bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory role of the Gα1 subunit in controlling cellular morphogenesis in Dictyostelium
Authors: Dharmawardhane Suranganie , Cubitt Andrew B. , Clark Alexandra M. , Firtel Richard A.
Published: April 2021

To determine the function of the Dictyostelium Gα1 subunit during aggregation and multicellular development, we analyzed the phenotypes of gα1 null cells and strains overexpressing either wild-type Gα1 or two putative constitutively active mutations of Gα1. Strains overexpressing the wild-type or mutant Gα1 proteins showed very abnormal culmination with an aberrant stalk differentiation. The similarity of the phenotypes between Gα1 overexpression and expression of a putative constitutively active Gα1 subunit suggests that these phenotypes are due to increased Gα1 activity rather than resulting from a non-specific interference of other pathways. In contrast, gα1 null strains showed normal morphogenesis except that the stalks were thinner and longer than those of wild-type culminants. Analysis of cell-type-specific gene expression using lacZ reporter constructs indicated that strains overexpressing Gα1 show a loss of ecmB expression in the central core of anterior prestalk AB cells. However, expression of ecmB in anterior-like cells and the expression of prestalk A-specific gene ecmA and the prespore-specific gene SP60/cotC appeared normal. Using a Gα1/lacZ reporter construct, we show that Gα1 expression is cell-type-specific during the multicellular stages, with a pattern of expression similar to ecmB, being preferentially expressed in the anterior prestalk AB cells and anterior-like cells. The developmental and molecular phenotypes of Gα1 overexpression and the cell-type-specific expression of Gα1 suggest that Gα1-mediated signaling pathways play an essential role in regulating multicellular development by controlling prestalk morphogenesis, possibly by acting as a negative regulator of prestalk AB cell differentiation.

During the aggregation phase of development, gα1 null cells display a delayed peak in cAMP-stimulated accumulation of cGMP compared to wild-type cells, while Gα1 overexpressors and dominant activating mutants show parallel kinetics of activation but decreased levels of cGMP accumulation compared to that seen in wild-type cells. These data suggest that Gα1 plays a role in the regulation of the activation and/or adaptation of the guanylyl cyclase pathway. In contrast, the activation of adenylyl cyclase, another pathway activated by cAMP stimulation, was unaf fected in gα1 null cells and cell lines overexpressing wild-type Gα1 or the Gα1(Q206L) putative dominant activating mutation. However, the Gα1(G45V) putative constitutively active mutation showed significantly reduced adenylyl cyclase activity in response to cAMP. All Gα1 mutant cell lines aggregated normally; however, aggregates of cells expressing Gα1(G45V) developed ring-like structures that then developed a polarity and a small mound-like structure before forming a slug. Immunoprecipitation results suggest that the G45V phenotypes may be due to altered properties of this subunit and its association with the βg subunit.
XASH genes promote neurogenesis in Xenopus embryos
Authors: Ferreiro Beatriz , Kintner Chris , Zimmerman Kathy , Anderson David , Harris William A.
Published: April 2021

Neural development in Drosophila is promoted by a family of basic helix-loop-helix (bHLH) transcription factors encoded within the Achaete Scute-Complex (AS-C). XASH-3, a Xenopus homolog of the Drosophila AS-C genes, is expressed during neural induction within a portion of the dorsal ectoderm that gives rise to the neural plate and tube. Here, we show that XASH-3, when expressed with the promiscuous binding partner XE12, specifically activates the expression of neural genes in naive ectoderm, suggesting that XASH-3 promotes neural development. Moreover, XASH-3/XE12 RNA injections into embryos lead to hypertrophy of the neural tube. Interestingly, XASH-3 misexpression does not lead to the formation of ectopic neural tissue in ventral regions, suggesting that the domain of XASH proneural function is restricted in the embryo. In contrast to the neural inducer noggin, which permanently activates the NCAM gene, the activation of neural genes by XASH-3/XE12 is not stable in naive ectoderm, yet XASH-3/XE12 powerfully and stably activates NCAM, Neurofilament and type III β-tubulin gene expression in noggin-treated ectoderm. These results show that the XASH-3 promotes neural development, and suggest that its activity depends on additional factors which are induced in ectoderm by factors such as noggin.
β-catenin localization during Xenopus embryogenesis: accumulation at tissue and somite boundaries
Authors: Fagotto François , Gumbiner Barry M.
Published: April 2021

β-catenin is a cytoplasmic protein associated with cadherin adhesion molecules and has been implicated in axis formation in Xenopus (McCrea, P. D., Brieher, W. M. and Gumbiner, B. M. (1993) J. Cell Biol. 127, 477-484). We have studied its distribution in Xenopus embryos by immuno-fluorescence on frozen sections. Consistent with its function in cell-cell adhesion, β-catenin is present in every cell. However, high levels are expressed in certain regions and different tissues of the embryo. No simple correlation appears to exist between the levels of β-catenin with the expected strength of adhesion. High levels of β-catenin were found in regions undergoing active morphogenetic movements, such as the marginal zone of blastulae and gastrulae. This suggests that high expression of β-catenin could be involved in dynamic adhesion events. Surprisingly, β-catenin also accumulates on plasma membranes that probably do not establish direct or strong contacts with other cells. In particular, high amounts of β-catenin are found transiently at boundaries between tissue anlagen and at the intersomitic boundaries. This unexpected pattern of β-catenin expression raises the possibility that this molecule participates in developmental processes, perhaps independently of its classical role in cell-cell adhesion.
dishevelled is required during wingless signaling to establish both cell polarity and cell identity
Authors: Theisen Heidi , Purcell Judith , Bennett Mary , Kansagara Dipul , Syed Adeela , Marsh J. Lawrence
Published: April 2021

The dishevelled gene of Drosophila is required to establish coherent arrays of polarized cells and is also required to establish segments in the embryo. Here, we show that loss of dishevelled function in clones, in double heterozygotes with wingless mutants and in flies bearing a weak dishevelled transgene leads to patterning defects which phenocopy defects observed in wingless mutants alone. Further, polarized cells in all body segments require dishevelled function to establish planar cell polarity, and some wingless alleles and dishevelled; wingless double heterozygotes exhibit bristle polarity defects identical to those seen in dishevelled alone. The requirement for dishevelled in establishing polarity is cell autonomous. The dishevelled gene encodes a novel intracellular protein that shares an amino acid motif with several other proteins that are found associated with cell junctions. Clonal analysis of dishevelled in leg discs provides a unique opportunity to test the hypothesis that the wingless dishevelled interaction specifies at least one of the circumferential positional values predicted by the polar coordinate model. We propose that dishevelled encodes an intracellular protein required to respond to a wingless signal and that this interaction is essential for establishing both cell polarity and cell identity.
p75-deficient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF
Authors: Lee Kuo-Fen , Davies Alun M. , Jaenisch Rudolf
Published: April 2021

To understand the role of low-affinity neurotrophin receptor p75 in neural development, we previously generated mice carrying a null mutation in the p75 locus (Lee, K. F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A. H., Chao, M. V. and Jaenisch, R. (1992) Cell 69, 737 –749). To elucidate the mechanisms leading to deficits in the peripheral nervous system in p75 mutant mice, we have employed dissociated cultures to examine the responses of p75-deficient dorsal root ganglion (DRG) and superior cervical ganglion (SCG) neurons to different neurotrophins. We found that p75-deficient DRG and SCG neurons displayed a 2- to 3-fold decreased sensitivity to NGF at embryonic day 15 (E15) and postnatal day 3 (P3), respectively, ages that coincide with the peak of naturally occurring cell death. Furthermore, while p75-deficient E15 DRG neurons did not change their response specificity to BDNF, NT-3, and NT-4/5, P3 SCG neurons became more responsive to NT-3 at higher concentrations (nanomolar ranges). These results may help explain the deficits in the peripheral nervous system in p75 mutant mice and provide evidence that p75 can modulate neurotrophin sensitivity in some neurons.
Developmental expression of G proteins in a migratory population of embryonic neurons
Authors: Horgan A.M. , Lagrange M. T. , Copenhaver P. F.
Published: April 2021

Directed neuronal migration contributes to the formation of many developing systems, but the molecular mechanisms that control the migratory process are still poorly understood. We have examined the role of heterotrimeric G proteins (guanyl nucleotide binding proteins) in regulating the migratory behavior of embryonic neurons in the enteric nervous system of the moth, Manduca sexta. During the formation of the enteric nervous system, a group of approx. 300 enteric neurons (the EP cells) participate in a precise migratory sequence, during which the undifferentiated cells populate a branching nerve plexus that lies superficially on the visceral musculature. Once migration is complete, the cells then acquire a variety of positionspecific neuronal phenotypes. Using affinity-purified antisera against different G protein subtypes, we found no apparent staining for any G protein in the EP cells prior to their migration. Coincident with the onset of migration, however, the EP cells commenced the expression of one particular G protein, Goα. The intensity of immunostaining continued to increase as migration progressed, with Goα immunoreactivity being detectable in the leading processes of the neurons as well as their somata. The identity of the Goα-related proteins was confirmed by protein immunoblot analysis and by comparison with previously described forms of Goα from Drosophila. When cultured embryos were treated briefly with aluminium fluoride, a compound known to stimulate the activity of heterotrimeric G proteins, both EP cell migration and process outgrowth were inhibited. The effects of aluminium fluoride were potentiated by alpha toxin, a pore-forming compound that by itself caused no significant perturbations of migration. In preliminary experiments, intracellular injections of the non-hydrolyzable nucleotide GTPγ-S also inhibited the migration of individual EP cells, supporting the hypothesis that G proteins play a key role in the control of neuronal motility in this system. In addition, once migration was complete, the expression of Goα-related proteins in the EP cells underwent a subsequent phase of regulation, so that only certain phenotypic classes among the differentiated EP cells retained detectable levels of Goα immunoreactivity. Thus Go may perform multiple functions within the same population of migratory neurons in the course of embryonic development.
Mutational analysis of the Drosophila tolloid gene, a human BMP-1 homolog
Authors: Finelli Alyce L. , Bossie Cynthia A. , Xie Ting , Padgett Richard W.
Published: April 2021

Seven zygotically active genes have been identified in Drosophila that determine the fate of dorsal cells in the developing embryo. decapentaplegic (dpp), a member of the transforming growth factor-β (TGF-β) family, appears to play the central role in dorsal ectoderm formation, as mutations in this gene confer the most severe mutant phenotype of this group of genes. dpp’s activity is modulated by tolloid, which also has a role in the determination of dorsal cell fate. tolloid encodes a protein that contains a metalloprotease domain and regulatory domains consisting of two EGF motifs and five C1r/s repeats. We have generated several mutant tolloid alleles and have examined their interaction with a graded set of dpp point alleles. Some tolloid alleles act as dominant enhancers of dpp in a trans heterozygote, and are therefore antimorphic alleles. However, a tolloid deficiency shows no such genetic interaction. To characterize the nature of the tolloid mutations, we have sequenced eighteen tolloid alleles. We find that five of the seven alleles that act as dominant enhancers of dpp are missense mutations in the protease domain. We also find that most tolloid alleles that do not interact with dpp are missense mutations in the C-terminal EGF and C1r/s repeats, or encode truncated proteins that delete these repeats. Based on these data, we propose a model in which the tolloid protein functions by forming a complex containing DPP via protein-interacting EGF and C1r/s domains, and that the protease activity of TOLLOID is necessary, either directly or indirectly, for the activation of the DPP complex. The tolloid antimorphs, which have non-conservative amino acid changes in the protease domain, would still be capable of interacting with DPP, or a complex containing DPP, via these protein-interacting domains, but unable to perform wild-type function, since protease activity would be abolished. This results in effectively inhibiting DPP’s action by sequestration in an inactive complex. Since tolloid has highly conserved homologs in other phyla, the same mechanism of activating TGF-β-like molecules may be conserved and operate in other animals.
Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart
Authors: Yutzey Katherine E. , Rhee Jeanne T. , Bader David
Published: April 2021

A unique myosin heavy chain cDNA (AMHC1), which is expressed exclusively in the atria of the developing chicken heart, was isolated and used to study the generation of diversified cardiac myocyte cell lineages. The pattern of AMHC1 gene expression during heart formation was determined by whole-mount in situ hybridization. AMHC1 is first activated in the posterior segment of the heart when these myocytes initially differentiate (Hamburger and Hamilton stage 9+). The anterior segment of the heart at this stage does not express AMHC1 although the ventricular myosin heavy chain isoform is strongly expressed beginning at stage 8+. Throughout chicken development, AMHC1 continues to be expressed in the posterior heart tube as it develops into the diversified atria. The early activation of AMHC1 expression in the posterior cardiac myocytes suggests that the heart cells are diversified when they differentiate initially and that the anterior heart progenitors differ from the posterior heart progenitors in their myosin isoform gene expression. The expression domain of AMHC1 can be expanded anteriorly within the heart tube by treating embryos with retinoic acid as the heart primordia fuse. Embryos treated with retinoic acid prior to the initiation of fusion of the heart primordia express AMHC1 throughout the entire heart-forming region and fusion of the heart primordia is inhibited. These data indicate that retinoic acid treatment produces an expansion of the posterior (atrial) domain of the heart and suggests that diversified fates of cardiomyogenic progenitors can be altered.
daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo
Authors: Vaessin Harald , Brand Michael , Jan Lily Yeh , Jan Yuh Nung
Published: April 2021

The first steps of neuronal precursor formation require several genes that encode transcription regulators with the helix-loop-helix (HLH) motif, including the proneural genes of the achaete-scute complex AS-C (achaete (ac), scute (sc) and lethal of scute (l’sc)) and daughterless (da). The da protein dimerizes with AS-C products in vitro to form DNA-binding proteins. Previous studies have shown that the AS-C genes are expressed initially in discrete clusters of ectodermal cells (the proneural clusters) and then more strongly in the neuronal precursors that arise from these clusters and delaminate from the epidermal layer. In this paper, we studied the distribution of da protein with an antibody raised against Da. We found that Da is ubiquitously but non-uniformly distributed. Within the ectodermal layer, its level is neither elevated (as in the case of AS-C genes) nor reduced (as in the case of emc product) in the proneural cluster. It is, however, at higher levels in many neuronal precursors. We further studied the requirement of da in neuronal precursor development by using a variety of markers for neuronal precursors. Our results reveal the existence of at least two stages in neuronal precursor formation. da is not required for the initial appearance of nascent neuronal precursors but is required for these cells to express multiple neuronal precursor genes and to produce the normal number of neurons.
Isolated rat cortical progenitor cells are maintained in division in vitro by membrane-associated factors
Authors: Temple Sally , Davis Andrew A.
Published: April 2021

Ventricular zone cells in the developing CNS undergo extensive cell division in vivo and under certain conditions in vitro. The culture conditions that promote cell division have been studied to determine the role that contact with cell membrane associated factors plays in the proliferation of these cells. Progenitor cells have been taken from the ventricular zone of developing rat cerebral cortex and placed into microwells. Small clusters of these cells can generate large numbers of neurons and non-neuronal progeny. In contrast, single progenitor cells largely cease division, approximately 90% acquiring neuron-like characteristics by 1 day in vitro. DiI-labeled, single cells from embryonic day 14 cortex plated onto clusters of unmarked progenitor cells have a significantly higher probability (approximately 3-fold) of maintaining a progenitor cell phenotype than if plated onto the plastic substratum around 100 μm away from the clusters. Contact with purified astrocytes also promotes the progenitor cell phenotype, whereas contact with meningeal fibroblasts or balb3T3 cells promotes their differentiation. Membrane homogenates from cortical astrocytes stimulate significantly more incorporation of BrdU by E14 cortical progenitor cells than membrane homogenates from meningeal fibroblasts. These data indicate that the proliferation of rat cortical progenitor cells can be maintained by cell-type specific, membrane-associated factors.
The Drosophila miti-mere gene, a member of the POU family, is required for the specification of the RP2/sibling lineage during neurogenesis
Authors: Bhat Krishna Moorthi , Schedl Paul
Published: April 2021

The Drosophila POU gene miti-mere (previously known as pdm2) has a complex spatial and temporal pattern of expression during early development; initially it is expressed in gap-gene-like pattern, then in 14 stripes and finally in a subset of the cells in the developing CNS and PNS. To study the function of this gene during develop-ment, we generated a ‘synthetic anti-morphic mutation’ by expressing a truncated version of the miti protein from a constitutive hsp83 and an inducible hsp70 promoter. We show that these Δmiti transgenes behave like classical anti-morphic mutations. Using these dominant negative trans-genes, together with deletions and a duplication for the gene, we show that miti is required during segmentation and neurogenesis. We have also used temperature-shift experiments with the hsp70Δmiti transgene to demonstrate that miti function in segmentation is distinct and separable from its function during neurogenesis. In segmentation, miti appears to be required in the specification of the segments A2 and A6. In the CNS, miti is required for the elaboration of the NB4-2→GMC-1→RP2/sib lineage. miti is initially required in this lineage to establish the identity of the parental ganglion mother cell, GMC-1. miti must then be down-regulated to allow the asymmetric division of GMC-1 into the RP2 and its sibling cell.
Dax, a locust Hox gene related to fushi-tarazu but showing no pair-rule expression
Authors: Dawes Rachel , Dawson Iain , Falciani Francesco , Tear Guy , Akam Michael
Published: April 2021

We describe an unusual Antennapedia class homeobox gene from the grasshopper Schistocerca gregaria (Orthoptera, African Plague Locust). Its sequence is not sufficiently similar to that of any other insect Hom-Hox gene to identify it unambiguously, but short conserved elements suggest a relationship to the segmentation gene fushi-tarazu, (ftz). We term it Sg Dax (divergent Antenna-pedia class homeobox gene). Antibodies raised against the protein encoded by this gene reveal two phases of expression during embryogenesis. In the early embryo, it is a marker for the posterior part of the forming embryonic primordium, and subsequently for the posterior part of the growing germ band. In older embryos, it labels a subset of neural precursor cells in each trunk segment, very similar to that defined by the expression of fushi tarazu (ftz) in Drosophila. We suggest that Schistocerca Dax and Drosophila ftz are homologous members of a gene family whose members are diverging relatively rapidly, both in terms of sequence and role in early development.
Expression of the BDNF gene in the developing visual system of the chick
Authors: Herzog Karl-Heinz , Bailey Karen , Barde Yves-Alain
Published: April 2021

Using a sensitive and quantitative method, the mRNA levels of brain-derived neurotrophic factor (BDNF) were determined during the development of the chick visual system. Low copy numbers were detected, and BDNF was found to be expressed in the optic tectum already 2 days before the arrival of the first retinal ganglion cell axons, suggesting an early role of BDNF in tectal development. After the beginning of tectal innervation, BDNF mRNA levels markedly increased, and optic stalk transection at day 4 (which prevents subsequent tectal innervation) was found to reduce the contralateral tectal levels of BDNF mRNA. Comparable reductions were obtained after injection of tetrodotoxin into one eye, indicating that, already during the earliest stages of target encounter in the CNS, the degree of BDNF gene expression is influenced by activity-dependent mechanisms. BDNF mRNA was also detected in the retina itself and at levels comparable to those found in the tectum. Together with previous findings indicating that BDNF prevents the death of cultured chick retinal ganglion cells, these results support the idea that the tightly controlled expression of the BDNF gene might be important in the co-ordinated development of the visual system.
Characterization of Star and its interactions with sevenless and EGF receptor during photoreceptor cell development in Drosophila
Authors: Kolodkin Alex L. , Pickup Amanda T. , Lin David M. , Goodman Corey S. , Banerjee Utpal
Published: April 2021

Loss-of-function mutations in Star impart a dominant rough eye phenotype and, when homozygous, are embryonic lethal with ventrolateral cuticular defects. We have cloned the Star gene and show that it encodes a novel protein with a putative transmembrane domain. Star transcript is expressed in a dynamic pattern in the embryo including in cells of the ventral midline. In the larval eye disc, Star is expressed first at the morphogenetic furrow, then in the developing R2, R5 and R8 cells as well as in the posterior clusters of the disc in additional R cells. Star interacts with Drosophila EGF receptor in the eye and mosaic analysis of Star in the larval eye disc reveals that homozygous Star patches contain no developing R cells. Taken together with the expression pattern at the morphogenetic furrow, these results demonstrate an early role for Star in photoreceptor development. Additionally, loss-of-function mutations in Star act as suppressors of R7 development in a sensitized genetic background involving the Son of sevenless (Sos) locus, and overexpression of Star enhances R7 development in this genetic background. Based on the genetic interactions with Sos, we suggest that Star also has a later role in photoreceptor development including the recruitment of the R7 cell through the sevenless pathway.
Involvement of inositol 1,4,5-trisphosphate-mediated Ca2+release in early and late events of mouse egg activation
Authors: Xu Zhe , Kopf Gregory S. , Schultz Richard M.
Published: April 2021

Sperm-induced activation of mammalian eggs is associated with a transient increase in the concentration of intracellular Ca2+. The role of inositol 1,4,5-trisphosphate (IP3)mediated release of Ca2+ from intracellular stores during mouse egg activation was examined in the present study by determining the effects of microinjected monoclonal antibody (mAb) 18A10, which binds to the IP3 receptor and inhibits IP3-induced Ca2+ release, on endpoints of egg activation following insemination. The antibody inhibited in a concentration-dependent manner the ZP2 to ZP2f conversion that is involved in the zona pellucida block to polyspermy, as well as the ZP2 to ZP2f conversion promoted by microinjected IP3 in non-inseminated eggs. As anticipated, inseminated eggs that had been microinjected with the antibody were polyspermic. In addition, the antibody inhibited the fertilization-associated decrease in H1 kinase activity and pronucleus formation, and the concentration dependence for inhibition of these events was similar to that observed for inhibiting the ZP2 to ZP2f conversion. Last, the antibody inhibited the fertilization-induced recruitment of maternal mRNAs and post-translational modifications of proteins. In each case, eggs microinjected with the mAb 4C11, which also binds to the IP3 receptor but does not inhibit IP3-induced Ca2+ release, had no inhibitory effect on fertilization and egg activation. Results of these studies suggest that IP3-mediated Ca2+ release is essential for both early and late events of mouse egg activation.
Topographic specificity of corticospinal connections formed in explant coculture
Authors: Kuang Rong Zhen , Merline Michael , Kalil Katherine
Published: April 2021

The corticospinal pathway connects layer V pyramidal neurons in discrete regions of the sensorimotor cortex to topographically matching targets in the spinal cord. In rodents initial pathway errors occur transiently during early postnatal development, such that visual cortical axons project inappropriately into the corticospinal tract. Nevertheless, only sensorimotor axons form corticospinal connections, which are topographically ordered in hamsters from the earliest stages of innervation. Previous work in vivo suggests that pathfinding is carried out by primary cortical axons whereas target innervation occurs by extension of axon collaterals at appropriate locations. In vitro studies have provided evidence that chemotropic factors may selectively attract extension of neurites into specific targets. To investigate the basis for corticospinal target selection during development, we have used an in vitro explant coculture system. Sensorimotor and visual cortical explants from newborn hamsters were presented with inappropriate targets from olfactory bulb and cerebellum and targets from the cervical (forelimb) and lumbar (hindlimb) enlargements of the early postnatal spinal cord. Under in vitro conditions, corticospinal target selection was highly specific and remarkably similar to corticospinal connectivity in vivo. Visual and sensorimotor cortical neurites extended nonselectively into the white matter of the spinal cord. However, only neurites from the sensorimotor cortex were able to extend into and arborize within the spinal gray. In the majority of cases, these connections were topographically appropriate, matching forelimb cortex to cervical cord and hindlimb cortex to lumbar cord. However, we found no evidence that chemotropic attraction was responsible for selection of appropriate targets by cortical neurites or that spinal target tissue promoted extension of cortical axon collaterals within the collagen matrix. These results suggest that the ability of cortical neurites to recognize correct spinal targets and form terminal arbors may require direct axon target interaction.
Retinoic acid promotes differentiation of photoreceptors in vitro
Authors: Kelley Matthew W. , Turner Jennifer K. , Reh Thomas A.
Published: April 2021

The results of several recent studies have demonstrated that cell commitment and differentiation in the developing vertebrate retina are influenced by cell-cell interactions within the microenvironment. Retinoic acid has been shown to influence cell fates during development of the nervous system, and retinoic acid has been detected in the embryonic retina. To determine whether retinoic acid mediates the differentiation of specific neuronal phenotypes during retinal histogenesis, we treated dissociated cell cultures of embryonic and neonatal rat retina with varying concentrations of all-trans or 9-cis retinoic acid and analyzed the effects on cell fate using neuron and photoreceptor-specific antibodies. Addition of exogenous retinoic acid caused a dose-dependent, specific increase in the number of cells that developed as photoreceptors in culture throughout the period of retinal neurogenesis. In the same cultures, retinoic acid also caused a dose-dependent decrease in the number of cells that developed as amacrine cells. Also, results of double-labeled immunohistochemical studies using bromodeoxyuridine demonstrated that the primary effect of retinoic acid was to influence progenitor cells to develop as newly generated rod photoreceptors. Since retinoic acid and at least one of the retinoic acid receptors (RARα) have been localized to the developing neural retina, these results suggest that retinoic acid may play a role in the normal development of photoreceptor cells in vivo.
Early event in maize leaf epidermis formation as revealed by cell lineage studies
Authors: Cerioli Sergio , Marocco Adriano , Maddaloni Massimo , Motto Mario , Salamini Francesco
Published: April 2021

The epidermal cells of the juvenile leaves of maize are covered by a wax layer. glossy mutants are known which reduce drastically wax deposition. We have used the somatically unstable glossy-1 mutable 8 allele to study the distribution on the epidermis of spontaneous revertant sectors of wild-type tissues. Sectors tend to start and end at positions that correlate with the location on the epidermis of the long costal cells of ribs. It is concluded that in the protoderm only a few cells have a role and position in the generation of each of the developmental modules located between leaf midrib and margin. The module consists of an epidermal strip of cells bordered by two lateral ribs. The module originates from at least 4 cells, with one cell being the progenitor of the other three. Data are provided describing the mode of longitudinal anticlinal epidermal cell divisions within the module that are responsible for the increase in leaf width. The results suggest the existence of a clonal type of development during early leaf epidermis formation.
Expression of baculovirus P35 prevents cell death in Drosophila
Authors: Hay Bruce A. , Wolff Tanya , Rubin Gerald M.
Published: April 2021

The baculovirus P35 protein functions to prevent apoptotic death of infected cells. We have expressed P35 in the developing embryo and eye of the fly Drosophila melanogaster. P35 eliminates most, if not all, normally occurring cell death in these tissues, as well as X-irradiation-induced death. Excess pupal eye cells that are normally eliminated by apoptosis develop into pigment cells when their death is prevented by P35 expression. Our results suggest that one mechanism by which viruses prevent the death of the host cell is to block a cell death pathway that mediates normally occurring cell death. Identification of molecules that interact biochemically or genetically with P35 in Drosophila should provide important insights into how cell death is regulated.
The role of tinman, a mesodermal cell fate gene, in axon pathfinding during the development of the transverse nerve in Drosophila
Authors: Gorczyca Michael G. , Phillis Randall W. , Budnik Vivian
Published: April 2021

During the development of peripheral nerves, pioneer axons often navigate over mesodermal tissues. In this paper, we examine the role of the mesodermal cell determination gene tinman on cells that provide pathfinding cues in Drosophila. We focus on a subset of peripheral nerves, the transverse nerves, that innervate abdominal segments. During wild-type embryonic development, the transverse nerve efferents associate with glial cells located on the dorsal aspect of the CNS midline (transverse nerve exit glia). These glial cells have cytoplasmic extensions that prefigure the transverse nerve pathway from the CNS to the body wall musculature prior to transverse nerve formation. Transverse nerve efferents extend along this scaffold to the periphery, where they fasciculate with projections from a peripheral neuron − the LBD. In tinman mutants, the transverse nerve exit glia appear to be missing, and efferent fibers remain stalled at the CNS midline, without forming transverse nerves. In addition, fibers of the LBD neurons are often truncated. These results suggest that the lack of exit glia prevents normal transverse nerve pathfinding. Another prominent defect in tinman is the loss of all dorsal neurohemal organs, FMRFamide-expressing thoracic structures which likely contain the homologs of the transverse nerve exit glia in the thoracic segments. Our results support the hypothesis that the exit glia have a mesodermal origin and that glia play an essential role in determining transverse nerve axon pathways.
Programming gene expression in developing epidermis
Authors: Byrne Carolyn , Tainsky Michael , Fuchs Elaine
Published: April 2021

As the major proteins of adult keratinocytes, keratins provide biochemical markers for exploring mouse epidermal embryogenesis. Here, we used a modified method of whole-mount in situ hybridization to track skin-specific expression of endogenous keratin mRNAs through-out embryogenesis. To monitor transcriptional regulation, we coupled this with β-galactosidase expression of a human epidermal keratin promoter-driven transgene. These studies have radically changed our perception of how the program of gene expression becomes established during epidermal development. Specifically, we have discovered that (1) basal keratin (K5 and K14) genes are first detected at E9.5 in a highly regional fashion, and surprisingly as early as the single layered ectodermal stage; (2) the early patterns do not correlate with morphogenesis per se, but rather with regional variations in the embryonic origin of underlying mesenchyme, supporting morphogenetic criteria that early inductive cues are mesenchymal; (3) epidermal keratin genes are expressed in periderm, supporting the notion that this layer arises from ectodermal stratification, even though it is simple epithelial-like in morphology and is subsequently sloughed during development; (4) later embryonic patterns of K5 and K14 gene expression parallel proliferative capacity and not stratification; and (5) K1 and K10 mRNAs are first detected as early as E13.5, and their patterns correlate with differentiation and not stratification.

These patterns of epidermal gene expression led us to explore whether potential transcriptional regulators of these genes are expressed similarly. We show that AP2 (but not Sp1) cRNAs hybridize in a pattern similar to, but preceding that of basal keratin cRNAs. Finally, using gene expression in cultured cells, we demonstrate that AP2 has a strong inductive effect on basal keratin expression in a cellular environment that does not normally possess AP2 activity.
The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD
Authors: Kopan Raphael , Nye Jeffrey S. , Weintraub Harold
Published: April 2021

We show that Myf-5 and mNotch mRNA are both present in the presomitic mesoderm before muscle cell commitment and before muscle structural gene activation. The failure of presomitic mesoderm to respond to Myf-5 and express myogenic properties implies that there may be a mechanism in presomitic mesoderm to suppress muscle differentiation. Here we show that ectopic expression of the intracellular domain of mNotch (mNotchIC) functions as a constitutively activated repressor of myogenesis both in cultured cells and in frog embryos. Mutagenesis experiments indicate that the target for inactivation by mNotch is the MyoD basic helix-loop-helix domain. mNotchIC contains a nuclear localization signal and localizes to the nucleus. Removal of the nuclear localization signal (NLS) reduces nuclear localization and diminishes the inhibition of myogenesis caused by Myf-5 or MyoD. Additional experiments show that the CDC10/SWI6/ankyrin repeats are also necessary for myogenic inhibition.
Ks1, an epithelial cell-specific gene, responds to early signals of head formation in Hydra
Authors: Weinziger Ruth , Salgado Luis M. , David Charles N. , Bosch Thomas C. G.
Published: April 2021

As a molecular marker for head specification in Hydra, we have cloned an epithelial cell-specific gene which responds to early signals of head formation. The gene, designated ks1, encodes a 217-amino acid protein lacking significant sequence similarity to any known protein. KS1 contains a N-terminal signal sequence and is rich in charged residues which are clustered in several domains. ks1 is expressed in tentacle-specific epithelial cells (battery cells) as well as in a small fraction of ectodermal epithelial cells in the gastric region subjacent to the tentacles. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid increase in the level of ks1 mRNA in head-specific epithelial cells and also induces ectopic ks1 expression in cells of the gastric region. Sequence elements in the 5′-flanking region of ks1 that are related to TPA-responsive elements may mediate the TPA inducibility of ks1 expression. The pattern of expression of ks1 suggests that a ligand-activated diacylglycerol second messenger system is involved in head-specific differentiation.
Early tagma-specific commitment of Drosophila CNS progenitor NB1-1
Authors: Prokop Andreas , Technau Gerhard M.
Published: April 2021

The developing central nervous system of many species expresses distinct segment-specific characteristics. We recently described the entire embryonic lineage of Drosophila neuroblast NB1–1 and showed that the compo-sition of this lineage differs between the thoracic and abdominal tagmata with respect to the presence or absence of specific glial and neuronal components (Udolph, G., Prokop, A., Bossing, T. and Technau, G. M. (1993) Devel-opment 118, 765–775). Here, we demonstrate by heterotopic transplantations that tagma specificity of NB1-1 is deter-mined in the neuroectoderm at the early gastrula stage (stage 7). Heterogenetic transplantation and mutant analysis show that the activity of the homeotic genes Ubx or abd-A is required for the expression of the abdominal variant of the lineage. Heat induction of Ubx or abd-A expression or their derepression in Polycomb mutant embryos can override thoracic determination several hours after gastrulation (stage 10/11). At that stage antibody stainings reveal both proteins to be present in NB1-1 during normal development. Possible mechanisms confer-ring the early tagma-specific determination are discussed.
HOM-C/Hox genes and four interacting loci determine the morphogenetic properties of single cells in the nematode male tail
Authors: Chow King L. , Emmons Scott W.
Published: April 2021

The copulatory structure of the C. elegans male tail includes a set of nine bilaterally symmetrical pairs of sense organs known as rays. Each ray comprises three cells, which are generated by a stereotyped cell sublineage expressed by 18 epidermal ray precursor cells. A pattern formation mechanism in the epidermis guides the specification of morphogenetic differences between the rays necessary for correct organelle assembly at specific positions within the epidermis. Expression of these ray dif-ferences was altered in mutations we described previously, resulting in displaced and fused rays. Here we show that two genes of the C. elegans HOM-C/Hox gene complex play a role in the pattern formation mechanism. Increasing or decreasing the gene dosage of mab-5, an Antennapedia homolog, and egl-5, an Abdominal B homolog, results in displacement and fusion of specific rays. These changes are interpreted as anterior or posterior transformations in ray identities. Mutations in the genes previously described are dominant modifiers of these effects. This suggests that these genes act in the same morphogenetic pathway as mab-5 and egl-5. Several lines of evidence, including cell ablation experiments, argue that the identity of each ray is specified cell-autonomously in the terminal cells of the ray lineages. mab-5 and egl-5, therefore, specify the morphogenetic properties of differentiating cells, without change in cell lineage or apparent cell type. Modifier genes may act upstream of mab-5 and egl-5 to regulate their expression. Alternatively, they may act at the same step in the pathway, as cofactors, or they may be target genes. Target genes could include genes specifying cell recognition and adhesion molecules governing ray organelle assembly.
Down-regulation of Drosophila Egf-r mRNA levels following hyperactivated receptor signaling
Authors: Sturtevant M. A. , O’Neill J. W. , Bier E.
Published: April 2021

Internalization of ligand-receptor complexes is a well-documented mechanism for limiting the duration and magnitude of a signaling event. In the case of the EGF-Receptor (EGF-R), exposure to EGF or TGF-α results in internalization of up to 95% of the surface receptor pool within 5 minutes of exposure to ligand. In this report, we show that levels of Drosophila Egf-r mRNA are strongly down-regulated in epidermal cells likely to have recently undergone high levels of EGF-R signaling. The cells in which Egf-r mRNA levels are down-regulated express the rhomboid gene, which is thought to locally amplify EGF-R signaling. Widespread Egf-r mRNA down-regulation can be induced by ubiquitous expression of rhomboid or by eliminating the Gap1 gene. These results suggest that cells engaged in intense EGF-R/RAS signaling limit the duration of the signal through a combination of short-acting negative feedback mechanisms such as receptor internalization followed by a longer lasting reduction in receptor transcript levels. Control of Egf-r mRNA levels by altering transcription or mRNA stability is a new tier of regulation to be considered in analysis of EGF-R signaling during development.
Mice with targeted disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve
Authors: Goddard, Judy M. , Rossel Mireille , Manley Nancy R. , Capecchi Mario R.
Published: April 2021

Mice were generated with targeted disruptions in the hoxb-1 gene. Two separate mutations were created: the first disrupts only the homeodomain and the second inactivates the first exon as well as the homeodomain. The phenotypes associated with these two mutant alleles are indistinguishable in surviving adult mice. The predominant defect in these mutant mice is a failure to form the somatic motor component of the VIIth (facial) nerve, possibly through a failure to specify these neurons. The phenotype of hoxb-1 mutant homozygotes closely resembles features of the clinical profile associated with humans suffering from Bell’s Palsy or Moebius Syndrome. These animals should therefore provide a useful animal model for these human diseases.
Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny
Authors: Köntges Georgy , Lumsden Andrew
Published: April 2021

To investigate the influence of hindbrain segmentation on craniofacial patterning we have studied the long term fate of neural crest (NC) subpopulations of individual rhom-bomeres (r), using quail-chick chimeras. Mapping of all skeletal and muscle connective tissues developing from these small regions revealed several novel features of the cranial neural crest. First, the mandibular arch skeleton has a composite origin in which the proximal elements are r1+r2 derived, whereas more distal ones are exclusively midbrain derived. The most proximal region of the lower jaw is derived from second arch (r4) NC. Second, both the lower jaw and tongue skeleton display an organisation which precisely reflects the rostrocaudal order of segmental crest deployment from the embryonic hindbrain. Third, cryptic intraskeletal boundaries, which do not correspond to anatomical landmarks, form sharply defined interfaces between r1+r2, r4 and r6+r7 crest. Cells that survive the early apoptotic elimination of premigratory NC in r3 and r5 are restricted to tiny contributions within the 2nd arch (r4) skeleton. Fourth, a highly constrained pattern of cranial skeletomuscular connectivity was found that precisely respects the positional origin of its constitutive crest: each rhombomeric population remains coherent throughout ontogeny, forming both the connective tissues of specific muscles and their respective attachment sites onto the neuro- and viscerocranium. Finally, focal clusters of crest cells, confined to the attachment sites of branchial muscles, intrude into the otherwise mesodermal cranial base. In the viscerocranium, an equally strict, rhombomere-specific matching of muscle connective tissues and their attachment sites is found for all branchial and tongue (hypoglossal) muscles. This coherence of segmental crest populations explains how cranial skeleto-muscular pattern can be implemented and conserved despite evolutionary changes in the shapes of skeletal elements.
Kinase-negative mutant epidermal growth factor receptor (EGFR) expression during embryonal stem cell differentiation favours EGFR-independent lineages
Authors: Wu Jie-Xin , Adamson Eileen D.
Published: April 2021

EGF receptors are expressed on most fetal and adult cells but their precise roles are not well known. We previously reported that, in P19 embryonal carcinoma cells, the expression of kinase-negative EGFR inhibits retinoic acid (RA)-induced differentiation to nervous tissue, suggesting that EGFR plays a role in differentiation (J.-X. Wu and E. D. Adamson (1993) Dev. Biol. 159, 208-222). Embryo stem (ES) cells differentiate into a wide range of tissue types after the removal of the cytokine LIF from the culture medium. We demonstrate here that the induction of some early markers of differentiation, tissue-type plasminogen activator (tPA), AFP and keratins 8 and 19 is inhibited, whilst brachyury and myosin are increased, in clones containing kinase-negative mutant EGFR. After an extended period of differentiation, the cell types present in mutant and control cultures differed. Mutant clones produced frequent cardiac and skeletal muscle as the predominant differentiated cell types in vitro; other cells types were sparse or absent. Teratocarcinomas formed by EGFR-Δkinase-expressing ES cells contained frequent skeletal and cardiac muscle as well as apoptotic nuclei, while normal ES cells produced no detectable muscle and less apoptoses. Since mutant differentiated cultures had slower growth rates and increased levels of cell death, we concluded that: (1) inactive EGFR does not allow some cell types to survive and/or proliferate; (2) tissues that do not require EGFR for their survival, development or function predominate in long-term mutant cultures; (3) EGFR activity is not necessary for cardiac and skeletal muscle or endoderm formation and (4) Impaired survival of EGF-dependent lineages leads to preferential selection of muscle in differentiating ES cells.