bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of intracellular changes in porcine eggs after fertilization and electroactivation
Authors: Sun F.Z. , Hoyland J. , Huang X. , Mason W. , Moor R.M.
Published: April 2021

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after elec-trostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude.

Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.
A transgenic model for studying development of the enteric nervous system in normal and aganglionic mice
Authors: Kapur Raj P. , Yost Cynthia , Palmiter Richard D.
Published: April 2021

The dopamine β-hydroxylase promoter has been shown to direct expression of the reporter gene product, β-galactosidase, to enteric neurons and putative embryonic neuroblasts in transgenic mice (Mercer et al., 1991; Kapur et al., 1991). In this paper, expression of the transgene, DβH-nlacZ, in the gastrointestinal tract is characterized in more detail in wild-type mice and mice which are also homozygous for the lethal spotted allele (ls). Expression of the transgene in wild-type embryos was first detected in scattered mesenchymal cells in the proximal foregut on embryonic day 9.5, and progressed distally until embryonic day 13.5 when the entire length of the gut was colonized by such cells. Several observations suggest that the mesenchymal cells which express the transgene (MCET) are, in fact, enteric neuroblasts, probably derived from the vagal neural crest. (1) The presence of MCET in progressively more caudal portions of the embryonic gut correlated with the neurogenic potential of isolated gastrointestinal segments grafted under the renal capsule. (2) Mitotic activity of MCET was demonstrated by incorporation of [3H]thymidine in utero. (3) The migratory behavior of MCET and/or their precursors was revealed in anastomotic subcapsular grafts of gut from transgenic and non-transgenic embryos; enteric ganglia of the latter were populated by MCET from the former. (4) Enteric expression of the transgene postnatally was restricted to intrinsic neurons that coexpressed other phenotypic markers of neuronal differentiation.

The pattern of transgene expression in ls/ls mice was identical to that seen in ls/+ and +/+ mice until embryonic day 12.5. At that stage, the cranial-to-caudal progression of gastrointestinal colonization by cells that expressed the transgene extended into the proximal colon of wild-type embryos. In dramatic contrast, caudal expression of the transgene in ls/ls embryos ended abruptly at the ileocecal valve. Colonization of the proximal and mid-colon proceeded during subsequent stages of development in ls/ls embryos, but at a retarded rate and in an irregular pattern. The terminal gut was never colonized in these mutant mice. These observations suggest that congenital aganglionosis coli which develops in ls/ls embryos results from a defect of non-neuroblastic mesenchyme that regionally impairs neuroblast migration.
Actin filaments, stereocilia and hair cells of the bird cochlea VI. How the number and arrangement of stereocilia are determined
Authors: Tilney Lewis G. , Cotanche Douglas A. , Tilney Mary S.
Published: April 2021

Beginning in 8-day embryos, stereocilia sprout from the apical surface of hair cells apparently at random. As the embryo continues to develop, the number of stereocilia increases. By 10 1/2 days the number is approximately the same as that encountered extending from mature hair cells at the same relative positions in the adult cochlea. Surprisingly, over the next 2–3 days the number of stereocilia continues to increase so that hair cells in a 12-day embryo have 1 1/2 to 2 times as many stereocilia as in adult hair cells. In short, there is an overshoot in stereociliary number. During the same period in which stereocilia are formed (9–12 days) the apical surface of each hair cell is filled with closely packed stereocilia; thus the surface area is proportional to the number of stereocilia present per hair cell, as if these features were coupled. The staircase begins to form in a 10-day embryo, with what will be the tallest row beginning to elongate first and gradually row after row begins to elongate by incorporation of stereocilia at the foot of the staircase. Extracellular connections or tip linkages appear as the stereocilia become incorporated into the staircase. After a diminutive staircase has formed, eg. in a 12-day embryo, the remaining stereocilia located at the foot of the staircase begin to be reabsorbed, a process that occurs during the next few days. We conclude that the hair cell determines the number of stereocilia to form by filling up the available apical surface area with stereocilia and then, by cropping back those that are not stabilized by extracellular linkages, arrives at the appropriate number. Furthermore, the stereociliary pattern, which changes from having a round crosssectional profile to a rectangular one, is generated by these same linkages which lock the stereocilia into a precise pattern. As this pattern is established, we envision that the stereocilia flow over the apical surface until frozen in place by the formation of the cuticular plate in the apical cell cytoplasm.
The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death
Authors: Yuan Junying , Horvitz H. Robert
Published: April 2021

Mutations in the gene ced-4 block almost all of the programmed cell deaths that normally occur during Caenorhabditis elegans development. We have cloned the ced-4 gene using a ced-4 mutation caused by the insertion of the transposon Tc4. When microinjected into a ced-4 animal, a 4.4 kb DNA fragment derived from the wild-type strain and corresponding to the region of the Tc4 insertion in the mutant ced-4(n1416) rescues the Ced-4 mutant phenotype. The ced-4 gene encodes a 2.2 kb RNA transcript. This mRNA is expressed primarily during embryogenesis, when most programmed cell deaths occur. The Ced-4 protein, as deduced from cDNA and genomic DNA clones, is 549 amino acids in length. Two regions of the putative Ced-4 protein product show some similarity to known calcium-binding domains.
Xenopus maternal RNAs from a dorsal animal blastomere induce a secondary axis in host embryos
Authors: Hainski Alexandra M. , Moody Sally A.
Published: April 2021

The initial steps of dorsal axis formation are controlled by localized maternal determinants in Drosophila, and a similar process has been proposed in Xenopus. The present study demonstrates that there are axis-inducing RNA molecules located in a specific dorsal midline, animal blastomere (D1.1) of the 16-cell-stage embryo. This blastomere, although in the animal hemisphere at cleavage stages, populates most of the dorsal lip of the blastopore, the region of Spemann’s organizer, during gastrulation, and is the major progenitor for dorsal mesodermal tissues. Cytosol from this blastomere causes ventral cells to take a more dorsal fate. RNA from this blastomere induces a secondary axis when injected into ventral blastomeres and restores the dorsal axis in UV-irradiated embryos. In Xenopus, activin B, goosecoid and Xwnt-8 RNAs can ectopically induce a dorsal axis; however, none is a maternal transcript. Therefore, the D1.1 blastomere probably contains dorsal determinant(s) that are either maternal members of these gene families, or other presently unknown molecule(s). Regardless of the identity of the determinant(s), this study presents the first indication that Xenopus maternal RNAs in the dorsal animal hemisphere are able to organize the dorsal axis.
The presence of hepatocyte growth factor in the developing rat
Authors: Defrances Marie C. , Wolf Helmut K. , Michalopoulos George K. , Zarnegar Reza
Published: April 2021

Hepatocyte growth factor (HGF), a heparin-binding polypeptide mitogen, stimulates DNA synthesis in adult rat and human hepatocytes and in several other cells of epithelial origin. Recently, it was determined that scatter factor (SF), a protein that has been shown to cause the dispersion and migration of epithelial cells in culture, is identical to HGF. Moreover, the receptor for HGF was identified as the product of the proto-onco-gene, c-MET, a tyrosine kinase-containing transmem-brane protein. c-MET expression has been reported in a variety of adult and embryonic mouse tissues. Simi-larly, we and others have demonstrated that HGF is expressed in various adult rat and human tissues. In the present study, the tissue distribution of HGF during rat development was determined by immunohistochemistry using an HGF-specific polyclonal antiserum. Between day 12 and day 19, immunoreactivity for HGF was present in various locations such as hematopoietic cells, somites, squamous epithelium of the esophagus and skin, periventricular germinal matrix of the brain, bronchial epithelium, renal collecting tubules and chon-drocytes. After day 19, HGF immunoreactivity was also present in the pancreas, submaxillary glands and neural tissues. In addition to immunolocalizing HGF in tissue sections, bioreactive and immunoreactive HGF was extracted and purified from rat fetuses. Other studies demonstrated the presence of HGF and c-MET mRNA in total fetal rat, and in fetal and neonatal rat liver. Addition of purified HGF to fetal and neonatal rat liver cultures enriched for hepatocytes stimulated DNA syn-thesis up to six-fold over controls. These findings strongly suggest a pivotal role for this potent regulator of growth and development.
Ultrabithorax is a regulator of β3 tubulin expression in the Drosophila visceral mesoderm
Authors: Hinz Uwe , Wolk Andrea , Renkawitz-Pohl Renate
Published: April 2021

β3 tubulin expression accompanies the specification and differentiation of the Drosophila mesoderm. The genetic programs involved in these processes are largely unknown. Our previous studies on the regulation of the β3 tubulin gene have shown that upstream sequences guide the expression in the somatic musculature, while regulatory elements in the first intron are necessary for expression in the visceral musculature. To further analyse this mode of regulation, which reflects an early embryonic specification program, we undertook a more detailed analysis of the regulatory capabilities of the intron. The results reveal not only a certain degree of redundancy in the cis-acting elements, which act at different developmental stages in the same mesodermal derivatives, but they also demonstrate in the visceral mesoderm, which forms a continuous epithelium along the body axis of the embryo, an early action of regulators guiding gene expression along the anterior-posterior axis of the embryo: an enhancer element in the intron leads to expression in a subdomain restricted along the anterior-posterior axis. This pattern is altered in mutants in the homeotic gene Ultrabithorax (Ubx), whereas ectopic Ubx expression leads to activity of the enhancer in the entire visceral mesoderm. So this element is likely to be a target of homeotic genes, which would define the 3 tubulin gene as a realisator gene under the control of selector genes.
Expression of the genes for TGF α, EGF and the EGF receptor during early pig development
Authors: Vaughan Tristan J. , James Peter S. , Pascall John C. , Brown Kenneth D.
Published: April 2021

Expression of mRNA for transforming growth factoralpha (TGF-α), epidermal growth factor (EGF) and the epidermal growth factor receptor (EGF-R) during early pig development was evaluated by reverse transcription-PCR. In the unfertilised pig oocyte, maternal transcripts for EGF, but not for TGF α or the EGF-R, were detected. Pig conceptuses were analysed at days 7, 8, 10, 12, 15, 17, 18 and 22 of pregnancy. EGF-R mRNA was detected at all stages of conceptus development analysed. Interestingly, TGF α mRNA was expressed by the developing blastocyst only at days 8, 10 and 12 of pregnancy, with the highest levels apparent at day 10. In contrast, EGF mRNA was first expressed by the postelongation conceptus at around day 15 of pregnancy with levels continuing to increase up to day 22. In the day-18 and day-22 conceptuses, this EGF message was shown to be primarily a product of the embryo-amnion and not the placental membranes. Furthermore, EGF was immunolocalised in the day-22 embryo to the developing lung bud, gut loop and amnion. In summary, the expression pattern of TGF α mRNA during early pig development is coincident with the onset of blastocyst elongation and suggests a possible role for TGF α during this period of cellular remodelling. The temporal and spatial expression of EGF mRNA and protein suggests a possible involvement for EGF in the establishment of the early organ systems.
An X-linked human collagen transgene escapes X inactivation in a subset of cells
Authors: Wu Hong , Fässler Reinhard , Schnieke Angelika , Barker Douglas , Lee Kwang-Ho , Chapman Verne , Francke Uta , Jaenisch Rudolf
Published: April 2021

Transgenic mice carrying one complete copy of the human 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle’s translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.
A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm
Authors: Nieto M. Angela , Gilardi-Hebenstreit Pascale , Charnay Patrick , Wilkinson David G.
Published: April 2021

Pattern formation in the hindbrain and paraxial mesoderm of vertebrates occurs by the formation of a series of repeated segments. These processes of segmentation appear different at the morphological level, since hindbrain segments, the rhombomeres, form by the subdivision of the neural epithelium into compartments, whereas the mesodermal somites form by the sequential aggregation of mesenchymal cells into epithelial balls. Previous studies have implicated genes encoding transcription factors in the development of hindbrain segments, but nothing is known of genes involved in the formation of somites. Cellular interactions and signal transduction must be an important aspect of hindbrain segmentation, so we have screened for tyrosine kinases expressed in rhombomere-restricted patterns in the developing mouse embryo. We have identified a receptor protein tyrosine kinase, Sek, that has high relative levels of expression in rhombomeres 3 and 5. This alternating pattern is established coincidentally, both spatially and temporally, with the expression of Krox-20, a zinc-finger gene expressed prior to the morphological formation of rhombomeres. In addition, Sek expression occurs in several other developing tissues, including a dynamic regulation in the developing forebrain, spinal cord, early mesoderm and anterior presomitic mesoderm (segmental plate). The latter expression occurs in two stripes that correlate with, and presage, the formation of somites. Sek expression initially occurs throughout the presumptive somite, then becomes restricted anteriorly, and finally is down-regulated as the definitive somite is formed. These data suggest that despite the morphological differences in the segmentation of the hindbrain and mesoderm, Sek is involved in the segmental patterning of both of these tissues.
Notch2: a second mammalian Notch gene
Authors: Weinmaster Gerry , Roberts Veronica J. , Lemke Greg
Published: April 2021

Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these ver- tebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.
Xenopus Hox-2 genes are expressed sequentially after the onset of gastrulation and are differentially inducible by retinoic acid
Authors: Dekker Erik-Jan , Pannese Maria , Houtzager Erwin , Timmermans Ans , Boncinelli Edoardo , Durston Antony
Published: April 2021

In this paper, we review experiments to characterise the developmental expression and the responses to all-trans retinoic acid (RA) of six members of the Hox-2 complex of homeobox-containing genes, during the early development of Xenopus laevis. We showed that the six genes are expressed in a spatial sequence which is colinear with their putative 3′ to 5′ chromosomal sequence and that five of them are also expressed rapidly after the beginning of gastrulation, in a 3′ to 5′ colinear temporal sequence. The sixth gene (Xhox2.9) has an exceptional spatial and temporal expression pattern. The six genes all respond to RA by showing altered spatiotemporal expression patterns, and are also RA-inducible, the sequence of the magnitudes of their RA responses being colinear with their 3′ to 5′ chromosomal sequence, and with their spatial and temporal expression sequences. Our data also reveal that there is a pre-existing anteroposterior polarity in the embryo’s competence for a response to RA. These results complement and extend previous findings made using murine and avian embryos and mammalian cell lines. They suggest that an endogenous retinoid could contribute to positional information in the early Xenopus embryo.
Gastrulation and the evolution of development
Authors: Wolpert L.
Published: April 2021

The original eukaryotic cell may have possessed the key processes necessary for metazoan development -cell differentiation, patterning and motility - and these are present in the cell cycle. Protozoa also possess key patterning processes. It remains a problem as to why there should be two main modes of development -one based on asymmetric cell division and the other on cellular interactions. The latter may be related to asexual reproduction.

The morphogenetic movements of gastrulation -as distinct from specifying the body plan -are highly conserved in a wide variety of organisms. This may reflect the requirement for patterning being specified in two dimensions, sheets of cells, and a third dimension being created by cell infolding.

The origin of the gastrula can be accounted for in terms of Haeckel’s gastrea theory -an early metazoan resembling the gastrula. Gastrulation in Cnidaria may resemble the primitive condition but there is nevertheless considerable diversity. While this may reflect, for example, yolkiness, it seems that there is little selection on developmental processes other than for reliability.

Thus it is possible that the embryo is privileged with respect to selection and this may help account for the evolution of novel processes like the origin of the neural crest.

Reliability is the key demand made on development This may be provided by apparent redundancy. Since many developmental processes involve switches and spatial patterning reliability is provided by parallel buffering mechanisms and not by negative feedback.
Lighting the fuse at fertilization
Authors: Whitaker Michael , Swann Karl
Published: April 2021

In most deuterostome eggs, fertilization is marked by an abrupt and transient increase in intracellular calcium concentration The transient takes the form of a propagating wave and is the signal for the onset of development For those interested in cell signalling, the two obvious questions to ask are how the wave is initiated and how it propagates through the egg cytoplasm Answers have come largely from experiments in frog, hamster, mouse and sea urchin eggs One explanation of signal transduction at fertilization makes an analogy with transmembrane signalling in somatic cells, where a family of G-protein-linked receptors pass activating signals across the plasma membrane Another, older idea is that it is the fusion of sperm and egg that is responsible for detonating the calcium explosion at fertilization We discuss the relative merits of the two ideas Both are plausible; the creative tension between them has led to experiments that broaden our view of signal transduction at fertilization
Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear
Authors: Mansour Suzanne L. , Goddard Judy M. , Capecchi Mario R.
Published: April 2021

We derived mice that carry a targeted insertion of a neor gene in the int-2 (Fgf-3) proto-oncogene coding sequences. The mutation was found to be recessive and mice that were homozygous for the insertion did not often survive to adulthood. The mutant mice had defects in the development of the tail and inner ear that could be correlated with disruption of int-2 expression in the posterior primitive streak and hindbrain or otic vesicle. While the tail phenotype was 100% penetrant, we found that the inner ear phenotype had reduced penetrance and variable expressivity. The variable expressivity could not be attributed to variability in the genetic background of the mutant allele or to leaky expression from the mutant allele. Thus, we conclude that even in a uniform genetic background, stochastic variation in the expression of a developmental circuit can result in dramatic differences in phenotypic consequences.
Regulation of wingless transcription in the Drososphila embryo
Authors: Ingham P. W. , Hidalgo A.
Published: April 2021

The segment polarity gene wingless (wg) is expressed in a complex pattern during embryogenesis suggesting that it plays multiple roles in the development of the embryo. The best characterized of these is its role in cell pattening in each parasegment, a process that requires the activity of other segment polarity genes including patched (ptc) and hedgehog (hh). Here we present further evidence that ptc and hh encode components of a signal transduction pathway that regulate the expression of wg transcription following its activation by pair-rule genes. We also show that most other aspects of wg expression are independent of this regulatory network.
A different type of amphibian mesoderm morphogenesis in Ceratophrys ornata
Authors: Purcell Susan M. , Keller Ray
Published: April 2021

Ceratophrys ornata, the Argentinean horned frog, has a significantly different pattern of early morphogenesis than does the most studied amphibian, Xenopus laevis. Time-lapse videomicroscopy, scanning electron microscopy, histological sections and lineage tracers have shown that, in C. ornata, some prospective noto-chord, somite and tailbud mesoderm cells leave the sur-face epithelium of the archenteron by ingression. After gastrulation, SEM reveals cells with constricted apices and a bottle shape in three zones on the archenteron roof and in a fourth zone around the blastopore. Prospective somitic tissue ingresses first from two lateral zones, followed by ingression of prospective notochord from the medial zone and tailbud mesoderm from the circumblastoporal zone. This is unlike X. laevis, in which no cells with constricted apices are present on the dorsal surface of the archenteron, nor do any cells ingress into the deep mesodermal layers from the surface layer.
Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition
Authors: Giorgi F. , Lucchesi P. , Morelli A. , Bownes M.
Published: April 2021

Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles-L186/Basc, fs(2)A17 and ap4-which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages.

In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.
The in situ localization of tenascin splice variants and thrombospondin 2 mRNA in the avian embryo
Authors: Tucker Richard P.
Published: April 2021

Tenascin and thrombospondin belong to the growing family of extracellular matrix glycoproteins believed to have an anti-adhesive function during development. Immunohistochemistry has been used to identify these proteins in the developing central nervous system, in the matrix surrounding peripheral neurons, and in connective tissue. The antibodies used in most of these studies, however, could not distinguish between different splice variants (tenascin) nor different genetic forms (thrombospondin). For this reason, we used the reverse transcriptase polymerase chain reaction to generate DNA probes that are specific to the transcripts of high Mr tenascin and thrombospondin 2. These probes were then used for an in situ hybridization study to determine the cellular origins of specific tenascin and thrombospondin forms throughout the development of the chick. The mRNA encoding high Mr tenascin was found associated with motile cells and in tissues undergoing dynamic modeling: migrating glia, epithelial glia used as a substratum for migrating neurons, the growing tips of lung buds, and during osteogenesis. In contrast, the mRNAs of low Mr tenascin were concentrated in areas of cartilage deposition and chondrocyte proliferation. Thrombospondin 2 mRNA was not detected in the developing central nervous system at any time during development by in situ hybridization. In contrast, it was found in embryonic mesenchyme, perichondrium, epimysium, and endothelial cells. Thrombospondin 2 mRNA was detected in poly(A) RNA isolated from embryonic spinal cord and cerebellum by polymerase chain reaction, though it was not detected in poly(A) RNA from the avascular retina. Thus, thrombospondin 2 mRNA may be present in the developing brain at low levels in endothelial cells or blood cells. These data support the notion that tenascin splice variants have distinct roles during development, and that thrombospondin 2 is more likely to be playing a role associated with the morphogenesis of connective tissue than neuronal development.
Postembryonic patterns of expression of cut, a locus regulating sensory organ identity in Drosophila
Authors: Blochlinger Karen , Jan Lily Yeh , Jan Yuh Nung
Published: April 2021

The cut locus is both necessary and sufficient to specify the identity of a class of sensory organs in Drosophila embryos. It is also expressed in and required for the development of a number of other embryonic tissues, such as the central nervous system, the Malpighian tubules and the tracheal system. We here describe the expression of cut in the precursors of adult sensory organs. We also show that cut is expressed in cells of the prospective wing margin and correlate the wing margin phenotype caused by two cut mutations with altered cut expression patterns. Finally, we observe cut-expressing cells in other adult tissues, including Malpighian tubules, muscles, the central nervous system and ovarian follicle cells.
Non-overlapping expression of CRBP I and CRABP I during pattern formation of limbs and craniofacial structures in the early mouse embryo
Authors: Gustafson Anne-Lee , Dencker Lennart , Eriksson Ulf
Published: April 2021

Retinoic acid (RA), a physiological metabolite of retinol (vitamin A), is thought to be of importance for pattern formation in the developing embryo. However, the mechanism by which RA is generated, as well as the site of its formation in the developing embryo, is still unknown. In this paper, we show that radiolabelled retinol, administered to pregnant mice, is accumulated in specific locations in the embryos. As revealed by immunohistochemistry using antibodies to cellular retinol-binding protein I (CRBP I), retinol accumulates in regions of the embryo expressing CRBP I. In limbs and craniofacial structures, CRBP I expression and retinol accumulation was seen in endoderm and surface ectoderm. Most mesenchymal cells of the limbs and craniofacial structures did not express detectable levels of CRBP I but instead expressed cellular retinoic acid-binding protein I (CRABP I). Previous results have demonstrated that CRABP I is involved in accumulation of RA in the embryo. Thus, the spatially closely related but non-overlapping domains of expression of CRBP I and CRABP I suggests a role of a retinol/RA pathway in epithelial-mesenchymal interactions during pattern formation of limbs and of craniofacial structures.
The mouse NCAM gene displays a biphasic expression pattern during neural tube development
Authors: Bally-Cuif Laure , Goridis Christo , Santoni Marie-Josée
Published: April 2021

The neural cell adhesion molecule (NCAM) is one of the most abundant cell adhesion molecules expressed in vertebrates and it is thought to play important roles as a regulator of morphogenetic processes, but little is known of its expression pattern in mammalian embryos. In this study, we have examined the developmental profile of NCAM gene expression in mouse embryos from gestational day 7.5 to 12.5, focusing on the developing neural tube. NCAM transcripts were first detected around day 8.5 in the somites and the forming neural tube. At this stage, NCAM transcripts were expressed in the neuroepithelium throughout the width of the neural groove and tube up to a rostral boundary within the hindbrain, whereas NCAM mRNA levels were very low or undetectable in the neuroepithelium of the head region. The positional restriction of NCAM expression was con-firmed by immunohistochemistry at the protein, and by polymerase chain reaction analysis at the RNA level. Expression in the neuroepithelium was transient as the level of NCAM transcripts declined in the germinal layer beyond day 8.5. By day 9.5, strong NCAM expression had appeared on the earliest postmitotic neurones along the entire neuraxis, and this pattern of expression in all regions with differentiating neurones was maintained until day 12.5. We conclude that NCAM expression in the neural tube occurs in two spatiotemporal distinct waves : a first wave in the proliferating neuroepithelium showing positional dependence along the rostrocaudal axis, and a second wave on essentially all neurones that have become postmitotic.
Multiple restricted lineages in the embryonic rat cerebral cortex
Authors: Grove Elizabeth A. , Williams Brenda P. , Li Da-Qing , Hajihosseini Mohammad , Friedrich André , Price Jack
Published: April 2021

We have labelled precursor cells in the embryonic rat cerebral cortex using BAG, a retroviral vector that expresses β -galactosidase. We had previously reported that labelled precursor cells gxenerate clusters of labelled cells that could be classified into four types by their morphological appearance and anatomical distribution (Price and Thurlow, 1988). In this study, we have used immunohistochemistry and intracellular dye labelling to identify the cell types that make up these clusters. We discovered that clusters are almost always composed of a single cell type. In addition to clusters composed entirely of neurones, we found four different types of glial cell clusters. In the grey matter, glial clusters are composed either of protoplasmic astrocytes, or of cells that have an astrocyte morphology, but no glial filaments. In the white matter, clusters are composed of either fibrous astrocytes or oligodendocytes. Our results indicate that each of these different cortical cell types is generated from a separate population of precursor cells.
The Polycomb group gene Posterior Sex Combs encodes a chromosomal protein
Authors: Martin Elisabeth C. , Adler Paul N.
Published: April 2021

The Posterior Sex Combs (Psc) gene of Drosophila has been studied at the molecular level both because it is a Polycomb group (Pc-G) gene and hence required for the maintenance of segmental determination, and because it is the Drosophila homolog of the murine bmi-1 onco-gene. Although genetic interactions indicated that Psc functioned as a Pc-G gene, the zygotic mutant pheno-type of Psc showed little evidence of segmental transformations. We have examined mutant embryos derived from a mutant maternal germ line and found a stronger mutant phenotype, indicating that the weak zygotic phenotype of Psc is due to maternal rescue. We have found that Psc RNA accumulates in developing oocytes and this maternal RNA is presumably responsible for the maternal rescue. We have studied the expression of the Psc gene at both the RNA and protein levels. On northern blots, we find evidence for two Psc mRNAs and, on western blots, we find evidence for two Psc proteins that are altered either in abundance or size in Psc mutants. The Psc protein accumulates in all regions of the embryo and also in many tissues in a variety of developmental stages. In all cases, it is nuclear, as is its mammalian homolog, the bmi-1 protein. On polytene chromosomes, we find Psc at 45 chromosomal loci where two other Pc-G proteins are present.
Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall
Authors: Gaunt Stephen J. , Blum Martin , De Robertis Eddy M.
Published: April 2021

After an earlier, transient phase of expression in the developing primitive streak of 6.4-to 6.8-day mouse embryos, the homeobox gene goosecoid is now shown to be expressed in a later phase of mouse development, from 10.5 days onwards. The later, spatially restricted domains of goosecoid expression are detected in the head, limbs and ventrolateral body wall. At all sites, the domains of expression are first detected in undifferen-tiated tissue, and then expression persists as these tissues undergo subsequent morphogenesis. For example, goosecoid expression is noted in the first branchial arch at 10.5 days, and then expression persists as this tissue undergoes morphogenesis to form the lower jaw and the body of the tongue. Expression in tissues around the first branchial cleft persists as these undergo morphogenesis to form the base of the auditory meatus and eustachian tube. Expression in tissues around the newly formed nasal pits persists as these elongate to form the nasal chambers. Expression in the ventral epithelial lining of the otic vesicle persists as this eventually gives rise to the non-sensory epithelium of the cochlea. Expression in the proximal limb buds and ventrolateral body wall persists as these tissues undergo morphogenesis to form proximal limb structures and ventral ribs respectively. Our findings lead us to suggest that the goosecoid gene product plays a role in spatial programming within dis-crete embryonic fields, and possibly lineage compart-ments, during organogenesis stages of mouse develop-ment.
Site-specific cleavage of basement membrane collagen IV during Drosophila metamorphosis
Authors: Fessler Liselotte I. , Condic Maureen L. , Nelson Robert E. , Fessler John H. , Fristrom James W.
Published: April 2021

Breakdown of basement membranes is an important step in the controlled rearrangement of cells during metamorphosis, cell migration, and metastatic spread of tumor cells. One of our two laboratories found a unique collagenous peptide that only appears during metamor-phosis of Drosophila melanogaster. The other laboratory previously reported that during 20-hydroxyecdysone-induced eversion of Drosophila imaginal discs a glyco-protein named gp125 arises (Birr et al., 1990). We show that these two peptides are identical and that they are formed from basement membrane collagen IV. Cleav-age occurs at an imperfection of this homotrimeric col-lagen helix between residues 755/756 in the sequence CALDE/IKMPAK. The peptide is the carboxyl frag-ment, 100,647 Mr, as derived from the amino acid sequence of the collagen 1(IV) chain. The correspond-ing amino fragment was also recovered from a disul-fide-linked aggregate. This specific cleavage supports the concept of highly targeted, controlled breakdown of basement membranes during metamorphosis. Further-more, these cuts occur at strategic sites of the predicted supramolecular network of collagen IV molecules of Drosophila basement membranes.
Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans
Authors: Garriga Gian , Desai Chand , Horvitz H. Robert
Published: April 2021

The two serotonergic HSN motor neurons of the nema-tode Caenorhabditis elegans innervate the vulval mus-cles and stimulate egg laying by hermaphrodites. By analyzing mutant and laser-operated animals, we find that both epithelial cells of the developing vulva and axons of the ventral nerve cord are required for HSN axonal guidance. Vulval precursor cells help guide the growth cone of the emerging HSN axon to the ventral nerve cord. Vulval cells also cause the two HSN axons to join the ventral nerve cord in two separate fascicles and to defasciculate from the ventral nerve cord and branch at the vulva. The axons of either the PVP or PVQ neurons are also necessary for the HSN axons to run in two separate fascicles within the ventral nerve cord. Our observations indicate that the outgrowth of the HSN axon is controlled in multiple ways by both neuronal and nonneuronal cells.
Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo
Authors: Ogawa Minetaro , Nishikawa Satomi , Yoshinaga Kazuya , Hayashi Shin-Ichi , Kunisada Takahiro , Nakao Junji , Kina Tatsuo , Sudo Tetsuo , Kodama Hiroaki , Nishikawa Shin-Ichi
Published: April 2021

The protooncogene c-kit encodes a receptor type tyro-sine kinase and is allelic with the W locus of mice. SLF, the c-Kit ligand which is encoded by the Sl locus, has growth promoting activity for hemopoietic stem cells. Previous studies demonstrated that c-Kit is functionally required for the proliferation of hemopoietic progenitor cells at various differentiation stages in adult bone marrow. However, the absence of functional SLF and c-Kit in fetuses with mutant alleles of Sl and W loci pro-duces only minor effects on the myeloid and early ery-throid progenitor cells in the fetal liver, although the level of the late erythroid progenitor cells is significantly affected. We used an anti-c-Kit monoclonal antibody to investigate the expression and function of c-Kit in murine fetal hemopoietic progenitor cells. Flow-cyto-metric analysis showed that hemopoiesis in the yolk sac and fetal liver started from cells that express c-Kit. The c-Kit expression decreased upon maturation into ery-throcytes in each organ. By fluorescence activated cell sorting, the c-Kit+ cell population was enriched with the hemopoietic progenitor cells clonable in vitro (CFU-E, BFU-E and GM-CFC). To elucidate whether c-Kit func-tions in these progenitor cells in vivo, we took advan-tage of the antagonistic anti-c-Kit monoclonal antibody, ACK2, which can block the function of c-Kit. Adminis-tration of ACK2 after 12.5 days of gestation rapidly eliminated BFU-E and GM-CFC as well as CFU-E from the fetal liver. However, the number of these progeni-tor cells in the yolk sac and fetal liver was less affected when the fetuses were given ACK2 before 12.5 days of gestation. Our results provide evidence that there are two waves of hemopoiesis in murine embryos relative to c-Kit dependency. The c-Kit has an essential role on the growth of hemopoietic progenitor cells in the fetal liver after 12.5 days of gestation, whereas the progenitor cells in the liver and yolk sac of the earlier embryo do not depend on c-Kit and its ligand SLF.
Distal-less is a downstream gene of Deformed required for ventral maxillary identity
Authors: O’Hara Edward , Cohen Barbara , Cohen Stephen M. , McGinnis William
Published: April 2021

The homeotic selector (HOM) proteins are required for the diversification of the anterior-posterior axis of the Drosophila body plan, assigning unique identities to regional domains of cells comprising one or a few parasegments or segments. The HOM proteins apparently accomplish this task by the transcriptional regulation of numerous downstream genes. At present few downstream genes are known, so models of how downstream genes mediate HOM functions are based more on intuition than information. Our results indicate that Distal-less is a downstream gene of the HOM gene Deformed, and Distal-less function is required for the elaboration of a subset of the maxillary epidermal identities specified by Deformed. The regulatory effect of Deformed on Distal-less is mediated by a ventral maxillary-specific enhancer located 3 of the Distal-less transcription unit. We propose that Deformed and Distalless, both of which encode homeodomain transcription factors that are persistently expressed in ventral maxillary cells, combinatorially specify a subsegmental code required for a group of cells to differentiate maxillary cirri.
Two types of pole cells are present in the Drosophila embryo, one with and one without splicing activity for the third P-element intron
Authors: Kobayashi Satoru , Kitamura Tomiichiro , Sasaki Hitoshi , Okada Masukichi
Published: April 2021

In Drosophila, it has been postulated that the third intron of the P-element is spliced only in germ-line cells. To test whether this postulate is applicable to pole cells, the progenitor cells of germ line, we carried out a histochemical assay to detect the splicing activity in embryos. The splicing activity was detected in pole cells and primordial germ cells. The activity increased to reach a maximum at 5-6 hours AEL (after egg laying), then decreased to an undetectable level by 8-9 hours AEL. The splicing activity showed a small second peak at 12-15 hours AEL. It was rather unexpected that not all pole cells were capable of splicing the third intron.

Almost all pole cells that had the splicing activity at 5-6 hours AEL penetrated the embryonic gonads and differentiated into primordial germ cells. Our findings suggest that pole cells are selected to penetrate the gonads while they are migrating from the proctodeal cavity to the gonads. Furthermore, these results suggest that the machinery to splice the P-element is active in some pole cells, and that this activity is used for processing transcripts of genes that play important roles in the differentiation of pole cells into primordial germ cells.