bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of cardiac muscle differentiation in isolated animal pole explants of Xenopus laevis embryos
Authors: Logan Malcolm , Mohun Tim
Published: April 2021

We have isolated a cDNA fragment encoding a portion of the myosin heavy chain α-isoform (XMHCα) in the amphibian, Xenopus laevis. The XMHCα transcript is highly enriched in adult heart RNA and is expressed exclusively in embryonic heart tissue. It therefore provides a tissue-specific marker for cardiac muscle differentiation during early embryogenesis. Using an RNAase protection assay, we can detect the onset of cardiac muscle differentiation in an anterior, ventral region of tailbud embryos, many hours before the appearance of a beating heart. Whole-mount in situ RNA hybridisation indicates that expression of the XMHCα gene is restricted to the developing heart pri-mordium. XMHCα gene expression can also be induced in isolated animal pole explants of blastulae by treatment with the growth factor, activin A. Induction is dose-dependent, requiring high doses of the growth factor compared with that required for myotomal (skeletal) muscle differentiation. In contrast, no XMHCα tran-scripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differ-entiation. Activin-induced explants show a similar temporal pattern of XMHCα gene expression to that found in normal embryogenesis. Furthermore, cells expressing this gene appear clustered in one or two foci within fused explant aggregates, which often show regular, spontaneous contractions after several days in culture. These results show that terminal differentiation of cardiac muscle can occur in growth factor-induced explants and may be distinguished from skeletal muscle differentiation by the dose and nature of the inducing factor.
Neurogenesis in the insect brain: cellular identification and molecular characterization of brain neuroblasts in the grasshopper embryo
Authors: Zacharias David , Leslie J. , Williams D. , Meier Thomas , Reichert Heinrich
Published: April 2021

Brain neuroblasts in the embryonic grasshopper were studied by toluidine blue staining, BrdU incorporation, and immunocytochemistry in whole-mounts as well as by reconstruction of stained serial sections. Large dividing neuroblasts are observed by the 25% stage. During early neurogenesis these neuroblasts generate their progeny through mechanisms similar to those that occur in the segmental ganglia; each neuroblast divides asymmetrically to produce a chain of ganglion mother cells, and each ganglion mother cell divides symmetrically to produce a pair of neurons. Approximately 130 mitotically active, large neuroblasts are found in each brain hemisphere at the 30–45% stages. Through morphogenetic movements that occur between the 30–35% stages these neuroblasts become located in positions which are predictive of the major brain regions that they give rise to. Many of the brain neuroblasts can be identified as individuals based on their stereotyped position in the neurogenic array. Immunocytochemical experiments with antibodies against, engrailed, fasciclin I and TERM-1 show that brain neuroblasts can also be characterized by their expression of cell-specific molecular labels. These studies indicate that many features of the complex mature insect brain derive from a surprisingly simple and stereotyped set of neuronal precursor cells. Thus, many of the concepts and methods that have been used to study neurogenesis in the simpler segmental ganglia may also be applicable to the insect brain.
Different neurotrophins are expressed and act in a developmental sequence to promote the survival of embryonic sensory neurons
Authors: Buchman Vladimir L. , Davies Alun M.
Published: April 2021

To investigate if different neurotrophins regulate the survival of neurons at successive developmental stages, we studied the effect of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neu-rotrophin-3 (NT-3) on the survival of mouse trigeminal neurons at closely staged intervals in development. We show that during the earliest stages of target field innervation trigeminal neurons display a transitory survival response to BDNF and NT-3. This response is lost as the neurons become NGF-dependent shortly before neuronal death begins in the trigeminal ganglion. BDNF and NT-3 mRNAs are expressed in the peripheral trigeminal target field before the arrival of the earliest axons and the onset of NGF mRNA expression. The levels of BDNF and NT-3 mRNAs peak during the early stages of target field innervation and decline shortly after the loss of neuronal responsiveness to BDNF and NT-3. Our study provides the first clear evidence that different target-derived neurotrophins can act sequentially to promote the survival of developing neurons.
Regulative capacity of the cranial neural tube to form neural crest
Authors: Scherson Talma , Serbedzija George , Fraser Scott , Bronner-Fraser Marianne
Published: April 2021

In avian embryos, cranial neural crest cells emigrate from the dorsal midline of the neural tube shortly after neural tube closure. Previous lineage analyses suggest that the neural crest is not a pre-segregated population of cells within the neural tube; instead, a single progenitor in the dorsal neural tube can contribute to neurons in both the central and the peripheral nervous systems (Bronner-Fraser and Fraser, 1989Neuron 3, 755–766). To explore the relationship between the ‘premigratory’ neural crest cells and the balance of the cells in the neural tube in the midbrain and hindbrain region, we have challenged the fate of these populations by ablating the neural crest either alone or in combination with the adjoining ventral portions of the neural tube. Focal injections of the vital dye, DiI, into the neural tissue bordering the ablated region demonstrate that cells at the same axial level, in the lateral and ventral neural tube, regulate to reconstitute a population of neural crest cells. These cells emigrate from the neural tube, migrate along normal pathways according to their axial level of origin and appear to give rise to a normal range of derivatives. This regulation following ablation suggests that neural tube cells normally destined to form CNS derivatives can adjust their prospective fates to form PNS and other neural crest derivatives until 4.5–6 hours after the time of normal onset of emigration from the neural tube.
Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain
Authors: Carpenter Ellen M. , Goddard Judy M. , Chisaka Osamu , Manley Nancy R. , Capecchi Mario R.
Published: April 2021

Targeted disruption of the murine hox-A1 gene results in severe defects in the formation of the hindbrain and associated cranial ganglia and nerves. Carbocyanine dye injections were used to trace afferent and efferent projections to and from the hindbrain in hox-A1 /hox-A1 mutant mice. Defects were observed in the position of efferent neurons in the hindbrain and in their projection patterns. In situ hybridization was used to analyze the transcription pattern of genes expressed within specific rhombomeres. Krox-20, int-2 (fgf-3), and hox-B1 all display aberrant patterns of expression in hox-A1 mutant embryos. The observed morphological and molecular defects suggest that there are changes in the formation of the hindbrain extending from rhombomere 3 through rhombomere 8 including the absence of rhom-bomere 5. Also, motor neurons identified by their axon projection patterns which would normally be present in the missing rhombomere appear to be respecified to or migrate into adjacent rhombomeres, suggesting a role for hox-A1 in the specification of cell identity and/or cell migration in the hindbrain.
Expression of Synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse
Authors: Littleton J. Troy , Bellen Hugo J. , Perin Mark S.
Published: April 2021

Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin’s cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. We have initiated the study of Synaptotag-min’s expression during Drosophila neurogenesis in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin (syt) message is present in the cell bodies of all peripheral nervous system neurons and many, if not all, central nervous system neurons during neurite outgrowth and synapse formation, and in mature neurons. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes to discrete regions of the neurophil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, we propose that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms under-lying these processes.
Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis)
Authors: Pesce Maurizio , Farrace Maria Grazia , Piacentini Mauro , Dolci Susanna , Felici Massimo De
Published: April 2021

Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30°C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417 –427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530 –536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831–838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281– 284).

In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4–5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture. These last results suggest a novel mechanism by which these two cytokines may affect the in vitro as well possibly in vivo development of mammalian PGCs.
Selective accumulation of MyoD and Myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones
Authors: Hughes Simon M. , Taylor Jane M. , Tapscott Stephen J. , Gurley Cathy M. , Carter William J. , Peterson Charlotte A.
Published: April 2021

Each of the myogenic helix-loop-helix transcription factors (MyoD, Myogenin, Myf-5, and MRF4) is capable of activating muscle-specific gene expression, yet distinct functions have not been ascribed to the individual proteins. We report here that MyoD and Myogenin mRNAs selectively accumulate in hindlimb muscles of the adult rat that differ in contractile properties: MyoD is prevalent in fast twitch and Myogenin in slow twitch muscles. The distribution of MyoD and Myogenin transcripts also differ within a single muscle and correlate with the proportions of fast glycolytic and slow oxidative muscle fibres, respectively. Furthermore, the expression of a transgene consisting of a muscle-specific cis-regulatory region from the myoD gene controlling lacZ was primarily associated with the fast glycolytic fibres. Alteration of the fast/slow fibre type distribution by thyroid hormone treatment or by cross-reinnervation resulted in a corresponding alteration in the MyoD/Myogenin mRNA expression pattern. These findings show that the expression of specific myogenic helix-loop-helix regulators is under the control of innervation and humoral factors and may mediate differential control of contractile protein gene expression in adult muscle.
Embryonic wing and leg motoneurons have intrinsically different survival properties
Authors: Mettling Clément , Camu William , Henderson Christopher E.
Published: April 2021

Although spinal motoneurons in the chick embryo are born in a rostro-caudal gradient, the timing of their naturally occurring cell death varies in the opposite sense: brachial motoneurons (MNs) die later than lumbar MNs. We used in vitro methods to determine whether this difference results from factors intrinsic or extrinsic to the MNs. Embryonic MNs were purified from E5 lumbar and brachial spinal cord by a method that enriches for MNs with heightened trophic requirements; they were cultured in serum-free medium. In the absence of muscle extract, death of both populations was rapid; similar results are obtained in ovo after limb ablation. In the presence of muscle extracts, however, lumbar MNs always died more rapidly than brachial MNs, as in the normal embryo. We tested the ability of wing and leg bud extracts to provide trophic factors for MNs in culture. MNs from a given level responded equally well to wing or leg extracts. However, dose-response studies showed that near-maximal survival of brachial MNs was obtained with lower concentrations of limb extract than those required for optimal survival of lumbar MNs. These results suggest that the delayed apoptosis in the brachial MN population is not a result of differing peripheral trophic support, but is intrinsically programmed, at least partially through a higher sensitivity to trophic factors.
The murine cripto gene: expression during mesoderm induction and early heart morphogenesis
Authors: Dono Rosanna , Scalera Lorenzo , Pacifico Francesco , Acampora Dario , Persico M. Graziella , Simeone Antonio
Published: April 2021

The murine cripto gene encodes a 171-aminoacid epidermal growth factor-related protein, with 93% similarity to its human counterpart in the ‘EGF-like’ domain. The murine cripto mRNA contains two B1 repeats in its 3 non-coding region and a 163-nucleotide homology to the human mRNA.

The mouse cripto gene is expressed at low level in specific organs of the adult animal such as spleen, heart, lung and brain. In situ hybridization analysis during murine embryogenesis (day 6.2 to day 10.5) reveals a very restricted expression pattern. cripto transcripts are first detected in a few epiblastic cells at day 6.5. During gastrulation, the transcripts are expressed in the forming mesoderm and later during development cripto gene expression is restricted to the truncus arteriosus of the developing heart. This expression pattern suggests a role for cripto gene in the determination of the epiblastic cells that subsequently give rise to the mesoderm.
Postembryonic proliferation of neuroendocrine cells expressing adipokinetic hormone peptides in the corpora cardiaca of the locust
Authors: Kirschenbaum Sheila R. , O’Shea Michael
Published: April 2021

Neuroendocrine glands that synthesize and secrete peptide hormones regulate the levels of these peptide messengers during development. In this article we describe a mechanism for regulating neuropeptide levels in the corpora cardiaca of the locust Schistocerca gregaria, a neuroendocrine gland structurally analogous to the vertebrate adenohypophysis. A set of five colocalized peptide hormones of the adipokinetic hormone family is synthesized in intrinsic neurosecretory cells in the corpora cardiaca. During postembryonic development there are progressive changes in the absolute and relative levels of these five peptide hormones. We show that the ability of the gland to increase peptide synthesis is due to a 100-fold increase in the number of cells which make up the gland. The gland grows by the addition of new cells derived from symmetrical division of undifferentiated precursor cells within the corpora cardiaca. We show, using double-label immunocytochemistry, that cells born in the glandular lobe mature into cells that express adipokinetic hormone peptides. The pattern of cell birth and peptide expression can account for the dramatic increase in postembryonic peptide levels.
Mutations affecting the cytoskeletal organization of syncytial Drosophila embryos
Authors: Sullivan William , Fogarty Patrick , Theurkauf William
Published: April 2021

Cytoplasmic organization, nuclear migration, and nuclear division in the early syncytial Drosophila embryo are all modulated by the cytoskeleton. In an attempt to identify genes involved in cytoskeletal functions, we have examined a collection of maternal-effect lethal mutations induced by single P-element transposition for those that cause defects in nuclear movement, organization, or morphology during the syncytial embryonic divisions. We describe three mutations, grapes, scrambled, and nuclear-fallout, which define three previously uncharacterized genes. Females homozygous for these mutations produce embryos that exhibit extensive mitotic division errors only after the nuclei migrate to the surface. Analysis of the microfilament and microtubule organization in embryos derived from these newly identified mutations reveal disruptions in the cortical cytoskeleton. Each of the three mutations disrupts the actin-based pseudocleavage furrows and the cellularization furrows in a distinct fashion. In addition to identifying new genes involved in cytoskeletal organization, these mutations provide insights into cytoskeletal function during early Drosophila embryogenesis.
Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope
Authors: Downs Karen M. , Davies Tim
Published: April 2021

We describe and illustrate a set of morphological landmarks for classifying mouse embryos by gross morphology from before gastrulation to the beginning of organogenesis. These landmarks are visible at the resolution of the dissecting microscope in embryos that are intact except for reflexion of Reichert’s membrane. Adoption of these criteria should facilitate interpretation of both the expression patterns of genes and the consequences of experimental manipulation of embryos during early postimplantation development.
expanded: a gene involved in the control of cell proliferation in imaginal discs
Authors: Boedigheimer Michael , Laughon Allen
Published: April 2021

The expanded gene was first identified by a spontaneous mutation that causes broad wings. We have identified an enhancer-trap insertion within expanded and used it to generate additional mutations, including one null allele. expanded is an essential gene, necessary for proper growth control of imaginal discs and, when mutant, causes either hyperplasia or degeneration depending on the disc. Wing overgrowth in expanded hypermorphs is limited to specific regions along the anterior-posterior and dorsal-ventral axis. expanded encodes a novel 1429 amino acid protein that is localized to the apical surface of disc cells and contains three potential SH3-binding sites. Together, these observations suggest that the Expanded protein engages in protein-protein interactions regulating cell proliferation in discs.
A novel H+ permeability dominating intracellular pH in the early mouse embryo
Authors: Baltz Jay M. , Biggers John D. , Lechene Claude
Published: April 2021

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electro-chemical gradient: its dependence on external K+ con-centration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.
Distribution of tudor protein in the Drosophila embryo suggests separation of functions based on site of localization
Authors: Bardsley Anne , McDonald Kent , Boswell Robert E.
Published: April 2021

Mutations in the tudor locus of Drosophila affect two distinct determinative processes in embryogenesis; segmentation of the abdomen and determination of the primordial germ cells. The distribution of tudor protein during embryogenesis, and the effect of various mutations on its distribution, suggest that tudor protein may carry out these functions separately, based on its location in the embryo. The protein is concentrated in the posterior pole cytoplasm (germ plasm), where it is found in polar granules and mitochondria. Throughout the rest of the embryo, tudor protein is associated with the cleavage nuclei. Mutations in all maternal genes known to be required for the normal functioning of the germ plasm eliminate the posterior localization of tudor protein, whereas mutations in genes required for the functioning of the abdominal determinant disrupt the localization around nuclei. Analysis of embryos of different maternal genotypes indicates that the average number of pole cells formed is correlated with the amount of tudor protein that accumulates in the germ plasm. Our results suggest that tudor protein localized in the germ plasm is instrumental in germ cell determination, whereas nuclear-associated tudor protein is involved in determination of segmental pattern in the abdomen.
Restricted expression of the hyaluronan receptor, CD44, during postimplantation mouse embryogenesis suggests key roles in tissue formation and patterning
Authors: Wheatley Susan C. , Isacke Clare M. , Crossley Philip H.
Published: April 2021

CD44 is a multifunctional adhesion protein that acts as a major receptor for the hygroscopic extracellular matrix component, hyaluronan. This receptor-ligand binding directly mediates at least some of the cell-cell and cell-matrix interactions ascribed to CD44. Other interactions involving CD44 may be modulated indirectly by its ability to bind growth factors and thereby to promote cell attachment. During vertebrate development, multiple cases of hyaluronan involvement in cell proliferation, cell migration and histogenesis have been documented. In addition, there is evidence suggesting a central role for cell surface glycoproteins and proteoglycans in mediating the action of polypeptide growth factors involved in tissue patterning. In view of this, we undertook to investigate expression of the CD44 protein during postimplantation mouse embryogenesis. Between 9.5 and 12.5 days of embryonic development, the pre-dominant form of CD44 protein corresponds to the hyaluronan-binding CD44H form. However, species with a higher Mr were also detected, implying that CD44 isoforms generated by alternative splicing of CD44 RNA are employed in normal development. Further, we used mouse embryos to perform whole-mount immunohistochemistry and examine the temporal and spatial distribution of this glycoprotein. CD44 is expressed at high levels in the heart, somites and condensing limb-bud mesenchyme at critical stages of morphogenesis. These sites correlate with regions where hyaluronan has been demonstrated to regulate morphogenetic events. Of novel interest, however, is the high expression of CD44 in regions that do not correlate with sites of known hyaluronan-mediated developmental events. These include instructive epithelia participating in epithelial-mesenchymal cell interactions such as the apical ectodermal ridge of the developing limb bud and the odontogenic placodes of the presumptive upper and lower jaws.
Two rhombomeres are altered in Hoxa-1 mutant mice
Authors: Mark Manuel , Lufkin Thomas , Vonesch Jean-Luc , Ruberte Esther , Olivo Jean-Christophe , Dollé Pascal , Gorry Philippe , Lumsden Andrew , Chambon Pierre
Published: April 2021

This study provides a detailed description of the anatomical defects in the Hoxa-1−/−mutant mice previously generated in our laboratory (T. Lufkin, A. Dierich, M. LeMeur, M. Mark and P. Chambon, 1991; Cell 66, 1105-1119). Three-dimensional reconstructions of the Hoxa-1−/− rhombencephalon reveals that it bears only five rhombomeric structures (ie. morphological segments) instead of the normal seven. The first three of these rhombomeres appear normal as judged from the distribution pattern of CRABPI transcripts in the neurectoderm and from the histological analysis of the cranial nerve components derived from these structures. In contrast, the neural-crest-cell-free region normally located opposite rhombomere 5 is lacking in Hoxa-1−/− embryos, and motor neurons of the facial and abducens nerves, which normally differentiate within rhombomeres 4, 5 and 6, are missing in Hoxa-1−/− fetuses. These morphological data, combined with the determination of the molecular positional identities of the rhombomeres 4 and 5 (P. Dollé, T. Lufkin, R. Krumlauf, M. Mark, D. Duboule and P. Chambon, 1993; Proc. Natl. Acad. Sci. USA, in press), suggest that rhombomere 4 is markedly reduced, whereas rhombomere 5 is almost absent. Thus, the remnants of rhombomeres 4 and 5 appear to be fused caudally with rhombomere 6 to form a single fourth rhombomeric structure. Moreover, the migration of neural crest cells contributing to the glossopharyngeal and vagus nerves occurs in a more rostral position, resulting in abnormalities of these cranial nerves, which were visualized by whole-mount anti-neurofilament immunostaining. The mutual relationship along the rostrocaudal axis between the otic pit and the neuroepithelial site of int-2 protein secretion (a putative otogenic cue) is not significantly changed in Hoxa-1−/− embryos. However, the abnormal relationship between the rhombencephalon and the epithelial inner ear may account for the aplasia and faulty differentiation of the membranous labyrinth, the disruption of the cartilaginous otic capsule and the disorganisation of some middle ear structures. This phenotype is compared with that of the Hoxa-1−/− mutants generated by O. Chisaka, T. S. Musci and M. R. Capecchi, 1992 (Nature 335, 516-520) and with that of the mice homozygous for the kreisler mutation.
Mechanisms of compartment formation: Evidence that non-proliferating cells do not play a critical role in defining the D/V lineage restriction in the developing wing of Drosophila
Authors: Blair Seth S.
Published: April 2021

The dorsoventral (D/V) lineage boundary in the developing wing disc of Drosophila restricts growing cells to the prospective dorsal or ventral compartments of the wing blade. This restriction appears along the prospective margin of the wing some time during the middle to late stages of wing disc growth. It has been proposed that the restriction is established and maintained by the formation of a zone of non-proliferating cells that acts as a barrier between cells in the dorsal and ventral compartments (O’Brochta and Bryant, Nature 313, 138-141, 1985). In the adult, however, no group of barrier cells has been identified between the compartments. This study will show the following. (1) A group of cells does exist that lies between the dorsal and ventral rows of margin bristle precursors; these cells, which express cut in the late third instar wing disc, are thus in an ideal position to act as barrier cells. (2) This cut-expressing region is split into dorsal and ventral regions by the expression of the dorsal-specific gene apterous. (3) The D/V lineage restriction defined by marked dorsal and ventral clones lies in the middle of the cut-expressing region and is exactly congruent with the boundary of apterous expression. (4) No group of barrier cells is observed between dorsal and ventral clones. (5) Clones often run along the boundary for long distances, suggesting that they can grow along the D/V boundary without crossing it. These results thus do not support the existence of a groups of cells acting as a barrier between dorsal and ventral compartments. Nor do they support a critical role for division rates near the D/V boundary in establishing or maintaining the lineage restriction.
δ-crystallin enhancer binding protein δEF1 is a zinc finger-homeodomain protein implicated in postgastrulation embryogenesis
Authors: Funahashi Jun-ichi , Sekido Ryohei , Murai Kasumi , Kamachi Yusuke , Kondoh Hisato
Published: April 2021

We investigated nuclear factors that bind to δ1-crystallin enhancer core and regulate lens-specific transcription. A nuclear factor δEF1, which binds to the essential element of the δ1-crystallin enhancer core, was molecularly cloned from the chicken by a southwestern method. The protein organization of δEF1 deduced from the cDNA sequence indicated that it has heterogeneous domains for DNA-binding, two widely separated zinc fingers and a homeodomain, analogous to Drosophila ZFH-1 protein. The C-terminal zinc fingers were found to be responsible for binding to the δ1-crystallin enhancer core sequence. δEF1 had proline-rich and acidic domains common to various transcriptional activators. During embryogenesis, δEF1 expression was observed in the postgastrulation period in mesodermal tissues; initially, in the notochord, followed by somites, nephrotomes and other components. The expression level changed dynamically in a tissue, possibly reflecting the differentiation states of the constituent cells. Besides mesoderm, δEF1 was expressed in the nervous system and the lens, but other ectodermal tissues and endoderm remained very low in δEF1 expression. Cotransfection experiments indicated that this factor acts as a repressor of δ1-crystallin enhancer. Possession of heterogeneous DNA-binding domains and its dynamic change of expression in embryogenesis strongly suggest that EF1 acts in multiple ways depending on the cell type and the gene under its regulation.
Relationship between expression of serendipity α and cellularisation of the Drosophila embryo as revealed by interspecific transformation
Authors: Ibnsouda Saad , Schweisguth François , de Billy Gérard , Vincent Alain
Published: April 2021

A dramatic reorganization of the cytoskeleton underlies the cellularisation of the syncytial Drosophila embryo. Formation of a regular network of acto-myosin filaments, providing a structural framework, and possibly a contractile force as well, appears essential for the synchronous invagination of the plasma membrane between adjacent nuclei. The serendipity alpha (sry α) gene is required for this complete reorganization of the microfilaments at the onset of membrane invagination. We compare here the structure and expression of sry α between D. pseudoobscura, D. subobscura and D. melanogaster. Interspersion of evolutionarily highly conserved and divergent regions is observed in the protein. One such highly conserved region shows sequence similarities to a motif found in proteins of the ezrin-radixinmoesin (ERM) family. Four 7-13 bp motifs are conserved in the 5 promoter region; two of these are also found, and at the same position relative to the TATA box, in nullo, another zygotic gene recently shown to be involved in cellularisation. The compared patterns of expression of D. melanogaster sry and nullo, and D. pseudoobscura sry reveal a complex regulation of the spatiotemporal accumulation of their transcripts. The D. pseudoobscura sry gene is able to rescue the cellularisation defects associated with a complete loss of sry α function in D. melanogaster embryos, even though species-specific aspects of its expression are maintained. Despite their functional homologies, the D. melanogaster and D. pseudoobscura sry RNAs have different subcellular localisations, suggesting that this specific localization has no conserved role in targeting the sry α protein to the apical membranes.
v-erbA and citral reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, in Xenopus embryogenesis
Authors: Schuh Timothy J. , Hall Bonnie L. , Kraft Joan Creech , Privalsky Martin L. , Kimelman David
Published: April 2021

Treatment of late blastula/early gastrula stage Xenopus embryos with all-trans retinoic acid results in disruption of the primary body axis through effects on both mesoderm and neuroectoderm. This effect of retinoic acid, coupled with the known presence of retinoic acid in Xenopus embryos has led to the proposal that retinoic acid may be an endogenous morphogen providing posi-tional information in early development. To further elucidate the role of retinoic acid in early Xenopus devel-opment, we have attempted to interfere with the retinoic acid signalling pathway both at the level of retinoic acid formation, by treatment with citral (3,7-dimethy-2,6-octadienal), and at the level of nuclear retinoic acid receptor function, by microinjection of v-erbA mRNA. The feasibility of this approach was demonstrated by the ability of citral treatment and v-erbA mRNA injection to reduce the teratogenic effects of exogenous retinol and retinoic acid, respectively, in early Xenopus develop-ment. Interestingly, v-erbA mRNA injection and citral treatment of gastrula stage embryos resulted in tadpoles with a similar set of developmental defects. The defects were chiefly found in tissues that received a contribution of cells from the neural crest, suggesting that at least a subset of neural crest cells may be sensitive to the endogenous level of retinoic acid. In accord with this proposal, it was found that the expression patterns of two early markers of cranial neural crest cells, Xtwi and XAP-2, were altered in embryos injected with v-erbA mRNA. These results indicate that structures in addition to the primary axis are regulated by retinoic acid sig-nalling during early Xenopus development.
Cell-cell interactions regulate skeleton formation in the sea urchin embryo
Authors: Armstrong Norris , Hardin Jeff , McClay David R.
Published: April 2021

In the sea urchin embryo, the primary mesenchyme cells (PMCs) make extensive contact with the ectoderm of the blastula wall. This contact is shown to influence production of the larval skeleton by the PMCs. A previous observation showed that treatment of embryos with NiCl2 can alter spicule number and skeletal pattern (Hardin et al. (1992)Development, 116, 671-685). Here, to explore the tissue sensitivity to NiCl2, experiments recombined normal or NiCl2-treated PMCs with either normal or NiCl2-treated PMC-less host embryos. We find that NiCl2 alters skeleton production by influencing the ectoderm of the blastula wall with which the PMCs interact. The ectoderm is responsible for specifying the number of spicules made by the PMCs. In addition, experiments examining skeleton production in vitro and in half-and quarter-sized embryos shows that cell inter-actions also influence skeleton size. PMCs grown in vitro away from interactions with the rest of the embryo, can produce larger spicules than in vivo. Thus, the epithelium of the blastula wall appears to provide spatial and scalar information that regulates skeleton production by the PMCs.
The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype
Authors: Valarché Isabelle , Tissier-Seta Jean-Philippe , Hirsch Marie-Rose , Martinez Salvador , Goridis Christo , Brunet Jean-François
Published: April 2021

Transcriptional regulation of the gene encoding the cell adhesion receptor NCAM (neural cell adhesion molecule), a putative effector molecule of a variety of morphogenetic events, is likely to involve important regulators of morphogenesis. Here we identify two mouse homeodomain proteins that bind to an upstream regulatory element in the Ncam promoter: Cux, related to Drosophila cut and human CDP, and Phox2, a novel protein with a homeodomain related to that of the Drosophila paired gene. In transient transfection experiments, Cux was found to be a strong inhibitor of Ncam promoter activity, and this inhibition could be relieved by simultaneously overexpressing Phox2. These results suggest that the Ncam gene might be a direct target of homeodomain proteins and provide a striking example of regulatory cross-talk between homeodomain proteins of different classes. Whereas the expression pattern of Cux/CDP includes many NCAM-negative sites, Phox2 expression was restricted to cells also expressing Ncam or their progenitors. The localisation data thus strongly reinforce the notion that Phox2 plays a role in transcriptional activation of Ncam in Phox2-positive cell types.

In the peripheral nervous system, Phox2 was strongly expressed in all ganglia of the autonomic nervous system and more weakly in some cranial sensory ganglia, but not in the sensory ganglia of the trunk. Phox2 transcripts were detected in the primordia of sympathetic ganglia as soon as they form. Phox2 expression in the brain was confined to spatially restricted domains in the hindbrain, which correspond to the noradrenergic and adrenergic nuclei once they are identifiable. All Phox2-expressing components of the peripheral nervous system are at least transiently adrenergic or noradrenergic. In the developing brain, Phox2 was expressed at all known locations of (nor)adrenergic neurones and of their precursors. These results suggest that Phox2, in addition to regulating the NCAM gene, may be part of the regulatory cascade that controls the differentiation of neurons towards this neurotransmitter phenotype.
Expression of tie-2, a member of a novel family of receptor tyrosine kinases, in the endothelial cell lineage
Authors: Schnürch Harald , Risau Werner
Published: April 2021

We are interested in the molecular mechanisms that are involved in the development of the vascular system. In order to respond to morphogenetic and mitogenic signals, endothelial cells must express appropriate receptors. To characterize endothelial cell-specific receptors, we have concentrated on receptor tyrosine kinases, because several lines of evidence suggested the importance of controlled phosphotyrosine levels in endothelial cells. A strategy based on PCR amplification using degenerate oligonucleotides and mouse brain capillaries as mRNA source, led to the identification of a novel receptor tyrosine kinase, which we designated tie-2. In situ hybridization using a tie-2-specific probe revealed an interesting spatial and temporal expression pattern. The gene was expressed specifically in the endothelial lineage. tie-2 transcripts were present in endothelial cell precursors (angioblasts) and also in endothelial cells of sprouting blood vessels throughout development and in all organs and tissues so far examined. tie-2 was down-regulated in the adult. Because of the unusual combination of immunoglobulin, EGF-like and fibronectin type III domains in the extracellular portion of tie-2 which is shared by TEK and tie, these molecules may be considered members of a new family of receptor tyrosine kinases. Signal transduction via this new class of tyrosine kinases could lead to a better understanding of the molecular mechanisms of blood vessel formation.
IMP-L2: an essential secreted immunoglobulin family member implicated in neural and ectodermal development in Drosophila
Authors: Garbe James C. , Yang Eric , Fristrom James W.
Published: April 2021

The Drosophila IMP-L2 gene was identified as a 20hydroxyecdysone-induced gene encoding a membranebound polysomal transcript. IMP-L2 is an apparent secreted member of the immunoglobulin superfamily. We have used deficiencies that remove the IMP-L2 gene to demonstrate that IMP-L2 is essential in Drosophila. The viability of IMP-L2 null zygotes is influenced by maternal IMP-L2. IMP-L2 null progeny from IMP-L2+ mothers exhibit a semilethal phenotype. IMP-L2 null progeny from IMP-L2 null mothers are 100% lethal. An IMP-L2 transgene completely suppresses the zygotic lethal phenotype and partially suppresses the lethality of IMP-L2 null progeny from IMP-L2 null mothers. In embryos, IMP-L2 mRNA is first expressed at the cellular blastoderm stage and continues to be expressed through subsequent development. IMP-L2 mRNA is detected in several sites including the ventral neuroectoderm, the tracheal pits, the pharynx and esophagus, and specific neuronal cell bodies. Staining of whole-mount embryos with anti-IMP-L2 antibodies shows that IMP-L2 protein is localized to specific neuronal structures late in embryogenesis. Expression of IMP-L2 protein in neuronal cells suggests a role in the normal development of the nervous system but no severe morphological abnormalities have been detected in IMP-L2 null embryos.
Programmed cell death in the Drosophila CNS is ecdysone-regulated and coupled with a specific ecdysone receptor isoform
Authors: Robinow Steven , Talbot William S. , Hogness David S. , Truman James W.
Published: April 2021

At adult emergence, the ventral CNS of Drosophila shows a group of approximately 300 neurons, which are unique in that they express 10-fold higher levels of the A isoform of the ecdysone receptor (EcR-A) than do other central neurons. This expression pattern is established early in metamorphosis and persists throughout the remainder of the pupal stage. Although these cells represent a heterogeneous group of neurons, they all share the same fate of undergoing rapid degeneration after the adult emerges from the pupal case. One prerequisite for this death is the decline of ecdysteroids at the end of metamorphosis. Treatment of flies with 20hydroxyecdysone blocks the death of the cells, but only if given at least 3 hours before the normal time of degeneration. The correlation of a unique pattern of receptor isoform expression with a particular steroid-regulated fate suggests that variations in the pattern of receptor isoform expression may serve as important switches during development.
A dual role for the protein kinase shaggy in the repression of achaete- scute
Authors: Simpson Pat , Ruel Laurent , Heitzler Pascal , Bourouis Marc
Published: April 2021

achaete and scute are expressed in a spatially restricted pattern and provide neural potential to cells, rhe domains of expression depend partly on extra- macrochaetae whose product is itself spatially restricted and acts as a negative post-translational regulator of achaete and scute. The protein kinase shaggy also represses achaete and scute at many sites hut may act via intermediate transcription factors. However shaggy and extramacrochaetae act synergistically and molecular studies suggest that they may be part of the same pathway, shaggy is functionally homologous to the mammalian glycogen synthase kinase-3 and analogy with the known physiology of this enzyme, suggests that this function of shaggy may result from the “constitu- tive” activity. At the site where a single neural precursor will develop, achaete and scute are initially expressed in a group of equivalent cells. ‘Hie genes Notch and Delta are part of a lateral signal required to single out one precursor cell and to silence achaete and scute expression in the other cells, shaggy is required downstream of Notch for transduction of the inhibitory signal., This second role of shaggy may be due to modulation of enzymatic activity during signalling.
A fate map of the epiblast of the early chick embryo
Authors: Hatada Yohko , Stern Claudio D.
Published: April 2021

We have used carbocyanine dyes (DiI and DiO) to generate fate maps for the epiblast layer of the chick embryo between stage X and the early primitive streak stage (stages 2 –3).

The overall distribution of presumptive cell types in these maps is similar to that described for other laboratory species (zebrafish, frog, mouse). Our maps also reveal certain patterns of movement for these presumptive areas. Most areas converge towards the midline and then move anteriorly along it. Interestingly, however, some presumptive tissue types do not take part in these predominant movements, but behave in a different way, even if enclosed within an area that does undergo medial convergence and anterior movement. The apparently independent behaviour of certain cell populations suggests that at least some presumptive cell types within the epiblast are already specified at preprimitive streak stages.
X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene
Authors: Tam Patrick P. L. , Zhou Sheila X. , Tan Seong-Seng
Published: April 2021

We have determined the timing of the inactivation and reactivation of the X chromosome in the mouse primordial germ cells (PGCs) by monitoring the expression of an X-linked HMG-lacZ reporter gene. PGCs were identified by their distinct alkaline phosphatase activity and they were first localised in the primitive streak and allantoic bud of the 7.5-day gastrulating embryo. Although inactivation of the transgene was found in some PGCs at these sites, at least 85% of the population were still expressing the lacZ gene. This suggests that, although X-inactivation has commenced during gastrulation, the majority of PGCs still possess two active X chromosomes. Transgene activity remained unchanged during the relocation of PGCs to the hindgut endoderm, but decreased abruptly when PGCs left the hindgut and migrated through the mesentery. X-inactivation was completed during the migration of PGCs, but not simultaneously for the whole population. The first wave of PGCs entering the genital ridge at 9.5 days did not immediately re-activate the silent transgene until about 24 hours later. Re-activation of the transgene took place in over 80% of PGCs entering the genital ridge at 10.5 –13.5 days p.c., preceding the entry into meiosis. About 90% of the meiotic germ cells in the 14.5 –15.5 day fetal ovary expressed the transgene. Similar profiles of transgene activity were observed in PGCs of embryos that have inherited the lacZ transgene from different parents, showing unequivocally that X-inactivation in the germ cell lineage is not related to parental legacy. In contrast to those germ cells in the genital ridges, a small population of PGCs that was left outside the genital ridges at 13.5 –15.5 days did not re-activate the silent X. This strongly suggests that reactivation of the silent X chromosome in the female germ cells is a response to local signals in the genital ridge.