bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin
Authors: Lorenzen James A. , Baker Scott E. , Denhez Fabienne , Melnick Michael B. , Brower Danny L. , Perkins Lizabeth A.
Published: April 2021

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, D-ERK. However, the molecular details of D-ERK translocation are largely unknown. In this regard, we have identified D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated D-ERK. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated D-ERK in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic D-ERK mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated D-ERK. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin β homolog Ketel, also reduce the nuclear localization of activated D-ERK. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated D-ERK.
The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos
Authors: Kos Robert , Reedy Mark V. , Johnson Randy L. , Erickson Carol A.
Published: April 2021

The winged-helix or forkhead class of transcription factors has been shown to play important roles in cell specification and lineage segregation. We have cloned the chicken homolog of FoxD3, a member of the winged-helix class of transcription factors, and analyzed its expression. Based on its expression in the dorsal neural tube and in all neural crest lineages except the late-emigrating melanoblasts, we predicted that FoxD3 might be important in the segregation of the neural crest lineage from the neural epithelium, and for repressing melanogenesis in early-migrating neural crest cells.

Misexpression of FoxD3 by electroporation in the lateral neural epithelium early in neural crest development produced an expansion of HNK1 immunoreactivity throughout the neural epithelium, although these cells did not undergo an epithelial/mesenchymal transformation. To test whether FoxD3 represses melanogenesis in early migrating neural crest cells, we knocked down expression in cultured neural crest with antisense oligonucleotides and in vivo by treatment with morpholino antisense oligonucleotides. Both experimental approaches resulted in an expansion of the melanoblast lineage, probably at the expense of neuronal and glial lineages. Conversely, persistent expression of FoxD3 in late-migrating neural crest cells using RCAS viruses resulted in the failure of melanoblasts to develop.

We suggest that FoxD3 plays two important roles in neural crest development. First, it is involved in the segregation of the neural crest lineage from the neuroepithelium. Second, it represses melanogenesis, thereby allowing other neural crest derivatives to differentiate during the early stages of neural crest patterning.
The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling
Authors: Catoira Romy , Timmers Antonius C. J. , Maillet Fabienne , Galera Christine , Penmetsa R. Varma , Cook ouglas , Dénarié Jean , Gough Clare
Published: April 2021

The symbiotic infection of the model legume Medicago truncatula by Sinorhizobium meliloti involves marked root hair curling, a stage where entrapment of the microsymbiont occurs in a chamber from which infection thread formation is initiated within the root hair. We have genetically dissected these early symbiotic interactions using both plant and rhizobial mutants and have identified a M. truncatula gene, HCL, which controls root hair curling. S. meliloti Nod factors, which are required for the infection process, induced wild-type epidermal nodulin gene expression and root hair deformation in hcl mutants, while Nod factor induction of cortical cell division foci was reduced compared to wild-type plants. Studies of the position of nuclei and of the microtubule cytoskeleton network of hcl mutants revealed that root hair, as well as cortical cells, were activated in response to S. meliloti. However, the asymmetric microtubule network that is typical of curled root hairs, did not form in the mutants, and activated cortical cells did not become polarised and did not exhibit the microtubular cytoplasmic bridges characteristic of the pre-infection threads induced by rhizobia in M. truncatula. These data suggest that hcl mutations alter the formation of signalling centres that normally provide positional information for the reorganisation of the microtubular cytoskeleton in epidermal and cortical cells.
A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects
Authors: Dor Yuval , Camenisch Todd D. , Itin Ahuva , Fishman Glenn I. , McDonald John A. , Carmeliet Peter , Keshet Eli
Published: April 2021

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.
The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc
Authors: Keisman Eric L. , Baker Bruce S.
Published: April 2021

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. We show that the gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). We find that the sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males.
The Arabidopsis ATK1 gene is required for spindle morphogenesis in male meiosis
Authors: Chen Changbin , Marcus Adam , Li Wuxing , Hu Yi , Calzada Jean-Philippe Vielle , Grossniklaus Ueli , Cyr Richard J. , Ma Hong
Published: April 2021

The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis, called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.
Interaction ofLEAFY,AGAMOUSandTERMINAL FLOWER1in maintaining floral meristem identity inArabidopsis
Authors: Parcy François , Bomblies Kirsten , Weigel Detlef
Published: April 2021

The Arabidopsis transcription factor LEAFY acts upstream of homeotic genes such as AGAMOUS to confer floral identity on meristems that arise after the transition to reproductive development. Compared to the genetic circuitry regulating the establishment of floral meristem identity, little is known about its maintenance. Previous experiments with leafy heterozygous plants and agamous mutants grown in conditions that reduce the floral inductive stimulus have shown that both genes are required to prevent reversion of floral to inflorescence meristems. Here, we present evidence that LEAFY maintains floral meristem identity independently of AGAMOUS, and that the primary role of LEAFY is either direct repression of shoot identity genes or repression of an intermediate factor that activates shoot identity genes. The latter conclusions were deduced from the phenotypes conferred by a gain-of-function transgene, LEAFY:VP16, that appears to act as a dominant negative, or antimorphic, allele during maintenance of floral meristem identity. These observations contrast with previous findings that LEAFY acts as a direct activator of floral homeotic genes, supporting the hypothesis that the transcriptional activity of LEAFY is dependent on specific co-regulators.
Delta-Notch signaling induces hypochord development in zebrafish
Authors: Latimer Andrew J. , Dong Xinhong , Markov Youlia , Appel Bruce
Published: April 2021

Different cell types that occupy the midline of vertebrate embryos originate within the Spemann-Mangold or gastrula organizer. One such cell type is hypochord, which lies ventral to notochord in anamniote embryos. We show that hypochord precursors arise from the lateral edges of the organizer in zebrafish. During gastrulation, hypochord precursors are closely associated with no tail-expressing midline precursors and paraxial mesoderm, which expresses deltaC and deltaD. Loss-of-function experiments revealed that deltaC and deltaD were required for her4 expression in presumptive hypochord precursors and for hypochord development. Conversely, ectopic, unregulated Notch activity blocked no tail expression and promoted her4 expression. We propose that Delta signaling from paraxial mesoderm diversifies midline cell fate by inducing a subset of neighboring midline precursors to develop as hypochord, rather than as notochord.
ThejingZn-finger transcription factor is a mediator of cellular differentiation in theDrosophilaCNS midline and trachea
Authors: Sedaghat Yalda , Miranda Wilson F. , Sonnenfeld Margaret J.
Published: April 2021

We establish that the jing zinc-finger transcription factor plays an essential role in controlling CNS midline and tracheal cell differentiation. jing transcripts and protein accumulate from stage 9 in the CNS midline, trachea and in segmental ectodermal stripes. JING protein localizes to the nuclei of CNS midline and tracheal cells implying a regulatory role during their development. Loss of jing-lacZ expression in homozygous sim mutants and induction of jing-lacZ by ectopic sim expression establish that jing is part of the CNS midline lineage. We have isolated embryonic recessive lethal jing mutations that display genetic interactions in the embryonic CNS midline and trachea, with mutations in the bHLH-PAS genes single-minded and trachealess, and their downstream target genes (slit and breathless). Loss- and gain-of-function jing is associated with defects in CNS axon and tracheal tubule patterning. In jing homozygous mutant embryos, reductions in marker gene expression and inappropriate apoptosis in the CNS midline and trachea establish that jing is essential for the proper differentiation and survival of these lineages. These results establish that jing is a key component of CNS midline and tracheal cell development. Given the similarities between JING and the vertebrate CCAAT-binding protein AEBP2, we propose that jing regulates transcriptional mechanisms in Drosophila embryos and promotes cellular differentiation in ectodermal derivatives.
Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population
Authors: Rathjen Joy , Haines Bryan P. , Hudson Kathryn M. , Nesci Antonietta , Dunn Stephanie , Rathjen Peter D.
Published: April 2021

During embryogenesis the central and peripheral nervous systems arise from a neural precursor population, neurectoderm, formed during gastrulation. We demonstrate the differentiation of mouse embryonic stem cells to neurectoderm in culture, in a manner which recapitulates embryogenesis, with the sequential and homogeneous formation of primitive ectoderm, neural plate and neural tube. Formation of neurectoderm occurs in the absence of extraembryonic endoderm or mesoderm and results in a stratified epithelium of cells with morphology, gene expression and differentiation potential consistent with positionally unspecified neural tube. Differentiation of this population to homogeneous populations of neural crest or glia was also achieved. Neurectoderm formation in culture allows elucidation of signals involved in neural specification and generation of implantable cell populations for therapeutic use.
Multiple functions of a Zic-like gene in the differentiation of notochord, central nervous system and muscle inCiona savignyiembryos
Authors: Imai Kaoru S. , Satou Yutaka , Satoh Nori
Published: April 2021

Multiple functions of a Zic-like zinc finger transcription factor gene (Cs-ZicL) were identified in Ciona savignyi embryos. cDNA clones for Cs-ZicL, a β-catenin downstream genes, were isolated and the gene was transiently expressed in the A-line notochord/nerve cord lineage and in B-line muscle lineage from the 32-cell stage and later in a-line CNS lineage from the 110-cell stage. Suppression of Cs-ZicL function with specific morpholino oligonucleotide indicated that Cs-ZicL is essential for the formation of A-line notochord cells but not of B-line notochord cells, essential for the CNS formation and essential for the maintenance of muscle differentiation. The expression of Cs-ZicL in the A-line cells is downstream of β-catenin and a β-catenin-target gene, Cs-FoxD, which is expressed in the endoderm cells from the 16-cell stage and is essential for the differentiation of notochord. In spite of its pivotal role in muscle specification, the expression of Cs-ZicL in the muscle precursors is independent of Cs-macho1, which is another Zic-like gene encoding a Ciona maternal muscle determinant, suggesting another genetic cascade for muscle specification independent of Cs-macho1. Cs-ZicL may provide a future experimental system to explore how the gene expression in multiple embryonic regions is controlled and how the single gene can perform different functions in multiple types of embryonic cells.
Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors
Authors: Zhu Changqi C. , Dyer Michael A. , Uchikawa Masanori , Kondoh Hisato , Lagutin Oleg V. , Oliver Guillermo
Published: April 2021

Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family, plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Here, we report that Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6, which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. We also show that the interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6. Using PCR, we identified a specific DNA motif that is bound by Six3 and we demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. We also demonstrated that this interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae led to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos resulted in failure of lens placode invagination and production of δ-crystallin-negative cells within the placode. These specific alterations were not seen when the mutated form of Six3 cDNA was used in similar experimental approaches, indicating that Six3 interaction with Groucho proteins plays an essential role in vertebrate eye development.
Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud
Authors: Grandel Heiner , Lun Klaus , Rauch Gerd-Jörg , Rhinn Muriel , Piotrowski Tatjana , Houart Corinne , Sordino Paolo , Küchler Axel M. , Schulte-Merker Stefan , Geisler Robert , Holder Nigel , Wilson Stephen W. , Brand Michael
Published: April 2021

A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.
Tiling of theDrosophilaepidermis by multidendritic sensory neurons
Authors: Grueber Wesley B. , Jan Lily Y. , Jan Yuh Nung
Published: April 2021

Insect dendritic arborization (da) neurons provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. We have examined the morphologies of Drosophila da neurons by using the MARCM (mosaic analysis with a repressible cell marker) system. We show that each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. We place these neurons into four distinct morphological classes distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. Our data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems. The dendritic domains of cells in different classes, by contrast, can overlap extensively. We have examined the cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, our data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites.
Vitamin D3 receptor ablation alters mammary gland morphogenesis
Authors: Zinser Glendon , Packman Kathryn , Welsh JoEllen
Published: April 2021

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D3 receptor (VDR), whose ligand 1,25-dihydroxyvitamin D3 is the biologically active form of vitamin D3, has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D3 endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca2+ which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D3 and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D3 signaling pathway participates in negative growth regulation of the mammary gland.
Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling
Authors: Polleux Franck , Whitford Kristin L. , Dijkhuizen Paul A. , Vitalis Tania , Ghosh Anirvan
Published: April 2021

During telencephalic development, cells from the medial ganglionic eminence (MGE) are thought to migrate to the neocortex to give rise to a majority of cortical GABAergic interneurons. By combining time-lapse video-microscopy, immunofluorescence and pharmacological perturbations in a new in vitro migration assay, we find that MGE-derived cells migrate through the entire extent of the cortex and into the CA fields of the hippocampus, but avoid the dentate gyrus. Migrating neurons initially travel within the marginal zone and intermediate zone, and can enter the cortical plate from either location. Tangential migration is strongly stimulated by BDNF and NT4 and attenuated by the Trk-family inhibitor, K252a, suggesting that migration is regulated by TrkB signaling. Furthermore, TrkB-null mice show a significant decrease in the number of calbindin-positive neurons migrating tangentially in the embryonic cortex. BDNF and NT4 cause rapid activation of PI3-kinase in MGE cells, and inhibition of PI3-kinase (but not of MAP kinase or PLCγ) dramatically attenuates tangential migration. These observations suggest that TrkB signaling, via PI3-kinase activation, plays an important role in controlling interneuron migration in the developing cerebral cortex.
TheWUSCHELandSHOOTMERISTEMLESSgenes fulfil complementary roles inArabidopsisshoot meristem regulation
Authors: Lenhard Michael , Jürgens Gerd , Laux Thomas
Published: April 2021

Continuous organ formation from the shoot apical meristem requires the integration of two functions: a set of undifferentiated, pluripotent stem cells is maintained at the very tip of the meristem, while their daughter cells in the periphery initiate organ primordia. The homeobox genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) encode two major regulators of meristem formation and maintenance in Arabidopsis, yet their interaction in meristem regulation is presently unclear. Here, we have addressed this question using loss- and gain-of-function approaches. We show that stem cell specification by WUS does not require STM activity. Conversely, STM suppresses differentiation independently of WUS and is required and sufficient to promote cell division. Consistent with their independent and distinct phenotypic effects, ectopic WUS and STM activities induce the expression of different downstream target genes. Finally, the pathways regulated by WUS and STM appear to converge in the suppression of differentiation, since coexpression of both genes produced a synergistic effect, and increased WUS activity could partly compensate for loss of STM function. These results suggest that WUS and STM share labour in the shoot apical meristem: WUS specifies a subset of cells in the centre as stem cells, while STM is required to suppress differentiation throughout the meristem dome, thus allowing stem cell daughters to be amplified before they are incorporated into organs.
parachute/n-cadherinis required for morphogenesis and maintained integrity of the zebrafish neural tube
Authors: Lele Zsolt , Folchert Anja , Concha Miguel , Rauch Gerd-Jörg , Geisler Robert , Rosa Frédéric , Wilson Steve W. , Hammerschmidt Matthias , Bally-Cuif Laure
Published: April 2021

N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects β-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.
Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos
Authors: Parsons Michael J. , Campos Isabel , Hirst Elizabeth M. A. , Stemple Derek L.
Published: April 2021

Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular α subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane β subunit. We have removed the function of dystroglycan in zebrafish embryos. In contrast to mouse, where dystroglycan mutations lead to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, we find that loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organisation. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organisation. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis.
Dpp signalling is a key effector of the wing-body wall subdivision of theDrosophilamesothorax
Authors: Cavodeassi Florencia , Rodríguez Isabel , Modolell Juan
Published: April 2021

During development, the imaginal wing disc of Drosophila is subdivided along the proximal-distal axis into different territories that will give rise to body wall (notum and mesothoracic pleura) and appendage (wing hinge and wing blade). Expression of the Iroquois complex (Iro-C) homeobox genes in the most proximal part of the disc defines the notum, since Iro-C– cells within this territory acquire the identity of the adjacent distal region, the wing hinge. Here we analyze how the expression of Iro-C is confined to the notum territory. Neither Wingless signalling, which is essential for wing development, nor Vein-dependent EGFR signalling, which is needed to activate Iro-C, appear to delimit Iro-C expression. We show that a main effector of this confinement is the TGFβ homolog Decapentaplegic (Dpp), a molecule known to pattern the disc along its anterior-posterior axis. At early second larval instar, the Dpp signalling pathway functions only in the wing and hinge territories, represses Iro-C and confines its expression to the notum territory. Later, Dpp becomes expressed in the most proximal part of the notum and turns off Iro-C in this region. This downregulation is associated with the subdivision of the notum into medial and lateral regions.
Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
Authors: Mann Fanny , Peuckert Christiane , Dehner Frank , Zhou Renping , Bolz Jürgen
Published: April 2021

The development of connections between thalamic afferents and their cortical target cells occurs in a highly precise manner. Thalamic axons enter the cortex through deep cortical layers, then stop their growth in layer 4 and elaborate terminal arbors specifically within this layer. The mechanisms that underlie target layer recognition for thalamocortical projections are not known. We compared the growth pattern of thalamic explants cultured on membrane substrates purified from cortical layer 4, the main recipient layer for thalamic axons, and cortical layer 5, a non-target layer. Thalamic axons exhibited a reduced growth rate and an increased branching density on their appropriate target membranes compared with non-target substrate. When confronted with alternating stripes of both membrane substrates, thalamic axons grew preferentially on their target membrane stripes. Enzymatic treatment of cortical membranes revealed that growth, branching and guidance of thalamic axons are independently regulated by attractive and repulsive cues differentially expressed in distinct cortical layers. These results indicate that multiple membrane-associated molecules collectively contribute to the laminar targeting of thalamic afferents. Furthermore, we found that interfering with the function of Eph tyrosine kinase receptors and their ligands, ephrins, abolished the preferential branching of thalamic axons on their target membranes, and that recombinant ephrin-A5 ligand elicited a branch-promoting activity on thalamic axons. We conclude that interactions between Eph receptors and ephrins mediate branch formation of thalamic axons and thereby may play a role in the establishment of layer-specific thalamocortical connections.
Development of chromaffin cells depends on MASH1 function
Authors: Huber Katrin , Brühl Barbara , Guillemot François , Olson Eric N. , Ernsberger Uwe , Unsicker Klaus
Published: April 2021

The sympathoadrenal (SA) cell lineage is a derivative of the neural crest (NC), which gives rise to sympathetic neurons and neuroendocrine chromaffin cells. Signals that are important for specification of these two types of cells are largely unknown. MASH1 plays an important role for neuronal as well as catecholaminergic differentiation. Mash1 knockout mice display severe deficits in sympathetic ganglia, yet their adrenal medulla has been reported to be largely normal suggesting that MASH1 is essential for neuronal but not for neuroendocrine differentiation. We show now that MASH1 function is necessary for the development of the vast majority of chromaffin cells. Most adrenal medullary cells in Mash1–/– mice identified by Phox2b immunoreactivity, lack the catecholaminergic marker tyrosine hydroxylase. Mash1 mutant and wild-type mice have almost identical numbers of Phox2b-positive cells in their adrenal glands at embryonic day (E) 13.5; however, only one-third of the Phox2b-positive adrenal cell population seen in Mash1+/+ mice is maintained in Mash1–/– mice at birth. Similar to Phox2b, cells expressing Phox2a and Hand2 (dHand) clearly outnumber TH-positive cells. Most cells in the adrenal medulla of Mash1–/– mice do not contain chromaffin granules, display a very immature, neuroblast-like phenotype, and, unlike wild-type adrenal chromaffin cells, show prolonged expression of neurofilament and Ret comparable with that observed in wild-type sympathetic ganglia. However, few chromaffin cells in Mash1–/– mice become PNMT positive and downregulate neurofilament and Ret expression. Together, these findings suggest that the development of chomaffin cells does depend on MASH1 function not only for catecholaminergic differentiation but also for general chromaffin cell differentiation.
Wnt7b regulates mesenchymal proliferation and vascular development in the lung
Authors: Shu Weiguo , Jiang Yue Qin , Lu Min Min , Morrisey Edward E.
Published: April 2021

Although the Wnt signaling pathway regulates inductive interactions between epithelial and mesenchymal cells, little is known of the role that this pathway plays during lung development. Wnt7b is expressed in the airway epithelium, suggesting a possible role for Wnt-mediated signaling in the regulation of lung development. To test this hypothesis, we have mutated Wnt7b in the germline of mice by replacement of the first exon with the lacZ-coding region. Wnt7blacZ–/– mice exhibit perinatal death due to respiratory failure. Defects in early mesenchymal proliferation leading to lung hypoplasia are observed in Wnt7blacZ–/– embryos. In addition, Wnt7blacZ–/– embryos and newborn mice exhibit severe defects in the smooth muscle component of the major pulmonary vessels. These defects lead to rupture of the major vessels and hemorrhage in the lungs after birth. These results demonstrate that Wnt7b signaling is required for proper lung mesenchymal growth and vascular development.
Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver
Authors: Kumaravelu Parasakthy , Hook Lilian , Morrison Aline M. , Ure Jan , Zhao Suling , Zuyev Sergei , Ansell John , Medvinsky Alexander
Published: April 2021

In the developing mouse embryo the first definitive(transplantable-into-the-adult) haematopoietic stem cells/long-term repopulating units (HSC/RUs) emerge in the AGM region and umbilical vessels on 10-11 days post coitum (d.p.c.). Here, by limiting dilution analysis, we anatomically map the development of definitive HSC/RUs in different embryonic tissues during early colonisation of the liver. We show that by day 12 p.c. the mouse embryo contains about 66 definitive HSC/RUs (53 in the liver, 13 in other tissues), whereas on the previous day the total number of definitive HSC/RUs in the entire conceptus is only about 3. Owing to the length of the cell cycle this dramatic increase in the number of definitive HSC/RUs in only 24 hours is unlikely to be explained purely by cell division. Therefore,extensive maturation of pre-definitive HSCs to a state when they become definitive must take place in the day 11-12 embryo. Here we firstly identify the numbers of HSCs in various organs at 11-13 d.p.c. and secondly, using an organ culture approach, we quantitatively assess the potential of the aorta-gonadmesonephros (AGM) region and the yolk sac to produce/expand definitive HSC/RUs during days 11-12 of embryogenesis. We show that the capacity of the AGM region to generate definitive HSC/RUs is high on 11 d.p.c. but significantly reduced by 12 d.p.c. Conversely, at 12 d.p.c. the YS acquires the capacity to expand and/or generate definitive HSCs/RUs, whereas it is unable to do so on 11 d.p.c. Thus, the final steps in development of definitive HSC/RUs may occur not only within the AGM region, as was previously thought, but also in the yolk sac microenvironment. Our estimates indicate that the cumulative activity of the AGM region and the yolk sac is sufficient to provide the day 12 liver with a large number of definitive HSC/RUs,suggesting that the large pool of definitive HSC/RUs in day 12 foetal liver is formed predominantly by recruiting `ready-to-use' definitive HSC/RUs from extra-hepatic sources. In accordance with this we observe growing numbers of definitive HSC/RUs in the circulation during days 11-13 of gestation,suggesting a route via which these HSCs migrate.
Transcription activates repressed domains in theDrosophilabithorax complex
Authors: Bender Welcome , Fitzgerald Daniel P.
Published: April 2021

A series of mutations have been recovered in the bithorax complex of D. melanogaster that transform the first segment of the abdomen into a copy of the second or third abdominal segment. These dominantUltraabdominal alleles are all associated with P element insertions which are transcribed in the first abdominal segment. The transcripts proceed past the end of the P element for up to 50 kb, extending through the regulatory regions for the second and third abdominal segments. Blocking transcription from the P element promoter reverts the mutant phenotype. Previously identified Ultraabdominal alleles, not associated with P elements, also show abnormal transcription of the same region.
The expression of TGFβ signal transducers in the hypodermis regulates body size inC. elegans
Authors: Wang Jianjun , Tokarz Rafal , Savage-Dunn Cathy
Published: April 2021

In C. elegans, a TGFβ-related signaling pathway regulates body size. Loss of function of the signaling ligand (dbl-1),receptors (daf-4 and sma-6) or Smads (sma-2, sma-3and sma-4) results in viable, but smaller animals because of a reduction in postembryonic growth. We have investigated the tissue specificity of this pathway in body size regulation. We show that different tissues are reduced in size by different proportions, with hypodermal blast cell size most closely proportional to body size. We show that SMA-3 Smad is expressed in pharynx, intestine and hypodermis, as has been previously reported for the type I receptor SMA-6. Furthermore, we find that SMA-3::GFP is nuclear localized in all of these tissues, and that nuclear localization is enhanced by SMA-6 activity. Interestingly, SMA-3 protein accumulation was found to be negatively regulated by the level of Sma/Mab pathway activity. Using genetic mosaic analysis and directed expression of SMA-3, we find that SMA-3 activity in the hypodermis is necessary and sufficient for normal body size. Asdbl-1 is expressed primarily in the nervous system, these results suggest a model in which postembryonic growth of hypodermal cells is regulated by TGFβ-related signaling from the nervous system to the hypodermis.
hrTis required for cardiovascular development in zebrafish
Authors: Szeto Daniel P. , Griffin Kevin J. P. , Kimelman David
Published: April 2021

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells fromDrosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal inhrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role ofhrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markersgata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.
eFGF is required for activation ofXmyoDexpression in the myogenic cell lineage ofXenopus laevis
Authors: Fisher Malcolm E. , Isaacs Harry V. , Pownall Mary E.
Published: April 2021

This paper addresses the molecular mechanisms that regulate the transcriptional activation of the myogenic regulatory factor XmyoD in the skeletal muscle lineage of Xenopus laevis. Using antisense morpholino oligonucleotide-mediated inhibition, we show that the signalling molecule embryonic fibroblast growth factor (eFGF), which is the amphibian homologue of FGF4, is necessary for the initial activation of XmyoD transcription in myogenic cells. We demonstrate that eFGF can activate the expression of XmyoD in the absence of protein synthesis, indicating that this regulation is direct. Our data suggest that regulation of XmyoD expression may involve a labile transcriptional repressor. In addition, we show that eFGF is itself an immediate early response to activin, a molecule that mimics the endogenous mesoderm-inducing signal. We propose a model for the regulation of XmyoD within the early mesoderm, and discuss the relevance that these findings have for the understanding of myogenic specification in higher vertebrates.
Lola regulates midline crossing of CNS axons inDrosophila
Authors: Crowner Daniel , Madden Knut , Goeke Scott , Giniger Edward
Published: April 2021

The pattern and level of expression of axon guidance proteins must be choreographed with exquisite precision for the nervous system to develop its proper connectivity. Previous work has shown that the transcription factor Lola is required for central nervous system (CNS) axons of Drosophila to extend longitudinally. We show here that Lola is simultaneously required to repel these same longitudinal axons away from the midline, and that it acts, in part, by augmenting the expression both of the midline repellant, Slit, and of its axonal receptor, Robo. Lola is thus the examplar of a class of axon guidance molecules that control axon patterning by coordinating the regulation of multiple, independent guidance genes, ensuring that they are co-expressed at the correct time, place and relative level.