bluuebunny/crossref_metadata_2025 huggingface dataset:
Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila αPS2βPS integrins
Authors: Fogerty Frances J. , Fessler Liselotte I. , Bunch Thomas A. , Yaron Yifah , Parker Carol G. , Nelson Robert E. , Brower Danny L. , Gullberg Donald , Fessler John H.
Published: April 2021

Genetic and other studies of Drosophila integrins have implicated these extracellular matrix receptors in various morphogenetic events, but identification of their endogenous ligands has been elusive. We report the biochemical purification and cloning of tiggrin, a novel extracellular matrix protein from Drosophila. This 255 ×103Mr polypeptide contains the potential integrin recognition sequence Arg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 amino acid motif. The tiggrin gene is at chromosome locus 26D12 and is expressed by embryonic hemocytes and fat body cells. Tiggrin protein is detected in matrices, especially at muscle attachment sites that also strongly express integrins. Tiggrin-coated surfaces support primary embryo cell culture and provide excellent substrates for αPS2βPS integrin-mediated cell spreading. Soluble RGD-peptides inhibit this cell spreading.
Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse Sertoli cells
Authors: Yomogida Kentaro , Ohtani Haruo , Harigae Hideo , Ito Etsuro , Nishimune Yoshitake , Engel James Douglas , Yamamoto Masayuki
Published: April 2021

GATA-1 is an essential factor for the transcriptional activation of erythroid-specific genes, and is also abundantly expressed in a discrete subset of cells bordering the seminiferous epithelium in tubules of the murine testis. In examining normal and germ-line defective mutant mice, we show here that GATA-1 is expressed only in the Sertoli cell lineage in mouse testis. GATA-1 expression in Sertoli cells is induced concomitantly with the first wave of spermatogenesis, and GATA-1-positive cells are uniformly distributed among all tubules during prepubertal testis development. However, the number of GATA-1-positive cells declines thereafter and were found only in the peripheral zone of seminiferous tubules in stages VII, VIII and IX of spermatogenesis in the adult mouse testis. In contrast, virtually every Sertoli cell in mutant W/Wv, jsd/jsd or cryptorchid mice (all of which lack significant numbers of germ cells) expresses GATA-1, thus showing that the expression of this transcription factor is negatively controlled by the maturing germ cells. These observations suggest that transcription factor GATA-1 is a developmental stage- and spermatogenic cycle-specific regulator of gene expression in Sertoli cells.
Neural selective activation and temporal regulation of a mammalian GAP-43 promoter in zebrafish
Authors: Reinhard Eva , Nedivi Elly , Wegner Jeremy , Skene J. H. Pate , Westerfield Monte
Published: April 2021

Neurons throughout the vertebrate nervous system selectively activate the gene for a growth cone component, GAP-43, during embryonic development, and then decrease its expression abruptly as they form synapses. Distal interruption of mature axons in the central nervous system (CNS) of fish and amphibians, but not in the mammalian CNS reverses the developmental down-regulation of GAP-43 expression. To explore functional conservation and divergence of cis-acting elements that regulate expression of the GAP-43 gene, we studied activation, in transgenic zebrafish embryos, of mammalian GAP-43 genomic sequences fused to a marker gene. The DNA fragments containing the GAP-43 promoter, including a short fragment of 386 base pairs, were preferentially activated in the embryonic fish nervous system at times when extensive neuronal differentiation and neurite outgrowth take place.

After 2 days of development, expression of the mammalian transgenes was specifically downregulated in the fish spinal cord but increased in more rostral regions of the CNS. This expression pattern was well correlated with the regulation of the endogenous fish GAP-43 gene revealed by in situ hybridization. Elements of the mammalian gene located a substantial distance upstream of the minimal promoter directed additional expression of the marker gene in a specific set of non-neural cells in zebrafish embryos. Our results indicate that cis-acting elements of the GAP-43 gene, and signaling pathways controlling these elements during embryonic development, have been functionally conserved in vertebrate evolution.
A zebrafish retinoic acid receptor expressed in the regenerating caudal fin
Authors: White Jay A. , Boffa Michael B. , Jones Barbara , Petkovich Martin
Published: April 2021

Retinoic acid (RA) is an important signalling molecule in vertebrate pattern formation both in developing and regenerating tissues. The effects of RA are due largely to regulation of gene transcription, mediated by retinoic acid receptors (RAR-α, RAR-β, RAR-γ) and retinoid X receptors (RXR-α, RXR-β, RXR-γ). We have been using zebrafish as a model of regeneration to study the role of retinoic acid and its receptors in vertebrate pattern formation. In this report, we describe the molecular cloning and characterization of one of the zebrafish RARs that is the predominant receptor in the regenerating caudal fin and corresponds most closely to the RAR-γ subtype isolated from mouse and human and to RAR-δ from newt. Zebrafish RAR-γ (zfRAR-γ) exhibits both structural and functional conservation with its mammalian counterparts. Studies utilizing both normal and regenerating caudal fins of the zebrafish have indicated that it is the RAR-γ subtype, compared to RAR-α or RAR-β, which is expressed at the highest levels in the tail fin. To localize the expression pattern of RAR-γ during fin regeneration, we have carried out whole-mount in situ hybridization. ZfRAR-γ transcripts, during fin regeneration, are localized in the blastemal tissue formed at the distal ends of the bony rays following amputation. Treatment of fish with RA during fin regeneration induces a number of striking morphological effects on the regenerate. When amputations are performed distal to the branch points or dichotomies, where a single ray bifurcates to extend two individual ‘daughter’ rays, RA treatment causes a dichotomy reduction where the two ‘daughter’ rays fuse to once again form a single ray. The single ray subsequently bifurcates in a comparatively normal manner. Our data suggest that exogenous RA can respecify pattern in the regenerating caudal fin and identifies the blastemae as possible RA target tissues.
Heterochronic effects of glossy15 mutations on epidermal cell identity in maize
Authors: Evans Matthew M. S. , Passas Hildrun J. , Poethig R. Scott
Published: April 2021

Vegetative development in maize is divided into a juvenile phase and an adult phase that differ in the expression of a large number of morphological, anatomical, and biochemical traits. Recessive mutations of Glossy15 cause a premature switch in the expression of some of these phase-specific traits. Mutant plants cease producing juvenile traits (e.g. epicuticular wax) and begin to produce adult traits (e.g. epidermal hairs) significantly earlier than their wild-type siblings. In glossy15-1 plants this switch generally occurs at leaf 2 or 3 rather than at the normal position of leaf 6 or 7. An analysis of the effect of glossy15 mutations on a variety of vegetative and reproductive traits revealed that these mutations only affect the character of the epidermis. They have no effect on the overall vegetative morphology of the plant, or on its reproductive development. This phenotype is the opposite of that of the gain-of-function mutations Teopod1, Teopod2 and Teopod3, all of which prolong the expression of a large number of juvenile traits. Double mutants between glossy15 and Teopod1 or Teopod2 indicate that Glossy15 is required for the effect of Teopod1 and Teopod2 on epidermal traits but not for other aspects of the Teopod phenotype. We conclude that Glossy15 initiates or maintains the expression of juvenile epidermal traits and suppresses the expression of adult epidermal traits, and that it acts downstream of the Teopod genes.
Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS
Authors: Echelard Yann , Vassileva Galya , McMahon Andrew P.
Published: April 2021

The protooncogene Wnt-1 encodes a short-range signal which is first expressed in, and appears to demarcate, the presumptive midbrain. Absence of Wnt-1 expression leads to the loss of this region of the brain. By the end of neural tube closure, expression of Wnt-1 extends down much of the dorsal midline of the central nervous system (CNS). Expression is exclusively limited to the CNS at this and later stages. We have investigated the regulation of Wnt-1 during mouse development. Analysis of the embryonic expression of Wnt-1-lacZ reporter constructs spanning nearly 30 kb of the Wnt-1 locus identified a 5.5 kb cis-acting 3′ enhancer element which confers correct temporal and spatial expression on the lacZ gene. Interestingly embryos express Wnt-1-lacZ transgenes in migrating neural crest cells which are derived from the dorsal CNS. Ectopic expression of the Wnt-1-lacZ transgenes may result from perdurance of β-galactosidase activity in migrating neural crest cells originating from a Wnt-1-expressing region of the dorsal CNS. Alternatively, ectopic expression may arise from transient de novo activation of the transgenes in this cell population. These results are a first step towards addressing how regional cell signaling is established in the mammalian CNS. In addition, transgene expression provides a new tool for the analysis of neural crest development in normal and mutant mouse embryos.
Slow emergence of a multithreshold response to activin requires cell-contact-dependent sharpening but not prepattern
Authors: Green Jeremy B. A. , Smith J. C. , Gerhart John C.
Published: April 2021

The growth factor activin elicits mesodermal fates when applied to prospective ectodermal cells of the Xenopus blastula stage embryo. Previous experiments with dissoci-ated cells showed that there are at least five different responses separated by closely spaced, sharp dose thresh-olds. Here we investigate this multithreshold activin response further using probes for genes expressed at early gastrula stages, namely Xbra, goosecoid, noggin, Xwnt-8 and Mix.1. We show that initial dose-response profiles are broad and smooth in contrast to the later threshold-bound patterns. For Xbra, goosecoid and noggin, the later expression ranges are subsets of earlier ones. Unexpectedly, Xwnt-8 is initially induced at high doses only, but later appears only in cells that have received a low dose of activin. Keeping the cells dissociated after activin treatment, rather than allowing them to reaggregate, prevents sustained expression of Xbra and Xwnt-8 but allows that of goosecoid and noggin. However, cell contact is required for sharpening the dose-response threshold of goosecoid. Finally, we show that a previously reported dorsoventral prepattern in the animal cap is also cell-contact dependent and it is not required for the multi-threshold response to activin.
Misexpression of the Drosophila argos gene, a secreted regulator of cell determination
Authors: Freeman Matthew
Published: April 2021

I have examined the effects on cells in the developing eye of over-expressing the argos gene. Transgenic flies carrying argos expressed under hsp70 and sevenless control sequences were analysed. All cell types in the developing eye (except bristles) are sensitive to argos concentration: over-expression leads to too few cells forming, the opposite phenotype to that seen in argos loss-of-function mutants. This effect was only seen with HS-argos flies: sev-argos flies, which over-express the protein at a lower level are not affected, suggesting that a considerable over-expression is required to disrupt cell fate. However, sev-argos is able to rescue argos eye mutations completely, indicating that the normal expression pattern is not critical for wild-type eye development. By transfecting argos into tissue culture cells, I show that the protein is secreted in a soluble form.
Morphological differences in Xenopus embryonic mesodermal cells are specified as an early response to distinct threshold concentrations of activin
Authors: Symes Karen , Yordán Cristina , Mercola Mark
Published: April 2021

The involution of presumptive mesoderm that occurs during amphibian gastrulation is a complex process requiring the coordinated action of a diverse range of cells. We show that cells with distinct morphologies, resembling each of those normally found in the involuting tissue of the Xenopus embryo, are induced in dispersed animal pole cells by different doses of the potent mesoderm-inducing factor activin. Each cell type is induced within a restricted dose range of activin concentrations, the boundaries of which are well demarcated shortly after activin treatment. In contrast, Brachyury and goosecoid, two genes thought to pattern the presumptive mesoderm, and the gene encoding platelet-derived growth factor receptor α, which is expressed in the mesoderm of gastrula stage embryos, are induced by broad, overlapping ranges of high activin con- centrations at such early times. Similarly, the response of the gene encoding platelet-derived growth factor A, which is expressed normally in ectoderm of gastrula stage embryos, diminishes gradually as the activin concentration increases. Dose windows for the expression of these four genes narrow and become distinct from one another in cell aggregates after several hours in culture, suggesting that activin prompts a dynamic program of gene expression in induced mesoderm.
Ectopic Hoxa-1 induces rhombomere transformation in mouse hindbrain
Authors: Zhang Maobin , Kim Hee-Joong , Marshall Heather , Gendron-Maguire Maureen , Lucas Debra A. , Baron Agnès , Gudas Lorraine J. , Gridley Thomas , Krumlauf Robb , Grippo Joseph F.
Published: April 2021

Homeobox genes are expressed with a specific spatial and temporal order, which is essential for pattern formation during the early development of both invertebrates and vertebrates. Here we show that widespread ectopic expression of the Hoxa-1 (Hox 1.6) gene directed by a human β-actin promoter in transgenic mice is embry-olethal and produces abnormal phenotypes in a subset of domains primarily located in anterior regions. Interestingly, this abnormal development in the Hoxa-1 transgenic mice is associated with ectopic expression of the Hoxb-1 (Hox 2.9) gene in select hindbrain regions. At gestation day 9.5, two domains of strong Hoxb-1 expression are found in the anterior region of the hindbrains of Hoxa-1 transgenic embryos. One region represents the normal pattern of Hoxb-1 expression in rhombomere 4 and its associated migrating neural crest cells, while another major domain of Hoxb-1 expression consistently appears in rhombomere 2. Similar ectopic domains of β-galactosidase activity are detected in dual transgenic embryos containing both β-actin/Hoxa-1 transgene and a Hoxb-1/lacZ reporter construct. Expression of another lacZ reporter gene that directs β-galactosidase activity predominately in rhombomere 2 is suppressed in the Hoxa-1 transgenic embryos. We have also detected weaker and variable ectopic Hoxb-1 expression in rhombomeres 1, 3 and 6. No ectopic Hoxb-1 expression is detected in rhombomere 5 and the expression of Hoxa-3 and Krox-20 in this region is unchanged in the Hoxa-1 transgenic embryos. While no obvious change in the morphology of the trigeminal or facial-acoustic ganglia is evident, phenotypic changes do occur in neurons that emanate from rhombomeres 2 and 3 in the Hoxa-1 transgenic embryos. Additionally, alterations in the pattern of Hoxa-2 and Hoxb-1 expression in a sub-population of neural crest cells migrating from the rhombomere 2 region are detected in these transgenics. Taken together, these data suggest that ectopic Hoxa-1 expression can reorganize select regions of the developing hindbrain by inducing partial transformations of several rhombomeres into a rhombomere-4-like identity.
Microtubule arrays of the zebrafish yolk cell: organization and function during epiboly
Authors: Solnica-Krezel Lilianna , Driever Wolfgang
Published: April 2021

In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer.

At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter.

We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endo-cytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to a delay in all epibolic movements.

We propose that the yolk cell microtubules contribute either directly or indirectly to all epibolic movements. However, the epibolic movements of the yolk syncytial layer nuclei and of the blastoderm are not coupled, and only movements of the yolk syncytial nuclei are absolutely dependent on microtubules. We hypothesize that the microtubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the mechanisms by which the two microtubule arrays might function during epiboly are discussed.
Clonal relationships and cell patterning in the root epidermis of Arabidopsis
Authors: Dolan Liam , Duckett Catherine M. , Grierson Claire , Linstead Paul , Schneider Katharina , Lawson Emily , Dean Caroline , Poethig Scott , Roberts Keith
Published: April 2021

The development of the post-embryonic root epidermis of Arabidopsis thaliana is described. Clonal analysis has identified three sets of initials that give rise to the columella root cap cells, epidermis and lateral root-cap cells, and the cells of the cortex and endodermis respectively. The mature epidermis is composed of two cell types, root hair cells (derived from trichoblasts) and non-hair cells (derived from atrichoblasts). These cells are arranged in sixteen or more discrete files. Each hair cell file overlies the anticlinal (radial) wall of the underlying cortical cells and is separated from the next by one or two non-hair files. The root hair forms as a tip-growing projection from the basal end of the trichoblast i.e. the end nearest the root meristem. The non-hair epidermal cells are significantly longer than the hair forming cells and are located over the outer peri-clinal (tangential) wall of the underlying cortical cells. The size difference between the two cell types is apparent in the cell division zone before hairs form. This suggests that the signals required for the differentiation of the root epidermis function in the meristem itself. Ectopic hairs are present in the ctr1 root epidermis suggesting that a Raf protein kinase may play a role in pattern formation/differentiation in the root epidermis and that ethylene may be a diffusible signal involved in specifying pattern in the root epidermis.
Avian marginal zone cells function as primitive streak inducers only after their migration into the hypoblast
Authors: Eyal-Giladi Hefzibah , Lotan Tamar , Levin Tovi , Avner Orit , Hochman Jacob
Published: April 2021

Hypoblast cells of posterior marginal zone origin have been shown previously to be the inducers of primitive streak in the avian embryo. Here we checked: (1) whether the above cells acquire their inductivity while still whithin the marginal zone; (2) can inductivity be found in supernatants of defined blastodermic regions; (3) can differences in the electrophoretic pattern be shown between inducing and non-inducing tissue fragments and their conditioned media, which might give a clue as to what the inductive substance is.

The following observations were made: 1.(a) Stage X chick posterior marginal zone cells prior to their migration into the hypoblast do not induce a primitive streak, when applied to a stage XIII competent epiblast central disc. (b) A posterior marginal zone fragment, when applied to an epiblast central disc, even after being preincubated for up to 9 hours in vitro, is still non-inductive. (c) Mechanically fragmented stage X posterior marginal zones when applied as a layer to epiblast central discs are non-inductive. (d) Hypoblastic tissue in strip form induces a primitive streak.2.Competent stage XIII epiblast central discs (chick) were incubated for 2 hours in supernatants of stage XIII epiblasts or hypoblasts. Whereas no inductive effect was exerted by the epiblast supernatant, primitive streaks developed in about 50% of the epiblast central discs incubated in the hypoblast supernatant.3.Electrophoretic analysis (quails) reveals a protein of 28×10—3 Mr that is enriched in both hypoblastic tissue and its incubation medium and not in the epiblast + marginal zone + area opaca and their incubation medium. These findings suggest a possible correlation between this protein and the induction process.
Molecular characterisation of the Polycomblike gene of Drosophila melanogaster, a trans-acting negative regulator of homeotic gene expression
Authors: Lonie Andrew , D’Andrea Richard , Paro Renato , Saint Robert
Published: April 2021

The Polycomblike gene of Drosophila melanogaster, a member of the Polycomb Group of genes, is required for the correct spatial expression of the homeotic genes of the Antennapaedia and Bithorax Complexes. Mutations in Polycomb Group genes result in ectopic homeotic gene expression, indicating that Polycomb Group proteins maintain the transcriptional repression of specific homeotic genes in specific tissues during development. We report here the isolation and molecular characterisation of the Polycomblike gene. The Polycomblike transcript encodes an 857 amino acid protein with no significant homology to other proteins. Antibodies raised against the product of this open reading frame were used to show that the Polycomb-like protein is found in all nuclei during embryonic development. Antibody staining also revealed that the Polycomblike protein is found on larval salivary gland polytene chromosomes at about 100 specific loci, the same loci to which the Polycomb and polyhomeotic proteins, two other Polycomb Group proteins, are found. These data add further support for a model in which Polycomb Group proteins form multimeric protein complexes at specific chromosomal loci to repress transcription at those loci.
Restoration of the organizer after radical ablation of Hensen’s node and the anterior primitive streak in the chick embryo
Authors: Psychoyos Delphine , Stern Claudio D.
Published: April 2021

The region of the amniote embryo corresponding to Spemann’s organizer in amphibians is Hensen’s node, which lies at the tip of the primitive streak during gastrulation. It is a special site in the embryo that can be defined by the presence of progenitors of several axial tissues (notochord, prechordal mesoderm, somites, gut endoderm), by characteristic cell movements, by specific patterns of gene expression (e.g. goosecoid, HNF-3β, Sonic hedgehog) and, most importantly, by its ability to induce a complete axis, including host-derived neural tissue, when transplanted to an ectopic site.

Here, we show that complete removal not only of the node but also of the anterior 40% of the primitive streak leads to the development of normal embryos containing cells with all the fates normally produced by the node. Cell movement pathways through the regenerated node are identical to those seen in the normal embryo. The patterns of expression of HNF-3β and Sonic hedgehog are also restored, as is their left/right asymmetry, but goosecoid expression is not. When the regenerated node is transplanted to an ectopic site, it induces a complete embryonic axis that includes a fully patterned, host-derived central nervous system.

Analysis of the properties of cells surrounding the site of ablation shows that they acquire these properties gradually. We suggest that the organizer is a region of the embryo that is defined by cell interactions and that the node normally inhibits the organizer state in neighbouring cells.
The Dictyostelium dual-specificity kinase splA is essential for spore differentiation
Authors: Nuckolls Glen H. , Osherov Nir , Loomis William F. , Spudich James A.
Published: April 2021

We have studied the structure and function of the Dictyostelium kinase splA. A truncated form of the splA protein exhibited primarily tyrosine kinase activity in vitro; however, it also autophosphorylated on serine and threonine residues. The kinase domain of splA exhibits approximately 38% identity to the CTR1 kinase of Arabidopsis, which is a member of the Raf family. Outside its kinase domain, splA shares homology with the byr2 kinase of S. pombe. By aligning the sequences of splA, byr2 and STE11, a homologue of byr2 in S. cerevisiae, we have identified a conserved motif that is also found in members of the Eph family of growth factor receptor tyrosine kinases. SplA is expressed throughout development with a peak during the mound stage of morphogenesis. Strains in which the splA gene had been disrupted completed fruiting body formation; however, spore cells spontaneously lysed before completing their differentiation. Northern analysis revealed the expression of the prespore marker cotB and the prestalk markers ecmA and ecmB in the mutant strain during development. The spore differentiation marker spiA was detected in the mutant spores both by northern and immunoblotting, but these cells failed to assemble spore coats. Immunoblot analysis of the developmental pattern of tyrosine phosphorylation revealed a protein that was phosphorylated in mutants but was not phosphorylated in the wild-type cells. SplA is a novel dual specificity kinase that regulates the differentiation of spore cells.
Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis
Authors: Kreidberg Jordan A. , Donovan Michael J. , Goldstein Stuart L. , Rennke Helmut , Shepherd Kenneth , Jones Rosemary C. , Jaenisch Rudolf
Published: April 2021

A mutation was targeted to the murine α3 integrin gene. Homozygous mutant mice survived to birth, but died during the neonatal period. The mutation caused abnormal kidney and lung development. Mutant kidneys displayed decreased branching of the medullary collecting ducts, although the number of nephrons was not altered. Proximal tubules exhibited two distinct subsets of abnormalities, with the epithelial cells either containing excess lysosomes or becoming microcystic. In addition, glomerular development was markedly affected. In mutant kidneys, the extent of branching of glomerular capillary loops was decreased, with capillary lumina being wider than normal. The glomerular basement membrane was disorganized and glomerular podocytes were unable to form mature foot processes. Branching of the bronchi in lungs of mutant mice was also decreased and the large bronchi extended to the periphery. These results indicate a role for integrin receptors in basement membrane organization and branching morphogenesis.
Failure of ventral body wall closure in mouse embryos lacking a procollagen C-proteinase encoded by Bmp1, a mammalian gene related to Drosophila tolloid
Authors: Suzuki Noboru , Labosky Patricia A. , Furuta Yasuhide , Hargett Linda , Dunn Ray , Fogo Agnes B. , Takahara Kazuhiko , Peters Donna M. P. , Greenspan Daniel S. , Hogan Brigid L. M.
Published: April 2021

The mouse bone morphogenetic protein1 (Bmp1) gene encodes a secreted astacin metalloprotease that cleaves the COOH-propeptide of procollagen I, II and III. BMP-1 is also related to the product of the Drosophila patterning gene, tolloid (tld), which enhances the activity of the TGFβ-related growth factor Decapentaplegic and promotes deveopment of the dorsalmost amnioserosa. We have disrupted the mouse Bmp1 gene by deleting DNA sequences encoding the active site of the astacin-like protease domain common to all splice variants. Homozygous mutant embryos appear to have a normal skeleton, apart from reduced ossification of certain skull bones. However, they have a persistent herniation of the gut in the umbilical region and do not survive beyond birth. Analysis of the amnion of homozygous mutant embryos reveals the absence of the fold that normally tightly encloses the physiological hernia of the gut. At the electron microscopic level, the extracellular matrix of the amnion contains collagen fibrils with an abnormal morphology, consistent with the incorporation of partially processed procollagen molecules. Metabolical labelling and immunofluorescence studies also reveal abnormal processing and deposition of procollagen by homozygous mutant fibroblasts in culture.
Flowering as metamorphosis: two sequential signals regulate floral initiation in Lolium temulentum
Authors: McDaniel Carl N. , Hartnett Laura K.
Published: April 2021

We investigated floral initiation in the long-day monocot Lolium temulentum, strain Ceres, by culturing apices explanted from photoperiodically induced plants at various times after one inductive long day onto medium with, and without, gibberellin. Apices cultured on the first day after the inductive long day usually required gibberellin in the medium to initiate floral morphogenesis while apices explanted on the second day after induction did not require gibberellin. Apices explanted on the first day after induction onto medium without gibberellin grew vegetatively for many days but a several-day exposure to culture medium with gibberellin at any time caused most apices to initiate floral morphogenesis. The gibberellin synthesis inhibitor, ancymidol, when applied to plants before apex excision and when present in the culture medium reduced floral initiation by more than 50% in the absence of added gibberellin in the medium, but it was ineffective in the presence of gibberellin. These results indicated that floral initiation in photoperiodically induced plants resulted from two signals acting at the apex. The first signal induced the apex into a florally determined state and then the second signal, gibberellin, elicited expression of the florally determined state. Leaf removal and culture of apices from plants previously treated with gibberellin provided evidence that the leaf-applied gibberellin did not itself act on the apex to cause floral determination or initiation. Rather, the exogenous gibberellin appeared to stimulate the production of a signal in the leaves that then led to floral initiation.
Location and behavior of dorsal determinants during first cell cycle in Xenopus eggs
Authors: Kikkawa Mika , Takano Kazuhiro , Shinagawa Atsunori
Published: April 2021

In Xenopus eggs, removal of small volumes of cytoplasm along with the surface (2-10% of the entire egg volume) causes very severe dorsal reduction (average DAI=1.4) when made at a site ventrally 30° off the vegetal pole at 20% time of first cell cycle (0.2 NT). The greatest dorsal reduction (average DAI=1.1) occurs when removal is done at the vegetal pole at 0.3 NT, and intermediate reductions (average DAI=2.2-2.6) when done at sites dorsally, dorsolaterally or laterally 30° off the vegetal pole at 0.4 NT. Removal at sites dorsally, dorsolaterally or laterally 60° off the vegetal pole provokes slight dorsal reduction (average DAI=3.5-3.9) when made at 0.4-0.5 NT. Removal at all sites after 0.4 NT causes a steady decrease in the extent of dorsal reduction. By contrast, removal of larger volumes of dorsal cytoplasm (16-50% of the entire egg volume) causes a steady increase in the extent of dorsal reduction during first cell cycle with its maximum effect at 1.0 NT (average DAI=3.1). The surgery for the cytoplasmic removal does not affect cortical rotation. We conclude from these results that dorsal determinants are concentrated first in a small region ventrally 30° off the vegetal pole by 0.2 NT, then move toward the vegetal pole during the period 0.2-0.3 NT and disperse to a broad region spanning over both the presumptive dorsal and ventral, but mainly the dorsal, hemispheres during the period 0.3-0.8 NT.
Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway
Authors: Shawber Carrie , Nofziger Donna , Hsieh James J.-D. , Lindsell Claire , Bögler Oliver , Hayward Diane , Weinmaster Gerry
Published: April 2021

Notch controls cell fate by inhibiting cellular differentiation, presumably through activation of the transcriptional regulator human C promoter Binding Factor (CBF1), which transactivates the hairy and Enhancer of split (HES-1) gene. However, we describe constitutively active forms of Notch1, which inhibit muscle cell differentiation but do not interact with CBF1 or upregulate endogenous HES-1 expression. In addition, Jagged-Notch interactions that prevent the expression of muscle cell specific genes do not involve the upregulation of endogenous HES-1. In fact, exogenous expression of HES-1 in C2C12 myoblasts does not block myogenesis. Our data demonstrate the existence of a CBF1-independent pathway by which Notch inhibits differentiation. We therefore propose that Notch signaling activates at least two different pathways: one which involves CBF1 as an intermediate and one which does not.
Incomplete sister chromatid separation is the mechanism of programmed chromosome elimination during early Sciara coprophila embryogenesis
Authors: Phalle Brigitte de Saint , Sullivan William
Published: April 2021

Sex in Sciara coprophila is determined by maternally supplied factors that control the number of paternal X chromosomes eliminated during the syncytial embryonic divisions. Confocal microscopy and FISH demonstrate that the centromeres of the X chromosomes separate at anaphase and remain functional during the cycle in which the X chromosomes are eliminated. However, a region of the sister chromatids fails to separate and the X chromosomes remain at the metaphase plate. This indicates that failure of sister chromatid separation is the mechanism of chromosome elimination. Elimination of the X chromosomes requires the presence of a previously discovered Controlling Element that acts in cis during male meiosis. Using an X-autosome translocation, we demonstrate that the Controlling Element acts at-a-distance to prevent sister chromatid separation in the arm of an autosome. This indicates that the region in which sister chromatid separation fails is chromosome-independent. Although chromosome elimination occurs in all somatic nuclei and is independent of location of the nuclei within the embryo, the decision to eliminate is made at the level of the individual nucleus. Programmed X chromosome elimination occurs at different cycles in male and female embryos. These observations support a model in which elements on the X chromosome are titrating maternally supplied factors controlling the separation of sister X chromatids.
Unequal cleavage in leech embryos: zygotic transcription is required for correct spindle orientation in a subset of early blastomeres
Authors: Bissen Shirley T. , Smith Constance M.
Published: April 2021

Leech embryos undergo invariant sequences of equal and unequal cell divisions to give rise to identifiable progeny cells. While many of the early cleavages are under maternal control, the divisions of a subset of early blastomeres (the large cells of the D′ lineage) are perturbed after the inhibition of zygotic transcription. Analysis of the different types of cells produced in embryos injected with the transcriptional inhibitor, α-amanitin, revealed that the symmetry of cell division is perturbed in these large D′derived cells during this early period of development. These cells, which would normally undergo a series of equal and unequal cleavages, always undergo equal cleavages after the inhibition of zygotic transcription. It appears that zygotically transcribed gene product(s) are required in the large cells of the D′ lineage to orient the mitotic spindles properly for these unequal cell cleavages.
The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered
Authors: Montagne Jacques , Groppe Jay , Guillemin Karen , Krasnow Mark A. , Gehring Walter J. , Affolter Markus
Published: April 2021

The adult Drosophila wing is formed by an epithelial sheet, which differentiates into two non-neural tissues, vein or intervein. A large number of genes, many of them encoding components of an EGF-receptor signaling pathway, have previously been shown to be required for differentiation of vein tissue. Much less is known about the molecular control of intervein differentiation. Here we report that the Drosophila homolog of the mammalian Serum Response Factor gene (DSRF), which encodes a MADS-box containing transcriptional regulator, is expressed in the future intervein tissue of wing imaginal discs. In adult flies carrying only one functional copy of the DSRF gene, additional vein tissue develops in the wing, indicating that DSRF is required to spatially restrict the formation of veins. In mitotic clones lacking DSRF, intervein tissue fails to differentiate and becomes vein-like in appearance. Genetic and molecular evidence demonstrates that DSRF is encoded by the blistered locus, which produces ectopic veins and blistered wings when mutant. Our results show that DSRF plays a dual role during wing differentiation. It acts in a dosage-dependant manner to suppress the formation of wing veins and is required cell-autonomously to promote the development of intervein cells. We propose that DSRF acts at a key step between regulatory genes that define the early positional values in the developing wing disc and the subsequent localized expression of interveinspecific structural genes.
In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product
Authors: Bertuccioli Claudio , Fasano Laurent , Jun Susie , Wang Sheng , Sheng Guojun , Desplan Claude
Published: April 2021

The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the Nterminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.
The regulation of Krox-20 expression reveals important steps in the control of peripheral glial cell development
Authors: Murphy Paula , Topilko Piotr , Schneider-Maunoury Sylvie , Seitanidou Tania , Evercooren Annick Baron-Van , Charnay Patrick
Published: April 2021

The zinc finger transcription factor gene Krox-20 is expressed in Schwann cells and is required for the myelination of peripheral nerves. We show that the regulation of Krox-20 expression in peripheral glial cells reveals three important steps in the development and differentiation of these cells. (i) Expression of Krox-20 in Schwann cells requires continuous neuronal signalling via direct axonal contact. Therefore Krox-20 appears to be a key component of the transduction cascade linking axonal signalling to myelination. (ii) Krox-20 inducibility is acquired by Schwann cells at the time that they are formed from their precursors. Diffusible factor(s) synthesised by the neural tube can mediate this transition and can be mimicked by NDFβ or a combination of CNTF and bFGF. Furthermore, the neural tube activity is blocked by a hybrid protein containing the NDF-binding domain of the ErbB4 receptor, strongly implicating NDF in the physiological transition. (iii) In sensory ganglia, the microenvironment is capable of negatively regulating Krox-20, presumably by preventing the conversion of satellite glial cells toward a Schwann celllike phenotype.
Integrin-dependent adhesive activity is spatially controlled by inductive signals at gastrulation
Authors: Ramos Joe W. , Whittaker Charles A. , DeSimone Douglas W.
Published: April 2021

Integrins mediate cell-ECM interactions essential for morphogenesis, however, the extent to which integrin adhesive activities are regulated in the embryo has not been addressed. We report that integrin-dependent cell adhesion to the Arg-Gly-Asp (RGD) containing central cell-binding domain of fibronectin is required for gastrulation in Xenopus. Although all cells of the early embryo retain the ability to attach to this region, only involuting cells arising from the dorsal and ventral lips of the blastopore are able to spread and migrate on fibronectin in vitro. This change in adhesive behavior is mimicked by treating animal cap cells with activin-A. Activin-induced changes in adhesion are independent of new transcription, translation, or changes in receptor expression at the cell surface. We demonstrate that ectopic expression of integrin α4β1 in animal cap cells results in attachment to the non RGD-containing V-region of fibronectin. Further, these cells acquire the ability to spread on the V-region following activin induction. Thus, α4β1 adhesion to the V-region, like endogenous integrin binding to the central cell-binding domain, is responsive to activin signalling. These data indicate that cell adhesion to the central cell-binding domain is regulated in both space and time, and is under the control of inductive signals that initiate gastrulation movements. We suggest that position-specific inductive interactions are likely to represent a novel and general mechanism by which integrin adhesion is modulated throughout development.
Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile
Authors: Rankin Tracy , Familari Mary , Lee Eric , Ginsberg Ann , Dwyer Nancy , Blanchette-Mackie Joan , Drago John , Westphal Heiner , Dean Jurrien
Published: April 2021

Mammalian oocytes synthesize and secrete a zona pellucida that surrounds the growing oocytes, ovulated eggs and preimplantation embryos. The extracellular zona matrix is composed of three glycoproteins (ZP1, ZP2, ZP3) that are involved in folliculogenesis, species-specific fertilization, and passage of the early embryo down the oviduct. We have established a mouse line in which Zp3 has been inactivated by homologous recombination with an insertional mutation. Neither Zp3 transcripts nor ZP3 protein was detected in female mice homozygous for the mutation (Zp3−/−), whereas both ZP1 and ZP2 were present in mutant oocytes. Homozygous mutant Zp3−/− mice had follicles with germinal-vesicle-intact oocytes but that lacked a zona pellucida matrix and had a disorganized corona radiata. Although mutant oocytes underwent germinal vesicle breakdown (GVBD) prior to ovulation, the cumulus-oocyte complex was markedly disrupted and the oocytes were often separate from the cumulus cells. After hormone-induced ovulation, cumulus masses were present in the oviducts of homozygous mutant mice, but zona-free eggs were observed in only half of the females and, in these, less than 10% of the normal mumber of eggs were detected. No zona-free 2-cell embryos were recovered from homozygous mutant Zp3−/− female mice after mating with males proven to be fertile, and none became visibly pregnant or produced offspring. These results demonstrate that a genetic defect in a zona pellucida gene causes infertility and, given the conserved nature of the zona pellucida, a similar phenotype is expected in other mammals.
Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties
Authors: Joliot Alain , Trembleau Alain , Raposo Graça , Calvet Sophie , Volovitch Michel , Prochiantz Alain
Published: April 2021

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.