L. , Missias A. , Reinoso B. S.
Published: April 2021

Here, we review our studies in rats of target recognition by developing cortical axons focusing on their innervation of the basilar pons, a major hindbrain target. The corticopontine projection develops by a ‘delayed interstitial budding’ of collaterals from layer 5 corticospinal axons, rather than by a direct ingrowth of primary axons or by bifurcation of the growth cone. Branches form de novo from the axon cylinder in the pathway overlying the basilar pons and extend directly into it. Cocultures of cortex and basilar pons in 3-dimensional collagen matrices show that a diffusible chemotropic signal released by the basilar pons directs the growth of collateral branches from layer 5 axons in a target and neuron specific manner. ‘Delayed’ co-cultures suggest that a diffusible, pontine-derived signal also initiates the selective branching of layer 5 axons. In vivo experiments support this chemotropic mechanism. First, corticospinal axons form collateral branches at novel locations directly over ectopic aggregations of basilar pontine neurons induced by X-irradiation; no branches form at positions that would normally overlie the appropriate region of basilar pons which is absent because of the X-irradiation. Thus, the basilar pons, rather than local cues in the axon pathway, appears to control the location of corticospinal axon branching. Second, in a series of experiments in which different subsets of corticospinal axons are prevented from innervating the basilar pons, remaining corticospinal axons extend collaterals in a directed manner to regions of the basilar pons deprived of cortical input, a behavior consistent with a response to a diffusible chemoattractant emanating from these regions. In conclusion, our findings suggest that a diffusible, target-derived chemotropic molecule(s) underlies target recognition in this developing system by initiating the formation and directing the growth of pontine collateral branches of primary layer 5 corticospinal axons.
Motoneuronal development in the embryonic zebrafish
Authors: Eisen Judith S.
Published: April 2021

To learn how neurons find their appropriate targets, we have studied two populations of motoneurons in the embryonic zebrafish: primary motoneurons, individually identified cells whose growth cones pioneer the first nerve pathways in the muscle, and secondary motoneurons, cells which develop later and whose growth cones apparently extend along the axons of the primary motoneurons. Transplantation studies of single, identified primary motoneurons suggest that commitment of these cells to innervate their cell-specific muscle territories may be a multistep process in which they are first committed to be motoneurons and are later committed to extend axons along specific pathways. Ablation studies suggest that interactions among the primary motoneurons are unlikely to be necessary for proper pathfinding or commitment. However, interactions with the primary motoneurons may be important for proper development of the secondary motoneurons.
The developmental fate of the cephalic mesoderm in quail-chick chimeras
Authors: Couly Gérard F. , Coltey Pierre M. , Le Douarin Nicole M.
Published: April 2021

The developmental fate of the cephalic paraxial and prechordal mesoderm at the late neorula stage (3- somite) in the avian embryo has been investigated by using the isotopic, isochronic substitution technique between quail and chick embryos. The territories involved in the operation were especially tiny and the size of the transplants was of about 150 by 50 to 60 μm. At that stage, the neural crest cells have not yet started migrating and the fate of mesodermal cells exclusively was under scrutiny. The prechordal mesoderm was found to give rise to the following ocular muscles: musculus rectus ventraUs and medialis and musculus oblicus verttralis. The paraxial mesoderm was separated in two longitudinal bands: one median, lying upon the cephalic vesicles (median paraxial mesoderm - MPM); one lateral, lying upon the foregut (lateral paraxial mesoderm - LPM). The former yields the three other ocular muscles, contributes to mesencephalic meninges and has essentially skeletogenic potencies. It contributes to the corpus sphenoid bone, the orbitosphenoid bone and the otic capsules; the rest of the facial skeleton is of neural crest origin. At 3-somite stage, MPM is represented by a few cells only. The LPM is more abundant at that stage and has essentially myogenic potencies with also some contribution to connective tissue. However, most of the connective cells associated with the facial and hypobranchial muscles are of neural crest origin.

The more important result of this work was to show that the cephalic mesoderm does not form dermis. This function is taken over by neural crest cells, which form both the skeleton and dermis of the face. If one draws a parallel between the so-called “somitomeres” of the head and the trunk somites, it appears that skeletogenic potencies are reduced in the former, which in constrast have kept their myogenic capacities, whilst the forma­tion of skeleton and dermis has been essentially taken over by the neural crest in the course of evolution of the vertebrate head.
A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl
Authors: Delarue M. , Sanchez S. , Johnson K.E. , Darribere T. , Boucaut J.C.
Published: April 2021

We have determined the fate of presumptive mesodermal cells in the early Pleurodeles waltl gastrula. We labeled all cells in a gastrula with RLDx cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the presumptive regions are generally distributed according to classical fate maps for urodeles but that the boundaries between presumptive regions are indistinct, due to extensive intermingling between cells at the edges of grafted regions. We have shown that there is a high dorsal to low ventral gradient of mixing between superficial and deep cells.
Induction and repression of mammalian achaete-scute homologue (MASH) gene expression during neuronal differentiation of P19 embryonal carcinoma cells
Authors: Johnson Jane E. , Zimmerman Kathryn , Saito Tetsuichiro , Anderson David J.
Published: April 2021

MASH1 and MASH2, mammalian homologues of the Drosophila neural determination genes achaete-scute, are members of the basic helix-loop-helix (bHLH) family of transcription factors. We show here that murine P19 embryonal carcinoma cells can be used as a model system to study the regulation and function of these genes. MASHl and MASHl display complementary patterns of expression during the retinoic-acid-induced neuronal differentiation of P19 cells. MASHl mRNA is undetectable in undifferentiated P19 cells but is induced to high levels by retinoic acid coincident with neuronal differentiation. In contrast, MASHl mRNA is expressed in undifferentiated P19 cells and is repressed by retinoic acid treatment. These complementary expression patterns suggest distinct functions for MASHl and MASH2 in development, despite their sequence homology. In retinoic-acid-treated P19 cells, MASHl protein expression precedes and then overlaps expression of neuronal markers. However, MASHl is expressed by a smaller proportion of cells than expresses such markers.

MASHl immunoreactivity is not detected in differentiated cells displaying a neuronal morphology, suggesting that its expression is transient. These features of MASHl expression are similar to those observed in vivo, and suggest that P19 cells represent a good model system in which to study the regulation of this gene. Forced expression of MASHl was achieved in undifferentiated P19 cells by transfection of a cDNA expression construct. The transfected cells expressing exogenous MASHl protein contained E-box-binding activity that could be supershifted by an anti-MASHl antibody, but exhibited no detectable phenotypic changes. Thus, unlike myogenic bHLH genes, such as MyoD, which are sufficient to induce muscle differentiation, expression of MASHl appears insufficient to promote neurogenesis.
Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths
Authors: Behrendtsen Ole , Alexander Caroline M. , Werb Zena
Published: April 2021

The maintenance and developmental remodeling of extracellular matrix is crucial to such processes as uterine implantation and the cell migratory events of morphogenesis. When mouse blastocysts are placed in culture they adhere to extracellular matrix, and trophoblast giant cells migrate out onto the matrix and degrade it. The secretion of functional proteinases by developing mouse embryos increases dramatically at the time of implantation. By zymography we identified the major secreted gelatin-degrading proteinase, also known as type IV collagenase, as one migrating at 92 × 103Mr. Several casein-degrading proteinases were also secreted. The tissue inhibitor of metalloproteinases (TIMP) inhibited all of the embryo-derived proteinases detected by gelatin gel zymography, indicating that they are metalloproteinases, whereas TIMP did not inhibit all of the caseinases. Urokinase was also secreted. Addition of TIMP at 5·500 nM effectively inhibited the degradation of matrix by the trophoblast outgrowths. Blocking antibodies directed against 92 × 103Mr gelatinase abolished matrix degradation by the trophoblast cells. These observations suggest that several metalloproteinases are regulated in early development and that 92 × 103Mr gelatinase, in particular, has a rate-limiting function in degradation of the maternal extracellular matrix by trophoblast cells.
Metanephric mesenchyme contains multipotent stem cells whose fate is restricted after induction
Authors: Herzlinger Doris , Koseki Chizuko , Mikawa Takashi , Al-Awqati Qais
Published: April 2021

At least fourteen epithelial cell types of the mammalian nephron develop from the metanephric mesenchyme. To distinguish whether this single embryological primor-dium contains a heterogenous population of committed renal cell lines or a multipotent stem cell, the lac-Z gene was introduced into individual renal progenitors by retroviral mediated gene transfer. The differentiated fate of Zac-Z-tagged daughters derived from single metanephric mesenchymal cells was characterized after cytodifferentiation. We found that the metanephric mesenchyme contains multipotent stem cells that can generate at least three distinct cell types; glomerular, proximal and distal epithelia. After induction the fate of this multipotent cell becomes restricted to populate a single nephron segment.
Superman, a regulator of floral homeotic genes inArabidopsis
Authors: Bowman John L. , Sakai Hajime , Jack Thomas , Weigel Detlef , Mayer Ulrike , Meyerowitz Elliot M.
Published: April 2021

We describe a locus, SUPERMAN, mutations in which result in extra stamens developing at the expense of the central carpels in the Arabidopsis thaliana flower. The development of superman flowers, from initial primor-dium to mature flower, is described by scanning electron microscopy. The development of doubly and triply mutant strains, constructed with superman alleles and previously identified homeotic mutations that cause alterations in floral organ identity, is also described. Essentially additive phenotypes are observed in superman agamous and superman apetala2 double mutants. The epistatic relationships observed between either apetala3 or pistillata and superman alleles suggest that the SUPERMAN gene product could be a regulator of these floral homeotic genes. To test this, the expression patterns of AGAMOUS and APETALA3 were examined in superman flowers. In wild-type flowers, APETALA3 expression is restricted to the second and third whorls where it is required for the specification of petals and stamens. In contrast, in superman flowers, APETALA3 expression expands to include most of the cells that would normally constitute the fourth whorl. This ectopic APETALA3 expression is proposed to be one of the causes of the development of the extra stamens in superman flowers. The spatial pattern of AGAMOUS expression remains unaltered in superman flowers as compared to wild-type flowers. Taken together these data indicate that one of the functions of the wild-type SUPERMAN gene product is to negatively regulate APETALA3 in the fourth whorl of the flower. In addition, superman mutants exhibit a loss of determi-nacy of the floral meristem, an effect that appears to be mediated by the APETALA3 and PISTILLATA gene products.
Commitment of abdominal neuroblasts in Drosophila to a male or female fate is dependent on genes of the sex-determining hierarchy
Authors: Taylor Barbara J. , Truman James W.
Published: April 2021

Adult specific neurons in the central nervous system of holometabolous insects are generated by the postem-bryonic divisions of neuronal stem cells (neuroblasts). In the ventral nervous system of Drosophila melanogaster, sex-specific divisions by a set of abdominal neuroblasts occur during larval and early pupal stages. Animals mutant for several sex-determining genes were analyzed to determine the genetic regulation of neuroblast commitment to the male or female pattern of division and the time during development when these decisions are made. We have found that the choice of the sexual pathway taken by sex-specific neuroblasts depends on the expression of one of these genes, doublesex (dsx). In the absence of any functional dsx+ products, the sexspecific neuroblasts fail to undergo any postembryonic divisions in male or female larval nervous systems. From the analysis of intersexes generated by dominant alleles of dsx, it has been concluded that the same neuroblasts provide the sex-specific neuroblasts in both male and female central nervous systems. The time when neuroblasts become committed to generate their sex-specific divisions was identified by shifting tra-2ts flies between the male- and female-specifying temperatures at various times during larval development. Neuroblasts become determined to adopt a male or female state at the end of the first larval instar, a time when abdominal neuroblasts enter their first postembryonic S-phase.
Sequence and expression pattern ofpax-6are highly conserved between zebrafish and mice
Authors: Püschel Andreas W. , Gruss Peter , Westerfield Monte
Published: April 2021

Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice.

We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog, pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region, pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.
Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons
Authors: Pike Susan H. , Melancon Eloine F. , Eisen Judith S.
Published: April 2021

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.
Skeletal muscle phenotypes initiated by ectopic MyoD in transgenic mouse heart
Authors: Miner Jeffrey H. , Miller Jeffrey B. , Wold Barbara J.
Published: April 2021

Forced expression of the myogenic regulatory gene MyoD in many types of cultured cells initiates their conversion into skeletal muscle. It is not known, however, if MyoD expression serves to activate all or part of the skeletal muscle program in vivo during animal development, nor is it known how limiting the influences of cellular environment may be on the regulatory effects of MyoD. To begin to address these issues, we have produced transgenic mice which express MyoD in developing heart, where neither MyoD nor its three close relatives - myogenin, Myf-5, and MRF4/her-culin/Myf-6 - are normally expressed. The resulting gross phenotype in offspring from multiple, independent transgenic founders includes abnormal heart morphology and ultimately leads to death. At the molecular level, affected hearts exhibit activation of skeletal muscle-specific regulatory as well as structural genes. We conclude that MyoD is able to initiate the program that leads to skeletal muscle differentiation during mouse development, even in the presence of the ongoing cardiac differentiation program. Thus, targeted misexpression of this tissue-specific regulator during mammalian embryogenesis can activate, either directly or indirectly, a diverse set of genes normally restricted to a different cell lineage and a different cellular environment.
Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: a role for GH in embryonic and fetal development?
Authors: Garcia-Aragón Juanita , Lobie Peter E. , Muscat George E. O. , Gobius Kari S. , Norstedt Gunnar , Waters Michael J.
Published: April 2021

Although fetal growth is generally considered to be independent of pituitary growth hormone (GH), it is possible that pituitary GH plays a modulatory role in organ development or that a GH-like substance of non pituitary origin may influence fetal growth through the GH receptor. Accordingly, we have used immunohisto-chemisty, northern blot analysis, the reverse transcriptase-polymerase chain reaction and solution hybridization to study the ontogeny of the GH receptor/binding protein (BP) from the 12-day-old embryo (E12) to the E18 rat fetus.

GH receptor/BP immunoreactivity was observed in all major organ systems of the E18 rat fetus and was not preferentially associated with any germ layer derivative. A general increase in GH receptor/BP immunoreactivity was evident from E12 to E18, with a marked increase occurring between E16 and E18. Hemangioblastic tissue was, however, strongly or intensely immunoreactive at all stages of development, as was the placenta. Most noteworthy of the other tissues expressing GH receptor/BP immunoreactivity by day 18 were skeletal and smooth muscle, chondroprogenitor cells, epithelial lining cells, neuronal ganglia, ependymal cells and the adrenal cortex. In the placenta, the most prominent immunoreactivity was associated with decidual cells.

Total RNA was isolated from E12 to E18 rat fetuses and adult rat liver. Northern hybridization with a 35S-labelled rat GH receptor cRNA probe revealed that 3.9 kb and 1.2 kb transcripts complementary to the rat GH receptor ribopobe are present from at least E16. The existence of GH receptor mRNA at E12 and E14 was demonstrated by the polymerase chain reaction. Solution hybridization analysis demonstrated an exponential increase in the total embryonic GH receptor/BP mRNA during fetal development. However even by E18 levels were only 8% those of adult liver.

In conclusion, we have demonstrated the presence and defined the ontogeny of the GH receptor/BP in the rat foetus from mid gestation. These results, when combined with earlier studies of GH action on fetal tissues suggest a réévaluation of the role of GH in prenatal growth.
Evolutionary dissociation between cleavage, cell lineage and embryonic axes in sea urchin embryos
Authors: Henry Jonathan J. , Klueg Kristin M. , Raff Rudolf A.
Published: April 2021

Using vital dye staining and the microinjection of fluorescent cell lineage-autonomous tracers, the relationship between the first cleavage plane and the prospective larval dorsoventral axis was examined in several sea urchin species, including: Strongylocentrotus purpuratus, S. droebachiensis, Lytechinus pictus, Clypeaster rosaceus, Heliocidaris tuberculata and H. erythrogramma. The results indicate that there is no single relationship between the early cleavage pattern and the dorsoventral axis for all sea urchins; however, specific relationships exist for individual species. In S. purpuratus the first cleavage plane occurs at an angle 45 degrees clockwise with respect to the prospective dorsoventral axis in most cases, as viewed from the animal pole. On the other hand, in S. droebachiensis, L. pictus and H. tuberculata, the first cleavage plane generally corresponds with the plane of bilateral symmetry. There does not appear to be a predominant relationship between the first cleavage plane and the dorsoventral axis in C. rosaceus. In the direct-developing sea urchin H. erythrogramma the first cleavage plane bisects the dorsoventral axis through the frontal plane. Clearly, evolutionary differences have arisen in the relationship between cleavage pattern and developmental axes. Therefore, the mechanism of cell determination is not necessarily tied to any particular pattern of cell cleavage, but to an underlying framework of axial systems resident within sea urchin eggs and embryos.
Embryonic cytoplasmic extracts rescue murine androgenones to the blastocyst stage
Authors: Hagemann Lora J. , First Neal L.
Published: April 2021

Androgenones (paternally derived genome) show a significant inability to form a blastocoele cavity. Eighty percent of these embryos die or arrest at earlier stages. Factor(s) from both normal and partbenogenetic late preimplantation embryos injected into each blastomere of androgenetic 4-cell stage can rescue more than twice as many to the blastocyst stage (47.2% versus 19.2% for non-injected androgenones). This factor(s) becomes available beginning at the 4-cell stage and is titratable. Injected total cytoplasmic mRNA will also cause a rescue response. Isolating this specific factor message(s) will permit the eventual cloning of possibly the earliest parentally imprinted gene(s) expressed during development.
Expression of the Brachyury gene during mesoderm development in differentiating embryonal carcinoma cell cultures
Authors: Vidricaire Gaël , Jardine Karen , McBurney Michael W.
Published: April 2021

When aggregated and treated with dimethyl sulfoxide (DMSO), P19 embryonal carcinoma cells differentiate into cell types normally derived from the mesoderm and endoderm including epithelium and cardiac and skeletal muscle. The Brachyury gene is expressed transiently in these differentiating cultures several days before the appearance of markers of the differentiated cell types. The expression of Brachyury is not affected by DMSO but is induced by cell aggregation, which requires extracellular calcium. Expression of Brachyury is also induced by various members of the TGFβ family such as activin and bone morphogenetic proteins. D3 is a mutant clone of P19 cells selected for its failure to differentiate when aggregated in DMSO. Aggregated D3 cells express Brachyury mRNA suggesting that the mutation(s) responsible for the phenotype of D3 cells is downstream of the chain of events initiated by Brachyury expression.
The murine type II TGF-β receptor has a coincident embryonic expression and binding preference for TGF-β1
Authors: Lawler Sean , Candia Albert F. , Ebner Reinhard , Shum Lillian , Lopez Alfredo R. , Moses Harold L. , Wright Christopher V. E. , Derynck Rik
Published: April 2021

We have isolated cDNAs of the murine type II TGF-β receptor and have found a conserved cytoplasmic domain, but a less extensive homology in the extracellular receptor domain between the human and murine homologues. In situ hybridization analysis of the mouse fetus during mid gestation localized the expression of this receptor to various developing tissues, primarily in the mesenchyme and epidermis. This expression pattern correlates well with the expression of TGF-β in general and especially TGF-β1, suggesting that TGF-β1 exerts its developmental role through this receptor in an autocrine or paracrine fashion. Type II receptor expression was not detected in the central nervous system and developing cartilage. These tissues lack TGF-β1 expression but express TGF-β2 and/or TGF-β3, suggesting that they may exert their activities through separate receptor isoforms. In addition, the efficient binding of TGF-β1, but not TGF-β2, to the cloned type II receptor strengthens the likelihood that additional type II receptor isoforms exist which display preferential binding to TGF-β2 and have their own defined role in development.