bluuebunny/crossref_metadata_2025 huggingface dataset:
Dorso-ventral ectodermal compartments and origin of apical ectodermal ridge in developing chick limb
Authors: Altabef Muriel , Clarke Jonathan D. W. , Tickle Cheryll
Published: April 2021

We wish to understand how limbs are positioned with respect to the dorso-ventral axis of the body in vertebrate embryos, and how different regions of limb bud ectoderm, i.e. dorsal ectoderm, apical ridge and ventral ectoderm, originate. Signals from dorsal and ventral ectoderm control dorso-ventral patterning while the apical ectodermal ridge (AER) controls bud outgrowth and patterning along the proximo-distal axis. We show, using cell-fate tracers, the existence of two distinct ectodermal compartments, dorsal versus ventral, in both presumptive limb and flank of early chick embryos. This organisation of limb ectoderm is the first direct evidence, in vertebrates, of compartments in non-neural ectoderm. Since the apical ridge appears to be confined to this compartment boundary, this positions the limb. The mesoderm, unlike the ectoderm, does not contain two separate dorsal and ventral cell lineages, suggesting that dorsal and ventral ectoderm compartments may be important to ensure appropriate control of mesodermal cell fate. Surprisingly, we also show that cells which form the apical ridge are initially scattered in a wide region of early ectoderm and that both dorsal and ventral ectoderm cells contribute to the apical ridge, intermingling to some extent within it.
A novel α integrin subunit associates with β PS and functions in tissue morphogenesis and movement during Drosophila development
Authors: Stark Karen A. , Yee Gene H. , Roote Carol E. , Williams Erin L. , Zusman Susan , Hynes Richard O.
Published: April 2021

We have identified a novel a integrin subunit in Drosophila, that associates with bPS integrin. We report the temporal expression of the gene encoding this integrin subunit, which we have called aPS3, throughout development and the localization of its expression during embryogenesis. aPS3 RNA was localized to tissues undergoing invagination, tissue movement and morphogenesis such as salivary gland, trachea, midgut, dorsal vessel, midline of the ventral nerve cord, amnioserosa and the amnioproctodeal invagination. aPS3 DNA localized to the chromosomal vicinity of scab (scb), previously identified by a failure of dorsal closure. Embryos homozygous for the l19 allele of scb had no detectable aPS3 RNA and the 1035 allele of scb contains a P element inserted just 5¢ of the coding region for the shorter of the gene’s two transcripts. Furthermore, mutations in the scb locus exhibit additional defects corresponding to sites of aPS3 transcription, including abnormal salivary glands, mislocalization of the pericardial cells and interrupted trachea. Removal of both maternal and zygotic bPS produced similar defects, indicating that these two integrin subunits associate in vivo and function in the movement and morphogenesis of tissues during development in Drosophila. Phenotypic similarities suggest that laminin A is a potential ligand for this integrin, at least in some tissues.
SpMyb functions as an intramodular repressor to regulate spatial expression of Cyllla in sea urchin embryos
Authors: Coffman James A. , Kirchhamer Carmen V. , Harrington Michael G. , Davidson Eric H.
Published: April 2021

The Cyllla actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in Cyllla transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing Cyllla reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of Cyllla. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpura-tus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of Cyllla in the oral ectoderm and skeletogenic mesenchyme.
Vitamin A deficiency and mutations of RXRα, RXRβ and RARα lead to early differentiation of embryonic ventricular cardiomyocytes
Authors: Kastner Philippe , Messaddeq Nadia , Mark Manuel , Wendling Olivia , Grondona Jesus M. , Ward Simon , Ghyselinck Norbert , Chambon Pierre
Published: April 2021

Knock-out of the mouse RXRa gene was previously shown to result in a hypoplastic heart ventricular wall, histologically detectable in 12.5 dpc fetuses. We show here that a precocious differentiation can be detected as early as 8.5 dpc in ventricular cardiomyocytes of RXRα−/− mutants. This precocious differentiation, which is characterized by the presence of striated myofibrils, sarcoplasmic reticulum and intercalated disks, is found after 9.5 dpc in about 50% of RXRα−/− subepicardial myocytes. In contrast, wild-type subepicardial myocytes remain morphologically undifferentiated up to at least 16.5 dpc. A similar precocious differentiation was observed in 9.5 dpc subepicardial myocytes of several RXR0α−/− and RARα−/− mutants, as well as in vitamin A-deficient embryos. The proportion of differentiated subepicardial myocytes almost reached 100% in RXRα/RXRβ double null mutants, indicating a partial functional redundancy between RXRα and RXRβ. This differentiation defect was always paralleled by a decrease in the mitotic index. In addition, subepicardial myocytes of RXRα−/−, RXRα−/−1/RXRβ−/− or vitamin A deficient, but not of RXRβ−/− and RARα−/− embryos, were often flattened and more loosely connected to one another than those of WT embryos. Thus, retinoids are required at early stages of cardiac development to prevent differentiation, support cell proliferation and control the shape of ventricular myocytes, and both RXRs and RARs participate in the mediation of these functions.
SUP-17, a Caenorhabditis elegans ADAM protein related to Drosophila KUZBANIAN, and its role in LIN-12/NOTCH signalling
Authors: Wen Chenhui , Metzstein Mark M. , Greenwald Iva
Published: April 2021

LIN-12/NOTCH proteins mediate cell-cell interactions that specify cell fates. Previous work suggested that sup-17 facilitates lin-12 signalling in Caenorhabditis elegans. Here, we show that sup-17 encodes a member of the ADAM family of metalloproteases. SUP-17 is highly similar to Drosophila KUZBANIAN, which functions in Drosophila neurogenesis, and the vertebrate ADAM10 protein. Furthermore, we show by genetic analysis that the extracellular domain of LIN-12 appears to be necessary for sup-17 to facilitate lin-12 signalling and that sup-17 does not act downstream of lin-12. Finally, we show by cell ablation experiments that sup-17 can act cell autonomously to facilitate lin-12 activity. We discuss the implications of our observations for LIN-12/NOTCH signalling and how our results complement and extend results obtained from genetic analysis of kuz in Drosophila.
The Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved between flies and vertebrates
Authors: Bonini Nancy M. , Bui Quang T. , Gray-Board Gladys L. , Warrick John M.
Published: April 2021

The fly eyes absent (eya) gene which is essential for compound eye development in Drosophila, was shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, was defined: eya was found to be essential for eye formation by eyeless. Moreover, eya could itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Finally, we show that eya and eyeless together were more effective in eye formation than either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation.
Different levels of Ras activity can specify distinct transcriptional and morphological consequences in early Drosophila embryos
Authors: Greenwood Simon , Struhl Gary
Published: April 2021

The terminal portions of the Drosophila body pattern are specified by the localized activity of the receptor tyrosine kinase Torso (Tor) at each pole of the early embryo. Tor activity elicits the transcription of two ‘gap’ genes, tailless (tll) and huckebein (hkb), in overlapping but distinct domains by stimulating the Ras signal transduction pathway. Here, we show that quantitative variations in the level of Ras activity can specify qualitatively distinct transcriptional and morphological responses. Low levels of Ras activity at the posterior pole direct tll but not hkb transcription; higher levels drive transcription of both genes. Correspondingly, low levels of Ras activity specify a limited subset of posterior terminal structures, whereas higher levels specify a larger subset. However, we also show that the response to Ras activity is not uniform along the body. Instead, levels of Ras activity which suffice to drive tll and hkb transcription at the posterior pole fail to drive their expression in more central portions of the body, apparently due to repression by other gap gene products. We conclude that tll and hkb transcription, as well as the terminal structures, are specified by two inputs: a gradient of Ras activity which emanates from the pole, and the opposing influence of more centrally deployed gap genes which repress the response to Ras.
The Drosophila toucan (toc) gene is required in germline cells for the somatic cell patterning during oogenesis
Authors: Grammont, Muriel , Dastugue Bernard , Couderc Jean-Louis
Published: April 2021

We have characterized a new gene, called toucan, that is expressed and required in germline cells to promote proper differentiation of the somatic follicle cells. toucan mutant ovaries are defective in (i) the enclosure of newly formed germline cysts by the follicle cells, (ii) the formation of interfollicular stalks, (iii) the migration of the follicle cells over the oocyte and (iv) the formation of the eggshell. Overexpression of a toucan cDNA in the germline leads to the production of longer interfollicular stalks than wild-type ovaries, a phenotype that is the exact opposite of the toucan mutant phenotype. This observation shows that the formation of the interfollicular stalks depends not only on interactions among the somatic cells but also requires a germline signal. Moreover, dominant interactions have been observed between toucan and certain alleles of the daughterless, Notch and Delta genes, each of which is required in the somatic cells for the formation of egg chambers. toucan encodes for a large protein with a coiled-coil domain but has no other homology with known proteins. We propose that toucan participates in the production or localization of a germline-specific signal(s) that is required for the patterning of the follicular epithelium.
Regulation of the Twist target gene tinman by modular cis-regulatory elements during early mesoderm development
Authors: Yin Zhizhang , Xu Xiao-Lei , Frasch Manfred
Published: April 2021

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation. This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. We further show that the 180 bp enhancer also includes negatively acting sequences. Binding of Evenskipped to these sequences appears to reduce twistdependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.
Emergence of determined myotome precursor cells in the somite
Authors: Williams Brian A. , Ordahl Charles P.
Published: April 2021

Myotome and sclerotome precursor cells are derived, respectively, from cells in the dorsomedial and ventromedial regions of the somite. To assay changes in the specification of myotomal precursor cells during somite maturation, we implanted dorsomedial quadrant fragments, from staged quail somites, next to the notochords of host chick embryos, and superimposed two additional notochords on these implants. In this notochord signalling environment, dorsomedial quadrant cells that are developmentally plastic are expected to differentiate as cartilage, while cells determined to a myogenic fate are expected to differentiate as skeletal muscle. Large numbers of differentiated chondrocytes developed from dorsomedial quadrant grafts of all stages of paraxial mesoderm development tested, indicating that persistent chondrogenic potential in cells fated to form muscle and dermis can be elicited by notochord signals. Differentiated myocytes, however, appeared in two somite-stage-dependent phases. In the first phase, dorsomedial quadrants from segmental plate and early stage somites (II and IV) form small, disorganized clusters of individual myocytes. The frequency of first-phase myocluster formation increases as myogenic factor expression begins in the dorsomedial quadrant, indicating that myogenic determination assayed by this method is closely linked to the expression of myogenic factors in the dorsomedial quadrant. In the second phase, dorsomedial quadrants from somite stages XI-XIII consistently form morphologically organized muscle tissue containing large numbers of parallel-oriented, multinucleated myotubes. Mitotic labelling demonstrated that muscle precursors were determined to the muscle phenotype prior to withdrawal from the cell cycle. Thus, myogenic determination in cells of the dorsomedial quadrant is acquired at earlier stages of somite maturation than the ability to proliferate and form muscle tissue. These results are consistent with the hypothesis that successive lineages of myotome precursor cells with different mitotic and morphogenetic properties arise in the dorsomedial quadrant during somite maturation.
Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans
Authors: Grant Barth , Greenwald Iva
Published: April 2021

Previous work indicated that sel-1 functions as a negative regulator of lin-12 activity, and predicted that SEL-1 is a secreted or membrane associated protein. In this study, we describe cell ablation experiments that suggest sel-1 mutations elevate lin-12 activity cell autonomously. We also use transgenic approaches to demonstrate that the predicted signal sequence of SEL-1 can direct secretion and is important for function, while a C-terminal hydrophobic region is not required for SEL-1 function. In addition, by analyzing SEL-1 localization using specific antisera we find that SEL-1 is localized intracellularly, with a punctate staining pattern suggestive of membrane bound vesicles. We incorporate these observations, and new information about a related yeast gene, into a proposal for a possible mechanism for SEL-1 function in LIN-12 turnover.
Two Eph receptor tyrosine kinase ligands control axon growth and may be involved in the creation of the retinotectal map in the zebrafish
Authors: Brennan Caroline , Monschau Bruno , Lindberg Rick , Guthrie Brenda , Drescher Uwe , Bonhoeffer Friedrich , Holder Nigel
Published: April 2021

The isolation and characterisation of two zebrafish Eph receptor ligand cDNAs which we have called zfEphL3 and zfEphL4 is described. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours L3 is expressed throughout the midbrain including the region of the presumptive tectum whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoided membranes from Cos cells transfected with L3, whereas nasal axons did not. Both temporal and nasal axons avoided membranes from Cos cells transfected with L4. The expression patterns together with the functional data suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum.
TSO1 functions in cell division during Arabidopsis flower development
Authors: Liu Zhongchi , Running Mark P. , Meyerowitz Elliot M.
Published: April 2021

We describe an Arabidopsis mutant, tso1, which develops callus-like tissues in place of floral organs. The tso1 floral meristem lacks properly organized three cell layers, and the nuclei of these cells are irregular in size and shape. Further analyses reveal partially formed cell walls and increased DNA ploidy in tso1 floral meristem cells, indicating defects in mitosis and cytokinesis. Our finding that TSO1 is required for organ formation in floral tissues but not in other tissues indicates that TSO1 may encode a floral-specific cell division component, or that TSO1 function is redundant in nonfloral tissues.
Importance of nidogen binding to laminin γ1 for branching epithelial morphogenesis of the submandibular gland
Authors: Kadoya Yuichi , Salmivirta Katriina , Talts Jan Fredrik , Kadoya Kuniko , Mayer Ulrike , Timpl Rupert , Ekblom Peter
Published: April 2021

Epithelial-mesenchymal interactions are major driving forces for the development of most solid organs. The importance of these interactions was first shown for the embryonic submandibular gland more than 40 years ago. We here present evidence that interactions between two basement membrane components, nidogen (entactin) and laminin γ1 chain, could be important for epithelial-mes-enchymal interactions in this gland. Nidogen mRNA was detected by in situ hybridization in the mesenchyme, and yet the protein was detected in epithelial and endothelial basement membranes. The role of nidogen-laminin interactions for epithelial morphogenesis was studied by applying antibodies to submandibular gland organ cultures. Antibodies reacting strongly with the nidogen-binding site of laminin γ1 chain drastically perturbed branching epithelial morphogenesis. Electron microscopy of the epithelial-mesenchymal interface showed that blocking antibodies disrupted the formation of the basement membrane. Epidermal growth factor was shown to increase the expression of nidogen in mesenchyme, and could counteract the effect of the blocking antibodies. We suggest that nidogen could be an important mesenchymal factor for submandibular gland development.
Altered cellular proliferation and mesoderm patterning in Polycomb-M33-deficient mice
Authors: Coré Nathalie , Bel Sophie , Gaunt Stephen J. , Aurrand-Lions Michel , Pearce Jonathan , Fisher Amanda , Djabali Malek
Published: April 2021

In Drosophila, the trithorax-group and the Polycomb-group genes are necessary to maintain the expression of the homeobox genes in the appropriate segments. Loss-of-function mutations in those groups of genes lead to misexpression of the homeotic genes resulting in segmental homeotic transformations. Recently, mouse homologues of the Polycomb-group genes were identified including M33, the murine counterpart of Polycomb. In this report, M33 was targeted in mice by homologous recombination in embryonic stem (ES) cells to assess its function during development. Homozygous M33 (−/−) mice show greatly retarded growth, homeotic transformations of the axial skeleton, sternal and limb malformations and a failure to expand in vitro of several cell types including lymphocytes and fibroblasts. In addition, M33 null mutant mice show an aggravation of the skeletal malformations when treated to RA at embryonic day 7.5, leading to the hypothesis that, during development, the M33 gene might play a role in defining access to retinoic acid response elements localised in the regulatory regions of several Hox genes.
The C. elegans MEX-1 protein is present in germline blastomeres and is a P granule component
Authors: Guedes Susana , Priess James R.
Published: April 2021

In the nematode Caenorhabditis elegans, germ cells arise from early embryonic cells called germline blastomeres. Cytoplasmic structures called P granules are present in the fertilized egg and are segregated into each of the germline blastomeres during the first few cleavages of the embryo. Mutations in the maternally expressed gene mex-1 disrupt the segregation of P granules, prevent the formation of germ cells, and cause inappropriate patterns of somatic cell differentiation. We have cloned the mex-1 gene and determined the distribution pattern of the mex-1 gene products. The MEX-1 protein contains two copies of an unusual ‘finger’ domain also found in the PIE-1 protein of C. elegans. PIE-1 has been shown to be expressed in germline blastomeres, and is a component of P granules. We show here that MEX-1 also is present in germline blastomeres and is a P granule component, although MEX-1 is a cytoplasmic protein while PIE-1 is present in both the nucleus and cytoplasm. We further show that MEX-1 is required to restrict PIE-1 expression and activity to the germline blastomeres during the early embryonic cleavages.
The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells
Authors: Ito Kei , Awano Wakae , Suzuki Kazumi , Hiromi Yasushi , Yamamoto Daisuke
Published: April 2021

The mushroom body (MB) is an important centre for higher order sensory integration and learning in insects. To analyse the development and organisation of the MB neuropile in Drosophila, we performed cell lineage analysis in the adult brain with a new technique that combines the Flippase (flp)/FRT system and the GAL4/UAS system. We showed that the four mushroom body neuroblasts (MBNbs) give birth exclusively to the neurones and glial cells of the MB, and that each of the four MBNb clones contributes to the entire MB structure. The expression patterns of 19 GAL4 enhancer-trap strains that mark various subsets of MB cells revealed overlapping cell types in all four of the MBNb lineages. Partial ablation of MBNbs using hydroxyurea showed that each of the four neuroblasts autonomously generates the entire repertoire of the known MB substructures.
Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos
Authors: Pittack Catrin , Grunwald Gerald B. , Reh Thomas A.
Published: April 2021

During eye development, optic vesicles evaginate laterally from the neural tube and develop into two bilayered eye cups that are composed of an outer pigment epithelium layer and an inner neural retina layer. Despite their similar embryonic origin, the pigment epithelium and neural retina differentiate into two very distinct tissues. Previous studies have demonstrated that the developmental potential of the pigmented epithelial cells is not completely restricted; until embryonic day 4.5 in chick embryos, the cells are able to switch their phenotype and differentiate into neural retina when treated with fibroblast growth factors (FGF) (Park, C. M., and Hollenberg, M. J. (1989). Dev. Biol. 134, 201-205; Pittack, C., Jones, M., and Reh, T. A. (1991). Development 113, 577-588; Guillemot, F. and Cepko, C. L. (1992). Development 114, 743-754). These studies motivated us to test whether FGF is necessary for neural retina differentiation during the initial stages of eye cup development. Optic vesicles from embryonic day 1.5 chick were cultured for 24 hours as explants in the presence of FGF or neutralizing antibodies to FGF2. The cultured optic vesicles formed eye cups that contained a lens vesicle, neural retina and pigmented epithelium, based on morphology and expression of neural and pigmented epithelium-specific antigens. Addition of FGF to the optic vesicles caused the presumptive pigmented epithelium to undergo neuronal differentiation and, as a consequence, a double retina was formed. By contrast, neutralizing antibodies to FGF2 blocked neural differentiation in the presumptive neural retina, without affecting pigmented epithelial cell differentiation. These data, along with evidence for expression of several FGF family members and their receptors in the developing eye, indicate that members of the FGF family may be required for establishing the distinction between the neural retina and pigmented epithelium in the optic vesicle.
A Xenopus type I activin receptor mediates mesodermal but not neural specification during embryogenesis
Authors: Chang Chenbei , Wilson Paul A. , Mathews Lawrence S. , Hemmati-Brivanlou Ali
Published: April 2021

Activins and other ligands in the TGFb superfamily signal through a heteromeric complex of receptors. Disruption of signaling by a truncated type II activin receptor, XActRIIB (previously called XAR1), blocks mesoderm induction and promotes neuralization in Xenopus embryos. We report the cloning and characterization of a type I activin receptor, XALK4. Like truncated XActRIIB, a truncated mutant (tXALK4) blocks mesoderm formation both in vitro and in vivo; moreover, an active form of the receptor induces mesoderm in a ligand-independent manner. Unlike truncated XActRIIB, however, tXALK4 does not induce neural tissue. This difference is explained by the finding that tXALK4 does not block BMP4-mediated epidermal specification, while truncated XActRIIB inhibits all BMP4 responses in embryonic explants. Thus, the type I and type II activin receptors are involved in overlapping but distinct sets of embryonic signaling events.
The embryonic cerebellum contains topographic cues that guide developing inferior olivary axons
Authors: Chédotal Alain , Bloch-Gallego Evelyne , Sotelo Constantino
Published: April 2021

The formation of the olivocerebellar projection is supposed to be regulated by positional information shared between pre- and postsynaptic neurons. However, experimental evidence to support this hypothesis is missing. In the chick, caudal neurons in the inferior olive project to the anterior cerebellum and rostral ones to the posterior cerebellum. We here report in vitro experiments that strongly support the existence of anteroposterior polarity cues in the embryonic cerebellum.

We developed an in vitro system that was easily accessible to experimental manipulations. Large hindbrain explants of E7.5-E8 chick embryos, containing the cerebellum and its attached brainstem, were plated and studied using axonal tracing methods. In these cultures, we have shown that the normal anteroposterior topography of the olivocerebellar projection was acquired, even when the cerebellar lamella was detached from the brainstem and placed again in its original position. We also found that, following various experimental rotations of the anteroposterior axis of the cerebellum, the rostromedian olivary neurons still project to the posterior vermis and the caudolateral neurons to the anterior vermis, that now have inverted locations. Thus, the rotation of the target region results in the rotation of the projection. In addition, we have shown that the formation of the projection map could be due to the inability of rostromedian inferior olivary axons to grow in the anterior cerebellum. All these experiments strongly indicate that olivocerebellar fibers recognize within their target region polarity cues that organize their anteroposterior topography, and we suggest that Purkinje cells might carry these cues.
GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo
Authors: Ma Grace T. , Roth Matthew E. , Groskopf John C. , Tsai Fong-Ying , Orkin Stuart H. , Grosveld Frank , Engel James Douglas , Linzer Daniel I. H.
Published: April 2021

We previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that both of these factors regulate transcription of the placental lactogen I gene, as well as the related proliferin gene, in trophoblast giant cells in vivo. Placentas lacking GATA-3 accumulate placental lactogen I and proliferin mRNAs to a level 50% below that reached in the wild-type placenta. Mutation of the GATA-2 gene had a similar effect on placental lactogen I expression, but led to a markedly greater reduction (5- to 6-fold) in proliferin gene expression. Placentas lacking GATA-2 secrete significantly less angiogenic activity than wild-type placentas as measured in an endothelial cell migration assay, consistent with a reduction in expression of the angiogenic hormone proliferin. Furthermore, within the same uterus the decidual tissue adjacent to mutant placentas displays markedly reduced neovascularization compared to the decidual tissue next to wild-type placentas. These results indicate that GATA-2 and GATA-3 are important in vivo regulators of trophoblast-specific gene expression and placental function, and reveal a difference in the effect of these two factors in regulating the synthesis of related placental hormones.
Induction of deep layer cortical neurons in vitro
Authors: Bohner Amy P. , Akers Rebecca M. , McConnell Susan K.
Published: April 2021

Transplantation studies suggest that the laminar fates of cerebral cortical neurons are determined by environmental signals encountered just before mitosis. In ferret, E29 progenitor cells normally produce neurons of layers 5 and 6. When transplanted during S-phase into an older ventricular zone, E29 progenitors produce neurons that change their fates and migrate to layer 2/3; however, cells transplanted later in the cell cycle migrate to their normal deep-layer positions even in an older environment (McConnell and Kaznowski, 1991). Here we utilize three culture systems to investigate the nature of the environmental signals involved in laminar specification. E29 cells were first cultured at low density to ascertain whether cell contact and/or short-range cues are required for deep layer specification. Neurons transplanted after a short time in low-density culture failed to adopt their normal fates and migrated instead to the upper layers. When crude cell contacts were restored by pelleting E29 cells together, most transplanted neurons cells became specified to their normal deep layer fates. Finally, E29 cells were transplanted after being cultured in explants that maintained the architecture of the cerebral wall. Explants allowed normal deep layer specification to occur, as transplanted cells migrated to layers 5 and 6. These results suggest that short-range cues induce multipotent progenitors to produce deep layer neurons.
Establishing neuroblast-specific gene expression in the Drosophila CNS: huckebein is activated by Wingless and Hedgehog and repressed by Engrailed and Gooseberry
Authors: McDonald Jocelyn A. , Doe Chris Q.
Published: April 2021

The Drosophila ventral neuroectoderm produces a stereo-typed array of central nervous system precursors, called neuroblasts. Each neuroblast has a unique identity based on its position, pattern of gene expression and cell lineage. To understand how neuronal diversity is generated, we need to learn how neuroblast-specific gene expression is established, and how these genes control cell fate within neuroblast lineages. Here we address the first question: how is neuroblast-specific gene expression established? We focus on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. We show that Huckebein is a nuclear protein first detected in small clusters of neuroectodermal cells and then in a subset of neuroblasts. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Integration of these activation and repression inputs is required to establish the precise neuroectodermal pattern of huckebein, which is subsequently required for the development of specific neuroblast cell lineages.
Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain
Authors: Lee Scott M. K. , Danielian Paul S. , Fritzsch Bernd , McMahon Andrew P.
Published: April 2021

The developing vertebrate mesencephalon shows a rostrocaudal gradient in the expression of a number of molecular markers and in the cytoarchitectonic differentiation of the tectum, where cells cease proliferating and differentiate in a rostral to caudal progression. Tissue grafting experiments have implicated cell signalling by the mesencephalicmetencephalic (mid-hindbrain) junction (or isthmus) in orchestrating these events. We have explored the role of Wnt-1 and FGF8 signalling in the regulation of mesencephalic polarity. Wnt-1 is expressed in the caudal mesencephalon and Fgf8 in the most rostral metencephalon. Wnt-1 regulates Fgf8 expression in the adjacent metencephalon, most likely via a secondary mesencephalic signal. Ectopic expression of Fgf8 in the mesencephalon is sufficient to activate expression of Engrailed-2 (En-2) and ELF-1, two genes normally expressed in a decreasing caudal to rostral gradient in the posterior mesencephalon. Ectopic expression of Engrailed-1 (En-1), a functionally equivalent homologue of En-2 is sufficient to activate ELF-1 expression by itself. These results indicate the existence of a molecular hierarchy in which FGF8 signalling establishes the graded expression of En-2 within the tectum. This in turn may act to specify other aspects of A-P polarity such as graded ELF-1 expression. Our studies also reveal that FGF8 is a potent mitogen within the mesencephalon: when ectopically expressed, neural precursors continue to proliferate and neurogenesis is prevented. Taken together our results suggest that FGF8 signalling from the isthmus has a key role in coordinately regulating growth and polarity in the developing mesencephalon.
Postmitotic neurons migrate tangentially in the cortical ventricular zone
Authors: O’Rourke Nancy A. , Chenn Anjen , McConnell Susan K.
Published: April 2021

Patterns of cell movement play a key role in the establishment of the brain’s functional architecture during development. The migration of neuronal progenitor cells has been hypothesized to disperse clonally related cells among different areas of the developing cerebral cortex. To test this model, we explored the migratory patterns of cells in the proliferative zone of the intact cortex of the ferret. After focal injections of DiI, labeled cells migrated in all directions and over long distances within the ventricular and subventricular zones. These cells expressed the neuron-specific marker TuJ1 and did not incorporate BrdU after cumulative labeling. Our results reveal an extensive tangential dispersion of cortical cells mediated predominantly or exclusively by the non-radial migration of postmitotic neurons.
Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development
Authors: Macias D. , Gañan Y. , Sampath T. K. , Piedra M. E. , Ros M. A. , Hurle J. M.
Published: April 2021

Bone Morphogenetic Protein 2 (BMP-2) and Osteogenic Protein 1 (OP-1, also termed BMP-7) are members of the transforming growth factor β superfamily. In the present study, we have analyzed the effects of administering them locally at different stages and locations of the chick limb bud using heparin beads as carriers. Our results show that these BMPs are potent apoptotic signals for the undiffer-entiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. In addition, they promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog and ck-erg expression. Interestingly, the effects of these two BMPs on joint formation were found to be different. While the pre-dominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints revealed a distinct and complementary pattern of expression of these genes during the formation of the skeleton of the digits. While Op-1 exhibited an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, Bmp-2 expression was a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (i) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (ii) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones once the chondrogenic aggregates are established.
Developmental basis of homeosis in precociously germinating Brassica napus embryos: phase change at the shoot apex
Authors: Fernandez Donna E.
Published: April 2021

Precociously germinating Brassica napus (oilseed rape) embryos produce extra cotyledons or chimeric organs with sectors of cotyledon and leaf tissue, rather than leaves, at the shoot apex. To investigate this phenomenon in more detail, scanning electron microscopy was used to examine the development of organ primordia at the shoot apex. In situ hybridizations with molecular markers of the embryonic phase were used to assess the status of individ- ual cells in the shoot apex with regard to the transition between embryonic and vegetative phases. The results indicate that, under conditions that support precocious germination, primordia develop at the shoot apex in the mode characteristic of postgerminative growth, i.e. they arise sequentially in a spiral phyllotaxy. Cells in the rest of the embryo, however, can continue to express molecular markers of the embryonic phase for several weeks after the start of culture. When patterns of gene expression and the fate of individual primordia were compared, a strong correlation was found between organ identity and the status of cells in the vicinity of the meristem with regard to phase. Primordia that develop in situations where neighboring cells are in the embryonic phase always produce organs with cotyledon morphology. Primordia that develop in situations where neighboring cells have exited the embryonic phase produce leaves. Based on an examination of situations where chimeric organs are produced, I propose that short range interactions or signalling are likely to be involved in communicating information about phase to developing primordia.
VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth
Authors: Sze Ji Ying , Liu Yanxia , Ruvkun Gary
Published: April 2021

The POU homeobox gene unc-86 specifies many neuroblast and neural fates in the developing C. elegans nervous system. Genes regulated by unc-86 are mostly unknown. Here we describe a genetic strategy for the identification of downstream pathways regulated by unc-86. We activate UNC-86 transcription activity by inserting the VP16 activation domain into an unc-86 genomic clone that bears all regulatory sequences necessary for normal expression in C. elegans. unc-86/VP16 complements unc-86 mutations in the specification of neuroblast and neural cell fates, but displays novel genetic activities: it can suppress non-null mutations in the downstream genes mec-3 and mec-7 that are necessary for mechanosensory neuron differentiation and function. These data suggest that UNC-86/VP16 increases the expression of mec-3 and mec-7 to compensate Nfor the decreased activities of mutant MEC-3 or MEC-7 proteins. The suppression of mutations in downstream genes by an activated upstream transcription factor should be a general strategy for the identification of genes in transcriptional cascades. unc-86/VP16 also causes neural migration and pathfinding defects and novel behavioral defects. Thus, increased or unregulated expression of genes downstream of unc-86 can confer novel neural phenotypes suggestive of roles for unc-86-regulated genes in neural pathfinding and function. Genetic suppression of these unc-86/VP16 phenotypes may identify the unc-86 downstream genes that mediate these events in neurogenesis.
Preventing the loss of competence for neural induction: HGF/SF, L5 and Sox-2
Authors: Streit Andrea , Sockanathan Shanthini , Pérez Lidia , Rex Maria , Scotting Paul J. , Sharpe Paul T. , Lovell-Badge Robin , Stern Claudio D.
Published: April 2021

The response to neural induction depends on the presence of inducing signals and on the state of competence of the responding tissue. The epiblast of the chick embryo loses its ability to respond to neural induction by the organizer (Hensen’s node) between stages 4 and 4+. We find that the pattern of expression of the L5220 antigen closely mirrors the changes in competence of the epiblast in time and in space. For the first time, we describe an experiment that can extend the period of neural competence: when L5220 expression is maintained beyond its normal time by implanting HGF/SF secreting cells, the competence to respond to Hensen’s node grafts is retained. The host epiblast forms a non-regionalized neural tube, which expresses the pan-neural marker SOX-2 (a Sry-related transcription factor) but not any region-specific markers for the forebrain, hindbrain or spinal cord. Although HGF/SF secreting cells can mimic signals from Hensen’s node that maintain L5 expression, they cannot rescue the ability of the node to induce anterior structures (which is normally lost after stage 4). The ectoderm may acquire stable neural characteristics during neural induction by going through a hierarchy of states: competence, neuralization and regionalization. Our findings allow us to start to define these different states at a molecular level, and show that the competence to respond to neural induction is not entirely autonomous to the responding cells, but can be regulated by extracellular signalling molecules.
Bax promotes neuronal cell death and is downregulated during the development of the nervous system
Authors: Vekrellis Konstantinos , McCarthy Mary Jane , Watson Andrea , Whitfield Jonathan , Rubin Lee L. , Ham Jonathan
Published: April 2021

The Bcl-2 and Bcl-x proteins suppress programmed cell death, whereas Bax promotes apoptosis. We investigated the pattern of expression of Bcl-2, Bax and Bcl-x during neuronal differentiation and development. All three proteins were widely expressed in neonatal rats but, in the adult, Bax levels were 20-to 140-fold lower in the cerebral cortex, cerebellum and heart muscle, whereas Bcl-x was not downregulated in any of the tissues examined. In the cerebral cortex and cerebellum, the decrease in Bax levels occurred after the period of developmental cell death. Further, microinjection of a Bax expression vector into cultured sympathetic neurons, which depend on nerve growth factor for survival, induced apoptosis in the presence of survival factor and increased the rate of cell death after nerve growth factor withdrawal. This effect could be blocked by co-injection of an expression vector for Bcl-xL or for the baculovirus p35 protein, an inhibitor of caspases (ICE-like proteases). These results suggest that, during development, the sensitivity of neurons to signals that induce apoptosis may be regulated by modulating Bax levels and that Bax-induced death requires caspase activity.
Protease nexin-1 is expressed at the mouse met-/mesencephalic junction and FGF signaling regulates its promoter activity in primary met-/mesencephalic cells
Authors: Küry Patrick , Schaeren-Wiemers Nicole , Monard Denis
Published: April 2021

The expression pattern of the serine protease inhibitor gene Protease nexin-1 (PN-1) has been analyzed by in situ hybridization during embryonic nervous system develop-ment. PN-1 was found to be specifically expressed at the junction between the mes-and metencephalon (mid-and hindbrain). Transgenic embryos expressing the bacterial lacZ gene under the control of different fragments of the PN-1 upstream regulatory region were used to demarcate an enhancer sufficient for expression at this putative segmental border. Primary cell cultures derived from the embryonic neural tube at the level of the met-/mesen-cephalic junction were used to demonstrate a specific effect on transcriptional activity by basic fibroblast growth factor and Engrailed transcription factors, providing evidence that PN-1 is a target gene of these factors. The results of this study place PN-1 into the signaling cascade(s) consid-ered to be important for the development of this junction and suggest a role for PN-1 in the establishment or main-tenance of the differences between these adjacent neu-romeres.