bluuebunny/crossref_metadata_2025 huggingface dataset:
All-or-none craniorachischisis in Loop-tail mutant mouse chimeras
Authors: Musci Teresa S. , Mullen Richard J.
Published: April 2021

Mouse embryos homozygous for the mutant gene Looptail (Lp) are characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail. In the present study, experimental chimeric mice containing mixtures of genetically mutant (from Lp/ + ×Lp/ + matings) and genetically normal cells were produced. Our aim was to determine whether a ‘rescue,’ phenotypic gradient, or intermediate expression (i.e. alternating areas of open and closed neural tube) would be observed in these chimeras. We report our analyses of Loop-tail mutant chimeras (n=82) by gross examination, progeny testing and quantitative analysis of glucose phosphate isomerase (GPI) isozyme levels. An all-or-none craniorachischisis in Loop-tail mutant chimeras was observed. Two multicolored adult chimeras, without any gross evidence of a neural tube defect, were shown to be homozygous Loop-tail chimeras (Lp/Lp↔+/+) by progeny testing. These results indicate that the normal phenotype can be expressed in the presence of mutant cells. Conversely, six neonates with craniorachischisis were shown to be chimeras by GPI analyses. These results show that the full mutant phenotype can be expressed even when one-third to one-half of the cells are genotypically wild-type. This study did not determine which tissue is primarily responsible for the defective neurulation in this mutant, but suggests that a ‘threshold’ mechanism underlies the Loop-tail mutant phenotype. In some chimeras that threshold is not reached and the neural tube remains open, whereas in other chimeras the threshold is reached and the neural tube closes completely.
Invasive equine trophoblast expresses conventional class I Major Histocompatibility Complex antigens
Authors: Donaldson William L. , Zhang Chonghui H. , Oriol Julio G. , Antczak Douglas F.
Published: April 2021

Monoclonal antibodies and alloantisera were used in an indirect immunohistochemical assay to determine the expression of class I and class II Major Histocompatibility Complex (MHC) antigens by equine placental cells and the endometrial tissues at the fetal-maternal interface. MHC class I antigens were expressed at high density on the surface of the trophoblast cells of the chorionic girdle at days 32–36, just prior to their invasion of the endometrium. The mature gonadotrophin-secreting cells of the endometrial cups, which are derived from the chorionic girdle cells, had greatly reduced levels of MHC class I antigen expression while no MHC class I antigens were detectable on the non-invasive trophoblast cells of the allantochorion, except in small isolated patches. MHC class I antigens inununo-precipitated from chorionic girdle cells with either monoclonal antibodies or alloantisera had a relative molecular mass of 44 000, which was identical to that of MHC class I antigens precipitated from lymphocytes with the same reagents. MHC class H antigens were not detected on any trophoblast cells, although they were expressed at high levels by the endometrial glandular and lumenal epithelium immediately bordering the endometrial cups. MHC class I antigens were also expressed at high levels by endometrial tissues in the area of the cups. The high level of MHC class I antigen expression by endometrial glands within and bordering the cups was in sharp contrast to the greatly reduced class I antigen expression by the mature endometrial cup cells themselves. Polymorphic, paternal, MHC class I antigens that are expressed by the invasive trophoblast cells of the chorionic girdle probably provide the antigenic signal for maternal anti-fetal alloantibody production in early horse pregnancy. Reduced expression of these antigens by terminally differentiated endometrial cup cells may represent a strategy for the protection of the conceptus from maternal immunological attack and destruction that is distinct from prevention of primary maternal sensitization.
NGF mRNA expression in developing cutaneous epithelium related to innervation density
Authors: Harper Sarah , Davies Alun M.
Published: April 2021

To determine if the initial level of NGF mRNA in developing cutaneous epithelium is correlated with its final innervation density, we measured the concentration of NGF mRNA in the epithelia of the maxillary, mandibular and ophthalmic territories of trigeminal ganglion in the embryonic mouse. At the onset of neuronal death in the ganglion there were marked differences in the concentration of NGF mRNA in these epithelia: the level was highest in the epithelium of the densely innervated maxillary territory, it was lower in the epithelium of the moderately innervated mandibular territory and was lowest in the epithelium of the sparsely innervated ophthalmic territory. These regional differences in the level of NGF mRNA during the early stages of target field innervation suggest that the level of NGF production in target field cells, rather than regional differences in the access of innervating neurons to NGF, governs the number of neurons that survive. Because the same percentage cell death occurs in each of the subsets of trigeminal neurons that innervate the maxillary, mandibular and ophthalmic territories, regional differences in NGF synthesis are not responsible for establishing differences in innervation density, rather they maintain differences that arise earlier in development.
Regulations in the induction of the organized neural system in amphibian embryos
Authors: Yamada Tuneo
Published: April 2021

Some of the recent data on the induction of the neural system in amphibian embryos are reviewed, utilizing a model, according to which two basic events regulate in this system: (1) ectodermal dorsalization, which occurs all over the induced region of the ectoderm and is responsible for the neural and mesectodermal pathways and (2) caudalization, which occurs only on the posterior level of dorsalized ectoderm and is responsible for the posterior mode of induced differentiation, functioning as a gradient with the apex at the posterior end of the embryo. Dorsalization of ectoderm can be caused by treatment with Con A or TPA, both of which are potential mitogens. Not only after the treatment with TPA, but also during normal dorsalization, the activation of protein kinase C occurs in responding cells. The possibility is suggested that an early step of mitogenic transmembrane signal transduction induced by a growth factor regulates dorsalization in intact embryos. Ectodermal dorsalization is responsible for the appearance of neuronal and glial cell lineages, and independent of the ECM network formed on the internal surface of the responding ectoderm during gastrulation. In caudalization, a series of experiments suggests that the regulatory role is played by the transcript of the mesodermal posterior homeobox gene, Xhox 3. The expression of this gene in time and location closely coincides with the pattern of convergent extension, one type of morphogenetic movement, which is expressed in a posterior-anterior gradient. This directed cell motility is responsible for the formation of the body axis of vertebrates, and was shown to be involved in caudalization by earlier induction experiments in urodele embryos. Thus clues have been obtained for regulation in dorsalization and caudalization, paving the way for understanding the inductive action of the organizer.
Development and persistence of catecholaminergic neurons in cultured explants of fetal murine vagus nerves and bowel
Authors: Baetge G. , Schneider K. A. , Gershon M. D.
Published: April 2021

Transient catecholaminergic (TC) cells have been found to appear in the vagal pathway and bowel of fetal mice and rats. It has been proposed that these cells are migrating vagal crest-derived precursors of enteric neurons that lose their catecholaminergic properties when they terminally differentiate. In the current experiments, segments of fetal mouse gut were explanted before (day E9) TC cells or any neural markers could be detected in situ. Tyrosine hydroxylase (TH)-immuno-reactive neurons developed in vitro in 4/12 such explants; therefore, cells with a catecholaminergic potential are present in the gut of at least some animals prior to the in situ expression of this phenotype. The neurogenic potential of cells in the vagal pathway was similarly tested by studying cultures of explanted vagus nerves (day Ell). These studies revealed that neural precursors were present in the vagi and gave rise in vitro to neurons that displayed acetylcholinesterase (AChE) activity and neuron-specific enolase (NSE) immunoreactivity. A subset of these neural precursors were capable of migrating and formed satellite ganglia at a distance from the explants. Coincident expression of NSE and TH immunoreactivities was observed, indicating that at least some of the neurons that developed in vitro were derived from TC cells. Vagal TC cells, therefore, are neurogenic. Catecholaminergic cells did not disappear from cultured explants of vagus nerves or gut provided that these tissues contained TC cells at the time of explantation. Instead, catecholaminergic neurons developed and persisted in vitro for as long as cultures were maintained. These neurons contained aromatic L-amino acid decarboxylase as well as TH, NSE and neurofilament immunoreactivities. In contrast, if the bowel was explanted after the in situ disappearance of TC cells, catecholaminergic cells did not arise in the cultures. These experiments indicate that the period of time during which a catecholaminergic phenotype is expressed by neural precursors in the fetal vagal pathway and gut is not fixed, but can be changed by altering the environment of the cells as occurs when the bowel is grown in vitro’, moreover, contact with non-neuronal cells within the bowel is not by itself sufficient to inactivate catecholaminergic expression. The nature of the signal responsible for loss of the catecholaminergic phenotype in situ remains to be determined; however, the persistence of catecholaminergic expression in vitro should facilitate the investigation of this signal. expression, enteric nervous system.
Hormone-sensitive stages in the sexual differentiation of laryngeal muscle fiber number in Xenopus laevis
Authors: Marin Melanie L. , Tobias Martha L. , Kelley Darcy B.
Published: April 2021

The number of muscle fibers in the vocal organ of the adult male African clawed frog, Xenopus laevis, exceeds that of adult females. This sex difference is the result of rapid fiber addition in males between the end of metamorphosis, post-metamorphic stage 0 (PM0) and PM2. At PM0, male and female frogs have similar numbers of laryngeal muscle fibers. Males then add more muscle fibers than females and achieve an adult value that is 1.7 times the female number. Males castrated at PM0 have the same fiber number as females. Ovariectomy at PM0 does not alter muscle fiber addition in females. Gonadectomy at PM2 has no effect on fiber addition in either sex. Females attain masculine muscle fiber number if their ovaries are replaced with a testis at metamorphosis. Exogenous testosterone treatment at PM0 significantly increases fiber number in females but not in males. Exogenous testosterone given at PM2 has no effect on fiber number in females but decreases fiber number in males. We conclude that the testes are necessary for the marked addition of laryngeal muscle fibers seen in male X. laevis between PM0 and PM2. The masculine pattern of muscle fiber addition can be induced in females provided with a testis. Androgen secretion from the testes most probably accounts for masculinization of laryngeal muscle fiber number. After PM2, androgens are no longer necessary for muscle fiber addition and cannot increase fiber number in females.
Correlated onset and patterning of proopiomelanocortin gene expression in embryonic Xenopus brain and pituitary
Authors: Hayes William Pär , Loh Y. Peng
Published: April 2021

To identify cellular interactions that underlie the spatially appropriate transcription of neural genes, we characterized the embryonic development of proopiomelanocortin (POMC) gene expression in Xenopus laevis using in situ hybridization histochemistry. This has led to the establishment of a unique model system for studying how a neuropeptide gene program in four distinct cell groups is set up in pituitary and forebrain.

The embryonic onset and patterning of POMC expression was found to be spatially and temporally correlated inside and outside the brain. The first POMC cells in the pituitary primordium and diencephalon were juxtaposed near the infundibulum at stage 29/30, indicating they undergo molecular differentiation much earlier than previously reported for this system. By stage 31/32, many more POMC cells appeared in the morphologically undifferentiated pituitary primordium and brain. In fact, these cells were seen throughout the presumptive anterior and intermediate lobes of the pituitary and posterior diencephalon at the same time that the pituitary primordium is translocating ventral to diencephalon. By stage 39/40, coordinated morphogenesis produced the adult pattern of POMC cells localized in distinct anterior and intermediate pituitary lobes and two diencephalic nuclei. We propose in light of these findings that embryonic cells in the pituitary primordium and brain are simultaneously induced to transcribe the POMC gene, possibly as a result of reciprocal brain-pituitary interactions.
Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments
Authors: Smith Rosamund C. , Bement William M. , Dersch Mark A. , Dworkin-Rastl Eva , Dworkin Mark B. , Capco David G.
Published: April 2021

Microinjection of oligodeoxynucleotides (ODNs) complementary to cellular mRNAs has been advanced as an experimental approach to degrade target mRNAs in vivo and thereby obtain information as to the function of their cognate proteins. It is shown here that ODNs can induce a variety of aberrations in cell metabolism and structure when injected into Xenopus oocytes. Examination of histological sections of ODN-injected oocytes revealed the frequent abnormal accumulation of heavily staining basophilic material in the area of the germinal vesicle (gv). Ultrastructural analysis detected further abnormalities including blebbing of the plasma membrane, anomalous cytoskeletal structures, hyperorganised annulate lamellae, hyperinvagination of the gv, and formation of irregular nucleoli within the gv. Analysis of newly synthesised proteins by [35S]methio-nine radiolabelling of oocytes demonstrated that ODN injection can trigger a general decrease in both label uptake and protein synthesis. Qualitative effects on protein synthesis could also be observed, particularly a decrease in synthesis of high molecular weight proteins. The severity of ODN-induced effects is dose-dependent and highly variable from ODN to ODN. The previously reported delay in progesterone-induced maturation observed in oocytes depleted of the maternal mRNA D7 by ODN-directed degradation (Smith R.C., Dworkin M.B. and Dworkin-Rastl E.(1988) Genes and Devpt. 2, 1296-1306) is most likely a result of nonspecific ODN effects in the oocyte. Oocytes injected with effective antisense D7 ODNs that do not display detectable side effects matured with normal kinetics.
The pattern of early cleavage of the marsupial frog Gastrotheca riobambae
Authors: del Pino Eugenia M. , Loor-Vela Sandra
Published: April 2021

Comparison of early development of the marsupial frog Gastrotheca riobambae with Xenopus laevis suggests that the cleavage pattern of Xenopus and the tight coupling of events at the midblastula transition are features of the accelerated development of small amphibian eggs with aquatic reproduction, rather than generalized features of amphibian development. The large eggs of the marsupial frog Gastrotheca riobambae not only display an atypical holoblastic pattern of cleavage and a very slow rate of development, but the events of the midblastula transition are uncoupled, suggesting that amphibians may have a diversity of developmental patterns. Early cleavage of the egg, which measures about 3 mm in diameter, occurs mostly by meridional and vertical furrows. The first cleavage cycle takes about 16 to 20 h and about four days may be required to reach the midblastula stage. Cleavage becomes asynchronous at about the third cleavage cycle evidenced by the formation of cleavage furrows. However, during cleavage (up to 342-cells), the majority of the nuclei divide synchronously and only 15 to 40 % of the nuclei of a given embryo have a different cleavage schedule. At the 8-cell stage, nucleoli become visible (approximately 24 h after amplexus), signaling that transcription of rRNA has started at this early stage. Cell motility was detected in three- to four-day old embryos and seems to be associated with changes in cell shape and with expansion of the blastocoel at this stage.
Postimplantation development of tetraploid mouse embryos produced by electrofusion
Authors: Kaufman M. H. , Webb S.
Published: April 2021

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell ‘fused’ embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5–14 days p.c.
Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins
Authors: Linsenmayer Thomas F. , Chen Qian , Gibney Eileen , Gordon Marion K. , Marchant Jeffrey K. , Mayne Richard , Schmid Thomas M.
Published: April 2021

To examine the regulation of collagen types EX and X during the hypertrophic phase of endochondral cartilage development, we have employed in situ hybridization and immunofluorescence histochemistry on selected stages of embryonic chick tibiotarsi. The data show that mRNA for type X collagen appears at or about the time that we detect the first appearance of the protein. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Data on later-stage embryos demonstrate that once hypertrophic chondrocytes initiate synthesis of type X collagen, they sustain high levels of its mRNA during the remainder of the hypertrophic program. This suggests that these cells maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type X collagen protein in the hypertrophic matrix also extends to the marrow cavity. Type EX collagen is found throughout the hypertrophic matrix, as well as throughout the younger cartilaginous matrices. But the mRNA for this molecule is largely or completely absent from the oldest hypertrophic cells. These data are consistent with a model that we have previously proposed in which newly synthesized type X collagen within the hypertrophic zone can become associated with type II/IX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).
Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism
Authors: Condic M. L. , Fristrom D. , Fristrom J. W.
Published: April 2021

Imaginal discs of Drosophila are simple epithelial tissues that undergo dramatic changes in shape during metamorphosis, including elongation to form adult appendages such as legs and wings. We have examined the cellular basis of leg disc morphogenesis by staining filamentous actin to outline cell boundaries in discs and observing cell shapes with scanning confocal laser microscopy (SCLM). Surprisingly, we found that prior to the onset of morphogenesis, cells in the dorsal-lateral regions of leg discs are compressed in the proximal-distal axis and greatly elongated circumferentially. These cells are also asymmetric in the apical-basal axis, being more elongated in the apical-most region of the cell than they are subapically, and frequently contacting different sets of neighbors apically and basally. Elongated cells were first observed in early third instar discs, and persisted through several rounds of cell division as the discs matured. During appendage elongation in vivo and trypsin-accelerated elongation in vitro, these highly asymmetric cells became isometric. As the apical cell profiles changed shape, apical and basal cell contacts came into register. Measurements of apical cell dimensions suggest that changes in cell shape account for most of the elongation in the basitarsal and tibial leg segments between 0 and 6h after puparium formation (AP). The conversion of a stable population of anisometric cells to isometric dimensions constitutes a novel mechanism for altering the proportions of an epithelial sheet during development.
Hairless is required for the development of adult sensory organ precursor cells in Drosophila
Authors: Bang Anne G. , Hartenstein Volker , Posakony James W.
Published: April 2021

Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either ‘lost’ (i.e. the shaft and socket fail to appear) or they exhibit a ‘double socket’ phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the ‘bristle loss’ phenotype results from greater loss of H+ function than the ‘double socket’ phenotype. The systematic study of H allelic combinations enabled us to identify genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted us to investigate the cellular development of sensilla in these same positions in larvae and pupae and thereby establish the developmental basis for the mutant phenotypes. We have found that H is required for at least two steps of adult sensillum development. In positions where ‘double socket’ microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting ‘bristle loss’ in adult H mutants, we were unable at the appropriate stages of development to detect sensillum-specific cell types, the precursor cell divisions that generate them, or the primary precursor cells themselves. Thus, the H ‘bristle loss’ phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development.
Patterns of epithelial expression of Fos protein suggest important role in the transition from viable to cornified cell during keratinization
Authors: Fisher Chris , Byers Margaret R. , Iadarola Michael J. , Powers Elaine A.
Published: April 2021

An antibody directed against the DNA-binding region of c-fos was used to localize the distribution of cells positive for Fos protein in epithelial tissues. The antibody consistently bound to the nuclei of epithelial cells in the late stages of differentiation, just prior to cornification. The epidermis, palate, buccal mucosa, gingiva, tongue, forestomach and vagina in estrus all produced this type of labelling, suggesting a burst of expression immediately before cell death and cornification. The differentiating cells of the hair follicle, including the hair and inner root sheath, were also labelled. Non-keratinized tissues including junctional epithelium, embryonic epidermis and diestrus vaginal epithelium showed little or no Fos labelling. With the onset of keratinization at 18 days gestation or with induction of estrus in ovariecto-mized mice with estradiol benzoate, the epidermis and vagina expressed Fos protein in the manner typical for keratinized tissues. The Erf Er mutant epidermis, a tissue that is blocked in its ability to keratinize, overexpresses Fos with Fos-positive cells appearing in virtually every cell layer. Gel shift analysis demonstrates the presence of a functional AP-1 complex in epidermal extracts that is recognized by our antibody. Our data suggest that the expression of Fos is intricately related to epithelial cell differentiation, specifically in relation to the process of cornification and cell death.
Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture
Authors: Wiles Michael V. , Keller Gordon
Published: April 2021

When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10 % fetal calf serum, more than 40 % of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). βH1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst βmaj-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissueculture plates and grown in the presence of IL-3, a longterm output of cells from the mast cell lineage is observed. These findings indicate that early hematopoietic precursor cells can be readily generated from ES cells under well-defined culture conditions, and thus provides a unique in vitro model with which to analyze the earliest events involved in the development of the hematopoietic system.
Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum
Authors: Davis Claytus A. , Holmyard Douglas P. , Millen Kathleen J. , Joyner Alexandra L.
Published: April 2021

We have raised an antiserum, designated αEnhb-1, to a portion of the mouse En-2 protein containing the homeodomain. The antiserum detects both the En-1 and En-2 proteins in mouse, chick and Xenopus embryos by Western blot analysis. Using whole-mount immunohistochemistry, combined in some cases with scanning electron microscopy, we have examined the distribution of the proteins in the early embryos of these species. The major features of expression were similar. The initial production of En protein occurred, just before or during the formation of the first somites, in a band of the anterior neural plate in the prospective mid/hindbrain region. Later in development En-1 protein accumulated in the ventral ectoderm of the developing mouse and chick limb buds, indicating that a dorsal-ventral polarity is present as soon as any limb bud swelling is apparent and that, at least in the mouse, this polarity is established independently of the apical ectodermal ridge. In all three species, aEnhb-1 bound to a subset of ventro-lateral differentiating neurons in the spinal cord and bindbrain and their pattern of birth in the mouse reflected the division of the hindbrain into rhombo-meres. En-1 protein also accumulated in a lateral stripe of dermatome in the mouse and chick, indicating a dorsal-ventral subdivision of this tissue. The results show that En expression is a good marker for pattern formation in a variety of tissues and will be useful in experimental studies designed to characterize further these processes.
Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter
Authors: Takahashi Yu , Nogawa Hiroyuki
Published: April 2021

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 fan. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.
Patterns of Evi-1 expression in embryonic and adult tissues suggest that Evi-1 plays an important regulatory role in mouse development*
Authors: Perkins Archibald S. , Mercer John A. , Jenkins Nancy A. , Copeland Neal G.
Published: April 2021

Evi-1 is a putative protooncogene first identified as a common site of retroviral integration in murine myeloid leukemias. It encodes a 145×103Afr nuclear DNA-binding protein that contains ten zinc-finger motifs separated into two domains, as well as an acidic domain. These features suggest that Evi-1 encodes a transcriptional regulatory protein. In Drosophila, zinc-finger proteins such as Kruppel are involved in body plan patterning, and exhibit a spatially restricted pattern of expression in the embryo. To determine if Evi-1 may be involved in morphogenetic processes in the mouse embryo, we have performed in situ hybridization and Northern blot analysis on embryonic and adult mouse tissues to delineate the spatial and temporal pattern of Evi-1 expression. Our results show that Evi-1 is expressed at high levels in a few tissues in the embryo and is widely expressed, albeit at generally low levels, in the adult. Regions that exhibit high-level expression in the embryo include: the urinary system and the Mullerian ducts; the bronchial epithelium of the lung; focal areas within the nasal cavities; the endocardial cushions and truncus swellings in the heart; and the developing limbs. Expression in the limb occurs at the highest levels from 9.5 to 12.5 days, is present in both hind and forelimbs, is absent at the apical ectodermal ridge, and does not appear to establish a gradient. This pattern of expression in the limb is reminiscent of other putative transcriptional factors such as Hox-5.2 and retinoic acid receptor-gamma, consistent with the hypothesis that particular combinations or networks of transcriptional regulatory proteins are required for morphogenesis. Overall, these results suggest that Evi-1 plays an important role in mouse development.
Spatial regulation of the gap gene giant during Drosophila development
Authors: Kraut Rachel , Levine Michael
Published: April 2021

We describe the regulated expression of the segmen-tation gene giant (gt) during early embryogenesis. The gt protein is expressed in two broad gradients in precellu-lar embryos, one in anterior regions and the other in posterior regions. Double immunolocalization studies show that the gt patterns overlap with protein gradients specified by the gap genes hunchback (hb) and knirps (kni). Analysis of all known gap mutants, as well as mutations that disrupt each of the maternal organizing centers, indicate that maternal factors are responsible for initiating gt expression, while gap genes participate in the subsequent refinement of the pattern. The maternal morphogen bicoid (bcd) initiates the anterior gt pattern, while nanos (nos) plays a role in the posterior pattern. Gene dosage studies indicate that different thresholds of the bcd gradient might trigger hb and gt expression, resulting in overlapping but noncoincident patterns of expression. We also present evidence that different concentrations of hb protein are instructive in defining the limits of kni and gt expression within the presumptive abdomen. These results suggest that gt is a bona fide gap gene, which acts with hb, Krüppel and kni to initiate striped patterns of gene expression in the early embryo.
Colonization of the post-umbilical bowel by cells derived from the sacral neural crest: direct tracing of cell migration using an intercalating probe and a replication-deficient retrovirus
Authors: Pomeranz Howard D. , Rothman Taube P. , Gershon Michael D.
Published: April 2021

Experiments were done to test the hypothesis that the avian gut is colonized by cells derived from both vagal and sacral regions of the neural crest. A fluorescent dye, dil (l,l-dioctadecyl-3,3,3′,3′-tetramethylmdocarbocya-nine perchlorate), and a replication-deficient retrovirus (LZ10; Galileo et al. 1990) were employed as tracers. Since LZ10 was constructed with lacZ of E. coli as a reporter gene, infected cells were identified by demonstrating β-galactosidase immunoreactivity. Dil and LZ10 were injected between the neural tube and surface ectoderm (before the migration of crest cells away from the injection sites) at vagal, truncal (dil only), or sacral axial levels. The bowel was examined 4 days later in order to allow crest-derived cells sufficient time to migrate to the gut. Following injections of either tracer into the vagal crest, labelled cells were found in the gizzard and duodenum.’When dil or LZ10 was injected into the sacral crest, labelled cells were seen in the postumbilical bowel and ganglion of Remak. In the hindgut, marked cells were concentrated in the mesenchyme, just internal to the serosa, and were never observed rostral to the umbilicus. No fluorescent cells were ever found in the bowel following truncal injections of dil, although such cells were observed in sympathetic ganglia. Labelled cells were always found in dorsal root ganglia, no matter which tracer or level of the crest was injected. In embryos injected with LZ10, infected cells in the gut and dorsal root ganglia displayed a neural crest marker (NC-1 immunoreactivity). These observations confirm that the gut is colonized by cells from the sacral as well as the vagal region of the neural crest and that the émigrés from the sacral crest are confined to the post-umbilical bowel.
An extensive 3′ cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-β family in Drosophila
Authors: Blackman Ronald K. , Sanicola Michele , Raftery Laurel A. , Gillevet Trudy , Gelbart William M.
Published: April 2021

The decapentaplegic (dpp) gene in Drosophila melanogaster encodes a TGF-β-like signalling molecule that is expressed in a complex and changing pattern during development. One of dpp’s contributions is to proximal-distal outgrowth of the adult appendages, structures derived from the larval imaginal disks. Appendage specific mutations of dpp fall in a 20 kb interval 3’ to the known dpp transcripts. Here, we directly test the hypothesis that these mutations define an extended 3’ cis-regulatory region. By analysis of germ-line transformants expressing a reporter gene, we show that sequences from this portion of the gene, termed the dppdisk region, are capable of directing expression comparable to that defined by RNA in situ hybridization. We localize two intervals of the dppdisk region that appear to account for much of the dpp spatial pattern in imaginal disks and discuss the positions of these important elements in terms of the genetics of dpp. Finally, we provide evidence to suggest that one of our constructs expresses β-galactosidase in the early imaginal disk primordia in the embryo, at approximately the time when they are set aside from surrounding larval epidermal tissues. Thus, dpp may be involved directly in the determination of the imaginal disks.
An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers
Authors: Jermyn K. A. , Williams J. G.
Published: April 2021

The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982Devi Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination.

We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst ‘O’ region is a preparatory step in the process.
Molecular cloning and expression of a novel homeobox gene AHox1 of the ascidian, Halocynthia roretzi
Authors: Saiga Hidetoshi , Mizokami Atsushi , Makabe Kazuhiro W. , Satoh Noriyuki , Mita Takashi
Published: April 2021

We have isolated a novel ascidian homeobox gene, designated AHoxl, by screening the genomic DNA of Halocynthia roretzi with the Bombyx mori Antennapedia type homeobox as a probe. The AHoxl gene encodes a protein that consists of 741 amino acids. The homeobox of AHoxl is interrupted by 2 introns each of which is about 300 bp in length and it shows about 70 % similarity at a deduced amino acid level to that of Drosophila H2.0. This suggests that AHoxl is one of the most diverged homeobox genes so far characterized. Northern blot hybridization with an AHoxl probe showed the presence of single transcripts approximately 2.8 kb in length in larvae, juveniles and some adult tissues. The expression of AHoxl is scarcely detected during the course of early development but it increases to a moderate level at the larval stage. After metamorphosis, the level of AHoxl expression increases as development proceeds. In situ hybridization to the juvenile 7 days after metamorphosis showed that the site of AHoxl expression is the epithelium of digestive tract. Among the adult tissues examined, digestive tract, digestive gland and coelomic cells were the major sites of the expression of AHoxl. In gonad, body wall muscle and pharyngeal epithelium, the expression of AHoxl is relatively weak. These results suggest that AHoxl is primarily expressed in the tissues of endodermal origin and that the gene expression may be associated with differentiation of the endodermal tissues.
Decrease in DNA methylase activity during preimplantation development in the mouse
Authors: Monk M. , Adams R. L. P. , Rinaldi A.
Published: April 2021

During early mouse development, there are large-scale changes in DNA methylation. These changes may be due to the availability or stability of the enzyme, DNA methyltransferase (methylase), which is responsible for maintenance of DNA methylation. A microassay for methylase activity in preimplantation embryos shows that the level of maternally inherited enzyme is extremely high in the egg and that this activity is stable for the first three cleavage divisions. However, from the 8-cell to the blastocyst stage, there is a marked and absolute decrease in enzyme activity.
Homologous patterns in the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria and the fly Drosophila melanogaster
Authors: Meier Thomas , Chabaud Fabienne , Reichert Heinrich
Published: April 2021

To determine the generality of developmental mechanisms involved in the construction of the insect nervous system, the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria was characterized at the level of identified neurons and nerve branches and then compared to that previously described from the fly Drosophila melanogaster. For this, immunocytochemistry using a neuron-specific antibody was carried out on staged grasshopper embryos. Our results show that initially a simple peripheral nerve scaffolding is established in each segment of the animal. This scaffolding consists of a pair of intersegmental nerves that are formed by identified afferent and efferent pioneer neurons and a pair of segmental nerves that are formed by afferent pioneers situated in limb buds. Subsequently, identified sets of sensory neurons differentiate in a stereotyped spatiotemporal pattern in dorsal, lateral and ventral clusters in each segment and project their axons onto these nerves. Although segmentspecific differences exist, serial homologs of the developing nerves and sensory neurons can be identified. A comparison of these results with those obtained from Drosophila shows that virtually the same pattern of peripheral nerves and sensory structures is formed in both species. This indicates that the construction of the peripheral nervous system in extremely divergent modern insects relies on conserved developmental mechanisms that evolved in ancestral insects over 300 million years ago.
Patterning the vertebrate head: murine Hox 2 genes mark distinct subpopulations of premigratory and migrating cranial neural crest
Authors: Hunt Paul , Wilkinson David , Krumlauf Robb
Published: April 2021

The structures of the face in vertebrates are largely derived from neural crest. There is some evidence to suggest that the form of the facial pattern is determined by the crest, and that it is specified before migration as to the structures that it is able to form. The neural crest is able to control the form of surrounding, non-neural crest tissues by an instructive interaction. Some of this cranial crest is derived from a region of the hindbrain that expresses Hox 2 homeobox genes in an overlapping and segment-restricted pattern.

We have found that neurogenic and mesenchymal neural crest expresses Hox 2 genes from its point of origin beside the neural plate, during migration and after migration has ceased and that rhombomeres 3 and 5 do not have any expressing neural crest beside them. Each branchial arch expresses a different combination or code of Hox genes in a segment-restricted way.

The surface ectoderm over the arches initially does not express Hox genes, and later adopts an expression pattern that reflects that of neural crest that has come to underlie it. We suggest that initially the neural plate and neural crest are spatially specified, while the surface ectoderm is unpatterned. Subsequently some positional information could be transferred to the surface ectoderm as a result of an interaction with the neural crest.

Given that the role of the homologous genes in insects is position specification, and that neural crest is imprinted before migration, we suggest that Hox 2 genes are providing part of this positional information to the neural crest and hence are involved in patterning the structures of the branchial arches.
The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro
Authors: Brown J. J. G. , Whittingham D. G.
Published: April 2021

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development.

As culture continued in the absence of glucose, embryos decompacted (≈82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.