bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of a second AP-2 transcription factor: AP-2β
Authors: Moser Markus , Imhof Axel , Pscherer Armin , Bauer Reinhard , Amselgruber Werner , Sinowatz Fred , Hofstädter Ferdinand , Schüle Roland , Buettner Reinhard
Published: April 2021

AP-2 has been characterized previously as a unique 52×103Mr transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2β. AP-2β binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2α transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2α and AP-2β signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2α expression was found mainly in the skin, eye and prostate and AP-2β expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.
A biochemical model for the initiation and maintenance of the quiescent center: implications for organization of root meristems
Authors: Kerk Nancy M. , Feldman Lewis J.
Published: April 2021

A new hypothesis for the formation of the quiescent center is presented. Reported data support a mechanism for the establishment and maintenance of the quiescent center. The quiescent center is located at the most distal part of the root proper, the most terminal location in the root proper on the path of polar transport from the shoot. Of the many substances polarly transported in the root, auxin is one of the best studied and has been shown to affect root meristem organization. In our mechanism, polar auxin is directly linked to quiescence through the action of ascorbate oxidase and ascorbic acid. Immunolocalization of auxin in the root tip of Zea mays showed that auxin levels in the quiescent center were high compared to the levels in the immediately surrounding meristematic cells. Isolated quiescent centers were shown to have high levels of ascorbate oxidase mRNA and ascorbate oxidase activity relative to proximal meristem tissue. Exogenous auxin caused an increase in ascorbate oxidase mRNA levels and ascorbate oxidase enzyme activity in cultured root tissue. Immunolocalization of ascorbate oxidase in Zea root tips showed high levels of the protein in the quiescent center relative to surrounding cells. This is the first report of a positive marker and activity for the quiescent center. Histochemical detection of ascorbic acid in Zea root tips showed that quiescent center cells have low or undetectable levels of ascorbic acid, presumably due to the high levels of ascorbate oxidase in the quiescent center. As ascorbic acid is a compound known to be necessary for the transition from G1 to S in the cell cycle, its low levels in the quiescent center may be directly responsible for holding these rarely dividing cells in the extended G1 state in which they are mainly found. We propose that our mechanism complements published mathematical modeling of the anatomical structure of root apices, and further propose that the control of relative growth rates in this focal region of the root apex by this mechanism is a determining aspect in generating anatomical patterning in the root apex.
Function of the Polycomb protein is conserved in mice and flies
Authors: Müller Jürg , Gaunt Stephen , Lawrence Peter A.
Published: April 2021

A key aspect of determination — the acquisition and propagation of cell fates — is the initiation of patterns of selector gene expression and their maintenance in groups of cells as they divide and develop. In Drosophila, in those groups of cells where particular selector genes must remain inactive, it is the Polycomb-Group of genes that keep them silenced. Here we show that M33, a mouse homologue of the Drosophila Polycomb protein, can substitute for Polycomb in transgenic flies. Polycomb protein is thought to join with other Polycomb-Group proteins to build a complex that silences selector genes. Since members of this group of proteins have their homologues in mice, our results suggest that the molecular mechanism of cell determination is widely conserved.
Functional analysis of the Antirrhinum floral homeotic DEFICIENS gene in vivo and in vitro by using a temperature-sensitive mutant
Authors: Zachgo Sabine , Andrade Silva Eugenia de , Motte Patrick , Tröbner Wolfgang , Saedler Heinz , Schwarz-Sommer Zsuzsanna
Published: April 2021

Flowers of the temperature-sensitive DEFICIENS (DEF) mutant, def-101, display sepaloid petals and carpelloid stamens when grown at 26°C, the non-permissive temperature. In contrast, when cultivated under permissive conditions at 15°C, the morphology of def-101 flowers resembles that of the wild type. Temperature shift experiments during early and late phases of flower development revealed that second and third whorl organ development is differentially sensitive to changes in DEF expression. In addition, early DEF expression seems to control the spatially correct initiation of fourth whorl organ development. Reduction of the def-101 gene dosage differentially affects organogenesis in adjacent whorls: at the lower temperature development of petals in the second whorl and initiation of carpels in the centre of the flower is not affected while third whorl organogenesis follows the mutant (carpelloid) pattern. The possible contribution of accessory factors to organ-specific DEF functions is discussed. In situ analyses of mRNA and protein expression patterns during def-101 flower development at 15°C and at 26°C support previously proposed combinatorial regulatory interactions between the MADS-box proteins DEF and GLOBOSA (GLO), and provide evidence that the autoregulatory control of DEF and GLO expression by the DEF/GLO heterodimer starts after initiation of all organ primordia. Immunolocalisation revealed that both proteins are located in the nucleus. Interestingly, higher growth temperature affects the stability of both the DEF-101 and GLO proteins in vivo. In vitro DNA binding studies suggest that the temperature sensitivity of the def-101 mutant is due to an altered heterodimerisation/DNA-binding capability of the DEF-101 protein, conditioned by the deletion of one amino acid within the K-box, a protein region thought to be involved in protein-protein interaction. In addition, we introduce a mutant allele of GLO, glo-confusa, where insertion of one amino acid impairs the hydrophobic carboxy-terminal region of the MADS-box, but which confers no strong phenotypic changes to the flower. The strong mutant phenotype of flowers of def-101/glo-conf double mutants when grown in the cold represents genetic evidence for heterodimerisation between DEF and GLO in vivo. The potential to dissect structural and functional domains of MADS-box transcription factors is discussed.
Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo
Authors: Worrad Diane M. , Turner Bryan M. , Schultz Richard M.
Published: April 2021

Using immunofluorescent labeling and laser-scanning confocal microscopy, we show that isoforms of histone H4 acetylated on lysine 5, 8 and/or 12 (H4.Ac5-12), as well as RNA polymerase II, become enriched at the nuclear periphery around the time of zygotic gene activation, i.e., the 2-cell stage, in the preimplantation mouse embryo. In contrast, DNA and H4 acetylated on lysine 16 are uniformly distributed throughout the cytoplasm. Culture of embryos with inhibitors of histone deacetylase tricho-statin A and trapoxin results in an increase in the (1) amount of acetylated histone H4 detected by immunoblotting, (2) intensity and sharpness of the peripheral staining for H4.Ac5-12, and (3) relative rate of synthesis of proteins that are markers for zygotic gene activation. The enhanced staining for H4.Ac5-12 at the nuclear periphery seems to require DNA replication, but appears independent of cytokinesis or transcription, since its development is inhibited by aphidicolin but not by either cytochalasin D or α-amanitin. Lastly, the restricted localization of H4.Ac 5-12 is not observed in the 4-cell embryo or at later stages of preimplantation development. These results suggest that changes in chromatin structure underlie, at least in part, zygotic gene activation in the mouse.
goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development
Authors: Rivera-Pérez Jaime A. , Mallo Moisés , Gendron-Maguire, Maureen , Gridley Thomas , Behringer Richard R.
Published: April 2021

goosecoid (gsc) is an evolutionarily conserved homeobox gene expressed in the gastrula organizer region of a variety of vertebrate embryos, including zebrafish, Xenopus, chicken and mouse. To understand the role of gsc during mouse embryogenesis, we generated gsc-null mice by gene targeting in embryonic stem cells. Surprisingly, gsc-null embryos gastrulated and formed the primary body axes; gsc-null mice were born alive but died soon after birth with numerous craniofacial defects. In addition, rib fusions and sternum abnormalities were detected that varied depending upon the genetic background. Transplantation experiments suggest that the ovary does not provide gsc function to rescue gastrulation defects. These results demonstrate that gsc is not essential for organizer activity in the mouse but is required later during embryogenesis for craniofacial and rib cage development.
frizzled regulates mirror-symmetric pattern formation in the Drosophila eye
Authors: Zheng Limin , Zhang Jianjun , Carthew Richard W.
Published: April 2021

Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. We have investigated the mechanisms by which this pattern formation occurs and our results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. We propose that frizzled mediates a bidirectional signal emanating from the equator.
pRb is necessary for inhibition of N-myc expression by TGF-β1 in embryonic lung organ cultures
Authors: Serra Rosa , Moses Harold L.
Published: April 2021

Theβ type transforming growth factors (TGF-β) are potent inhibitors of epithelial cell proliferation, and data suggest that growth inhibition by TGF-β1 is mediated through suppression of Myc family genes in certain cell types. Indirect evidence has indicated that the product of the retinoblastoma gene (pRb) may also be involved in this pathway. Previously, we have shown that TGF-β1 inhibits branching morphogenesis and N-myc expression in mouse embryonic lung cultures. The purpose of this study was to determine the role of pRb in the inhibition of branching morphogenesis and N-myc expression by TGF-β1. Treatment with TGF-β1 was shown to inhibit development of lungs from homozygous Rb null (Rb−/−) and heterozygous null (Rb+/−) mouse embryos to the same extent as lungs from wild-type (Rb+/+) embryos. However, TGF-β1 treatment did not suppress N-myc expression in Rb−/− as it did in Rb+/+ embryonic lung explants as determined by in situ hybridization and quantitative RT-PCR. The effect of TGF-β1 treatment on N-myc expression in lungs from Rb+/− embryos was intermediate between that seen in Rb+/+ and Rb−/− embryos. Embryonic lungs derived from transgenic mice expressing the SV40 large T-antigen in lung epithelium under the control of the surfactant protein C promoter also showed inhibition of development in response to TGF-β1 treatment. The data demonstrate that pRb is necessary for TGF-β1 suppression of N-myc expression but not for TGF-β1 inhibition of branching morphogenesis; therefore, suppression of N-myc is not necessary for inhibition of branching morphogenesis by TGF-β1.
Programmed cell death in the absence of c-Fos and c-Jun
Authors: Roffler-Tarlov Suzanne , Brown Jeremy J. Gibson , Tarlov E. , Stolarov Javor , Chapman Deborah L. , Alexiou Maria , Papaioannou Virginia E.
Published: April 2021

Programmed cell death, or apoptosis, is a normal process in the development of a variety of embryonic and adult tissues, and is also observed in several pathological conditions. Several recent studies, using both expression and functional assays, have implicated the transcription factor, AP-1, in the regulation of programmed cell death, and specifically implicate the genes c-fos and c-jun, as well as some other family members. If the products of the c-fos and/or c-jun genes are essential components in the cascade of events that leads to programmed cell death in mammalian cells, it follows that cell death would not occur in mice lacking functional copies of these genes. We have made use of null mutations in the c-fos and c-jun genes that were produced by gene targeting (Johnson, R. S., Spiegelman, B. M. and Papaioannou, V. E. (1992). Cell 71, 577-586; Johnson, R. S., Van Lingen, B., Papaioannou, V. E. and Spiegelman, B. M. (1993). Genes Dev. 7, 1309-1317) to investigate this possibility. Cell death was assayed using an in situ apoptosis assay in c-fos null embryos and adults, c-jun null embryos, and c-fos/c-jun double null embryos compared with control mice. The occurrence of cell death in c-fos null mice was also assessed in two experimental conditions that normally lead to neuronal cell death. The first was unilateral section of the sciatic nerve in neonates, which leads to the death of anterior horn cells of the spinal cord on the operated side. The second was a genetic cross combining the weaver mutation, which causes death of cerebellar granule cells, with the c-fos mutation. Our results show that programmed cell death occurs normally in developing embryonic tissues and adult thymus and ovary, regardless of the absence of a functional c-fos gene. Furthermore, absence of c-fos had no effect on neuronal cell death in the spinal cord following sciatic nerve section, or in heterozygous weavers’ cerebellae. Finally, the results show that programmed cell death can take place in embryos lacking both Fos and Jun.
The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function
Authors: Krizek Beth Allyn , Meyerowitz Elliot M.
Published: April 2021

The class B organ identity genes, APETALA3 and PISTILLATA, are required to specify petal and stamen identity in the Arabidopsis flower. We show here that the activities of these two genes are sufficient to specify petals and stamens in flowers, in combination with the class A and C genes, respectively. Flowers of plants constitutively expressing both PISTILLATA and APETALA3 under the control of the 35S promoter from cauliflower mosaic virus consist of two outer whorls of petals and inner whorls of stamens. These plants also exhibit vegetative phenotypes that are not present in either of the singly (APETALA3 or PISTILLATA) overexpressing lines. These phenotypes include leaf curling and the partial conversion of later-arising cauline leaves to petals. The presence of additional floral whorls in flowers ectopically expressing APETALA3 and PISTILLATA and the rescue of missing organs in class A mutants by ectopic B function suggest that APETALA3 and PISTILLATA play an additional role in proliferation of the floral meristem.
argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop
Authors: Golembo Myriam , Schweitzer Ronen , Freeman Matthew , Shilo Ben-Zion
Published: April 2021

Argos is a secreted molecule with an atypical EGF motif. It was recently shown to function as an inhibitor of the signaling triggered by the Drosophila EGF receptor (DER). In this work, we determine the contribution of Argos to the establishment of cell fates in the embryonic ventral ectoderm. Graded activation of DER is essential for patterning the ventral ectoderm. argos mutant embryos show expansion of ventral cell fates suggesting hyperactivation of the DER pathway. In the embryonic ventral ectoderm, argos is expressed in the ventralmost row of cells. We show that argos expression in the ventral ectoderm is induced by the DER pathway: argos is not expressed in DER mutant embryos, while it is ectopically expressed in the entire ventral ectoderm following ubiquitous activation of the DER pathway. argos expression appears to be triggered directly by the DER pathway, since induction can also be observed in cell culture, following activation of DER by its ligand, Spitz. Argos therefore functions in a sequential manner, to restrict the duration and level of DER signaling. This type of inhibitory feedback loop may represent a general paradigm for signaling pathways inducing diverse cell fates within a population of non-committed cells.
A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula
Authors: Ruffins Seth W. , Ettensohn Charles A.
Published: April 2021

Previous lineage tracing experiments have shown that the vegetal blastomeres of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomeres were labeled no later than the sixth cleavage division (60-64 cell stage). In an earlier study we showed that single cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that cells labeled in the central region of the vegetal plate of the mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary mesenchyme cell (SMC) type (Ruffins and Ettensohn (1993)Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal cell types and information concerning the movements of these cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal cell fates, single blastomeres of four cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomeres do not contribute equally to SMC derivatives in L. variegatus.
Loss of fibroblast growth factor receptors is necessary for terminal differentiation of embryonic limb muscle
Authors: Itoh Naoki , Mima Tatsuo , Mikawa Takashi
Published: April 2021

Early in embryogenesis, precursors of the limb muscula- ture are generated in the somite, migrate to the limb buds and undergo terminal differentiation. Although myogenic differentiation in culture is affected by several growth factors including fibroblast growth factor (FGF), it remains uncertain whether migration and differentiation of myogenic cells in vivo are directly regulated by such growth factors. To investigate the roles of FGF signaling in the regulation of myogenesis both in the somite and the limb bud, mosaic chicken embryos were generated that consist of somitic cells carrying transgenes expressing one of the following: FGF1, FGF4, the FGF receptor type-1 (FGFR1) or its dominant negative mutant (ΔFGFR1). Cells infected with virus producing FGF ligand migrated into the somatopleure without differentiating into myotomal muscle, but differentiated into muscle fibers when they arrived in the limb bud. In contrast, cells overexpressing FGFR1 migrated into the limb muscle mass but remained as undifferentiated myoblasts. Cells infected with the ΔFGFR1-producing virus failed to migrate to the somato- pleure but were capable of differentiating into myotomal muscle within the somites. These results suggest that the FGFR-mediated FGF signaling (1) blocks terminal differ- entiation of myogenic cells within the somite and (2) sustains myoblast migration to limb buds from the somite, and that (3) down-regulation of FGFRs or FGFR signaling is involved in mechanisms triggering terminal differen- tiation of the limb muscle mass during avian embryogen- esis.
Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts
Authors: Durbec Pascale L. , Larsson-Blomberg Lena B. , Schuchardt Anita , Costantini Frank , Pachnis Vassilis
Published: April 2021

c-ret encodes a tyrosine kinase receptor that is necessary for normal development of the mammalian enteric nervous system. Germline mutations in c-ret lead to congenital megacolon in humans, while a loss-of-function allele (ret.k −) causes intestinal aganglionosis in mice. Here we examine in detail the function of c-ret during neurogenesis, as well as the lineage relationships among cell populations in the enteric nervous system and the sympathetic nervous system that are dependent on c-ret function. We report that, while the intestine of newborn ret.k − mice is devoid of enteric ganglia, the esophagus and stomach are only partially affected; furthermore, the superior cervical ganglion is absent, while more posterior sympathetic ganglia and the adrenal medulla are unaffected. Analysis of mutant embryos shows that the superior cervical ganglion anlage is present at E10.5, but absent by E12.5, suggesting that c-ret is required for the survival or prolif-eration of sympathetic neuroblasts. In situ hybridization studies, as well as direct labelling of cells with DiI, indicate that a common pool of neural crest cells derived from the postotic hindbrain normally gives rise to most of the enteric nervous system and the superior cervical ganglion, and is uniquely dependent on c-ret function for normal develop-ment. We term this the sympathoenteric lineage. In contrast, a distinct sympathoadrenal lineage derived from trunk neural crest forms the more posterior sympathetic ganglia, and also contributes to the foregut enteric nervous system. Overall, our studies reveal previously unknown complexities of cell lineage and genetic control mechanisms in the developing mammalian peripheral nervous system.
Contact inhibition/collapse and pathfinding of neural crest cells in the zebrafish trunk
Authors: Jesuthasan Suresh
Published: April 2021

Neural crest cells in the trunk of vertebrate embryos have a choice of pathways after emigrating from the neural tube: they can migrate in either the medial pathway between somites and neural tube, or the lateral pathway between somites and epidermis. In zebrafish embryos, the first cells to migrate all choose the medial pathway. High resolution imaging of cells in living embryos suggests that neural crest cells do so because of repulsion by somites: cells take the medial pathway because the lateral somite surface triggers a paralysis and retraction of protrusions (contact inhibi-tion or collapse) when the medial surface does not. Partial deletion of somites, using the spadetail mutation allows pre-cocious entry into the lateral pathway, but only where somites are absent, supporting the notion that an inhibitory cue on somites delays entry. Growth cones of Rohon-Beard cells enter the lateral pathway before neural crest cells, demonstrating that there is no absolute barrier to migration. These data, in addition to providing a detailed picture of neural crest cells migrating in vivo, suggest that neural crest cells, like neuronal growth cones, are guided by a specific cue that triggers ‘collapse’ of active protru-sions.
A sperm-supplied factor required for embryogenesis in C. elegans
Authors: Browning Heidi , Strome Susan
Published: April 2021

The paternal-effect embryonic-lethal gene, spe-11, is required for normal development of early C. elegans embryos. Spe-11 embryos fail to complete meiosis, form a weak eggshell, fail to orient properly the first mitotic spindle, and fail to undergo cytokinesis. Here we report cloning and sequencing of the spe-11 gene, which encodes a novel protein. As predicted by the paternal-effect mutant phenotype, the gene is expressed during spermatogenesis but is not detectable in females undergoing oogenesis, and the protein is present in mature sperm. To investigate whether SPE-11’s essential function is during spermatogenesis or whether sperm-delivered SPE-11 functions in the newly fertilized embryo, we engineered animals to supply SPE-11 to the embryo through the oocyte rather than through the sperm. We found that maternal expression is sufficient for embryonic viability. This result demonstrates that SPE-11 is not required during spermatogenesis, and suggests that SPE-11 is a sperm-supplied factor that participates directly in development of the early embryo. In contrast to the many known maternal factors required for embryogenesis, SPE-11 is the first paternally contributed factor to be genetically identified and molecularly characterized.
A distinct cyclin A is expressed in germ cells in the mouse
Authors: Sweeney Claire , Murphy Martin , Kubelka Michal , Ravnik Stuart E. , Hawkins Chris F. , Wolgemuth Debra J. , Carrington Mark
Published: April 2021

In this paper, the existence of two A-type cyclins in the mouse is demonstrated. In the adult mouse, the expression of cyclin A1, which has greatest sequence identity with Xenopus cyclin A1, is restricted to germ cells. In contrast cyclin A2, which has greatest sequence identity with human cyclin A and Xenopus cyclin A2, is expressed in all tissues analysed. In order to explore the function of cyclin A1 in germ cells, its expression during the meiotic cell cycle and its associated kinase subunits have been characterised in the testis. The levels of cyclin A1 mRNA rise dramatically in late pachytene spermatocytes and become undetectable soon after completion of the meiotic divisions; thus its expression is cell cycle regulated. In lysates of germ cells from adult testes, cyclin A1 is present in p13suc1 precipitates, and cyclin A1 immunoprecipitates possess histone H1 kinase activity. Three kinase partners of cyclin A1 were identified: p34cdc2, a polypeptide of 39×103Mr that is related to p33cdk2and, in lesser quantities, p33cdk2. Cyclin A1 was also detected in oocytes; in metaphase I and metaphase II oocytes, a proportion of the cyclin A1 colocalises with the spindle, possibly suggestive of a functional interaction. These data indicate that mammalian germ cells contain cyclin A1-dependent kinases that either act as a substitute for, or in addition to, the cyclin A2-dependent kinases characterised in somatic tissues.
Hoxa-13 and Hoxd-13 play a crucial role in the patterning of the limb autopod
Authors: Fromental-Ramaint Catherine , Warott Xavier , Messadecq Nadia , LeMeur Marianne , Dolle Pascal , Chambon Pierre
Published: April 2021

Members of the Abdominal-B-related Hox gene subfamily (belonging to homology groups 9 to 13) are coordinately expressed during limb bud development. Only two genes from homology group 13 (Hoxa-13 and Hoxd-13) are specifically expressed in the developing distal region (the autopod), which displays the most complex and evolutionarily flexible pattern among limb ‘segments’. We report here that targeted disruption of the Hoxa-13 gene leads to a specific forelimb and hindlimb autopodal phenotype, distinct from that of the Hoxd-13 paralogous gene inactivation. In both limbs, Hoxa-13 loss of function results in the lack of formation of the most anterior digit and to altered morphogenesis of some ‘preaxial’ carpal/tarsal elements. We have generated mice with all possible combinations of disrupted Hoxa-13 and/or Hoxd-13 alleles, which allowed us to investigate the degree of functional specificity versus redundancy of the corresponding gene products in the developing limb autopod. The phenotype of any double mutant was much more severe than the sum of the phenotypes seen in the corresponding single mutants, indicating that these genes act in a partially redundant manner. Our major findings were: (1) an abnormal autopodal phenotype in Hoxa-13+/−/Hoxd-13+/− double heterozygous mutants, which mostly consists of subsets of the alterations seen in each individual homozygous mutant, and therefore appears to result from quantitative, rather than qualitative, homeoprotein deficiency; (2) partly distinct alterations in mutants harboring a single non-disrupted allele of Hoxa-13 or Hoxd-13, indicating that the remaining reduced protein amounts are not functionally equivalent; (3) a polydactyly in the forelimbs of Hoxa-13+/−/Hoxd-13−/− double mutants, consisting of seven symmetrically arranged, truncated and mostly non-segmented digits; (4) an almost complete lack of chondrified condensations in the autopods of double homozygous mutants, showing that the activity of group 13 Hox gene products is essential for autopodal patterning in tetrapod limbs.
par-6, a gene involved in the establishment of asymmetry in early C. elegans embryos, mediates the asymmetric localization of PAR-3
Authors: Watts Jennifer L. , Etemad-Moghadam Bijan , Guo Su , Boyd Lynn , Draper Bruce W. , Mello Craig C. , Priess James R. , Kemphues Kenneth J.
Published: April 2021

The generation of asymmetry in the one-cell embryo of Caenorhabditis elegans is necessary to establish the anterior-posterior axis and to ensure the proper identity of early blastomeres. Maternal-effect lethal mutations with a partitioning defective phenotype (par) have identified several genes involved in this process. We have identified a new gene, par-6, which acts in conjunction with other par genes to properly localize cytoplasmic components in the early embryo. The early phenotypes of par-6 embryos include the generation of equal-sized blastomeres, improper localization of P granules and SKN-1 protein, and abnormal second division cleavage patterns. Overall, this phenotype is very similar to that caused by mutations in a previously described gene, par-3. The probable basis for this similarity is revealed by our genetic and immunolocalization results; par-6 acts through par-3 by localizing or maintaining the PAR-3 protein at the cell periphery. In addition, we find that loss-of-function par-6 mutations act as dominant bypass suppressors of loss-of-function mutations in par-2.
Inhibition of motor axon growth by T-cadherin substrata
Authors: Fredette Barbara J. , Ranscht Jason Miller and Barbara
Published: April 2021

As spinal motor neurons project to their hindlimb targets, their growth cones avoid particular regions along their pathway. T-cadherin is discretely distributed in the avoided caudal sclerotome and on extrasynaptic muscle surfaces (B. J. Fredette and B. Ranscht (1994) J. Neurosci. 14, 7331-7346), and therefore, the ability of T-cadherin to inhibit neurite growth was tested in vitro. T-cadherin inhibited neurite extension from select neuron populations both as a substratum, and as a soluble recombinant protein. Anti-T-cadherin antibodies neutralized the inhibition. Spinal motor neurons were inhibited only during the stages of axon growth across the sclerotome and muscle innervation. Inhibitory responses corresponded to neuronal T-cadherin expression, suggesting a homophilic binding mechanism. These results suggest that T-cadherin is a negative guidance cue for motor axon projections.
Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation
Authors: Kroll Kristen L. , Amaya Enrique
Published: April 2021

We have developed a simple approach for large-scale trans-genesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Trans-genic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.
Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation
Authors: Devoto Stephen H. , Melançon Ellie , Eisen Judith S. , Westerfield Monte
Published: April 2021

We have examined the development of specific muscle fiber types in zebrafish axial muscle by labeling myogenic precursor cells with vital fluorescent dyes and following their subsequent differentiation and fate. Two populations of muscle precursors, medial and lateral, can be distinguished in the segmental plate by position, morphology and gene expression. The medial cells, known as adaxial cells, are large, cuboidal cells adjacent to the notochord that express myoD. Surprisingly, after somite formation, they migrate radially away from the notochord, becoming a superficial layer of muscle cells. A subset of adaxial cells develop into engrailed-expressing muscle pioneers. Adaxial cells differentiate into slow muscle fibers of the adult fish. We have named the lateral population of cells in the segmental plate, lateral presomitic cells. They are smaller, more irregularly shaped and separated from the notochord by adaxial cells; they do not express myoD until after somite formation. Lateral presomitic cells remain deep in the myotome and they differentiate into fast muscle fibers. Thus, slow and fast muscle fiber types in zebrafish axial muscle arise from distinct populations of cells in the segmental plate that develop in different cellular environments and display distinct behaviors.
Regulation and function of transcription factor GATA-1 during red blood cell differentiation
Authors: Briegel Karoline , Bartunek Petr , Stengl Gabi , Lim Kim-Chew , Beug Hartmut , Engel James Douglas , Zenke Martin
Published: April 2021

The tissue-specific transcription factor GATA-1 is a key regulator of red blood cell differentiation. One seemingly contradictory aspect of GATA-1 function is that, while it is abundant in erythroid progenitor cells prior to the onset of overt differentiation, it does not significantly activate known GATA-1 target genes in those cells. To investigate the mechanisms underlying GATA-1 function during the transition from early to late erythropoiesis, we have examined its expression and activity in normal avian erythroid progenitor cells before and after induction of differentiation. In these primary progenitor cells, GATA-1 protein was predominantly located in the cytoplasm, while induction of differentiation caused its rapid relocalization to the nucleus, suggesting that nuclear translocation constitutes an important regulatory step in GATA-1 activation. As an alternative way of addressing the same question, we also ectopically expressed a GATA-1/estrogen receptor fusion protein (GATA-1/ER) in red blood cell progenitors, where nuclear translocation of, and transcriptional activation by, this hybrid factor are conditionally controlled by estrogen. We found that hormone-activated GATA-1/ER protein accelerated red blood cell differentiation, and concomitantly suppressed cell proliferation. These phenotypic effects were accompanied by a simultaneous suppression of c-myb and GATA-2 transcription, two genes thought to be involved in the proliferative capacity of hematopoietic progenitor cells. Thus, GATA-1 appears to promote differentiation in committed erythroid progenitor cells both by inducing differentiation-specific genes and by simultaneously suppressing genes involved in cell proliferation.
The chick limbless mutation causes abnormalities in limb bud dorsal-ventral patterning: implications for the mechanism of apical ridge formation
Authors: Grieshammer Uta , Minowada George , Pisenti Jacqueline M. , Abbott Ursula K. , Martin Gail R.
Published: April 2021

In chick embryos homozygous for the limbless mutation, limb bud outgrowth is initiated, but a morphologically distinct apical ridge does not develop and limbs do not form. Here we report the results of an analysis of gene expression in limbless mutant limb buds. Fgf4, Fgf8, Bmp2 and Msx2, genes that are expressed in the apical ridge of normal limb buds, are not expressed in the mutant limb bud ectoderm, providing molecular support for the hypothesis that limb development fails in the limbless embryo because of the inability of the ectoderm to form a functional ridge. Moreover, Fgf8 expression is not detected in the ectoderm of the prospective limb territory or the early limb bud of limbless embryos. Since the early stages of limb bud outgrowth occur normally in the mutant embryos, this indicates that FGF8 is not required to promote initial limb bud outgrowth. In the absence of FGF8, Shh is also not expressed in the mutant limb buds, although its expression can be induced by application of FGF8-soaked beads. These observations support the hypothesis that Fgf8 is required for the induction of Shh expression during normal limb development. Bmp2 expression was also not detected in mutant limb mesoderm, consistent with the hypothesis that SHH induces its expression. In contrast, SHH is not required for the induction of Hoxd11 or Hoxd13 expression, since expression of both these genes was detected in the mutant limb buds. Thus, some aspects of mesoderm A-P patterning can occur in the absence of SHH and factors normally expressed in the apical ridge. Intriguingly, mutant limbs rescued by local application of FGF displayed a dorsalized feather pattern. Furthermore, the expression of Wnt7a, Lmx1 and En1, genes involved in limb D-V patterning, was found to be abnormal in mutant limb buds. These data suggest that D-V patterning and apical ridge formation are linked, since they show that the limbless mutation affects both processes. We present a model that explains the potential link between D-V positional information and apical ridge formation, and discuss the possible function of the limbless gene in terms of this model.
Developmental territories created by mutual antagonism between Wingless and Decapentaplegic
Authors: Theisen Heidi , Haerry Theodor E. , O’Connor Michael B. , Marsh J. Lawrence
Published: April 2021

Drosophila appendages develop from imaginal discs which become subdivided into distinct regions during normal pat- terning. At least 3 axes of asymmetry are required to produce a chiral appendage such as a leg. The A/P com- partments provide one axis of asymmetry in all discs. In leg and antennal discs, the anterior compartment becomes asymmetric in the D/V axis with decapentaplegic (dpp) expression defining dorsal anterior leg, and wingless (wg) expression defining ventral anterior leg. However, unlike wing discs, no D/V compartment has been demonstrated in legs or antennae. How are the dorsal anterior and ventral anterior territories defined and maintained? Here we show that wg inhibits dpp expression and dpp inhibits wg expression in leg and eye/antennal discs. This mutual repression provides a mechanism for maintaining separate regions of wg and dpp expression in a developing field. We propose the term ‘territory’ to describe regions of cells that are under the domineering influence of a particular morphogen. Territories differ from compartments in that they are not defined by lineage but are dynamically main- tained by continuous morphogen signaling. We propose that the anterior compartment of the leg disc is divided into dorsal and ventral territories by the mutual antagonism between WG and DPP signaling.
The human β-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene
Authors: Tewari Rita , Gillemans Nynke , Harper Alex , Wijgerde Mark , Zafarana Gaetano , Drabek Dubravka , Grosveld Frank , Philipsen Sjaak
Published: April 2021

The β-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5′HS1-5. A fully active 6.5 kb version of the LCR, called the μLCR, has been described. Expression of the β-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic ε-globin and fetal γ-globin promoters, resulting in the almost exclusive transcription of the β-globin gene in adult ery-thropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5′HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial β-galactosidase (lacZ) gene. The results show that the μLCR and 5′HS3 direct erythroid-specific, embryonic expression of this construct, while 5′HS1, 5′HS2 and 5′HS4 are inactive at any stage of development. Expression of the μLCR and 5′HS3 transgenes is repressed during fetal stages of development. The trans-genes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybrid-ization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a β-globin gene is required for LCR function as conditions become more stringent during development.
The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development
Authors: Yu Kweon , Sturtevant Mark A. , Biehs Brian , François Vincent , Padgett Richard W. , Blackman Ronald K. , Bier Ethan
Published: April 2021

TGF-β-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-β family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-β-like Decapenta-plegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.
The Drosophila Enhancer of zeste gene encodes a chromosomal protein: examination of wild-type and mutant protein distribution
Authors: Carrington Elizabeth A. , Jones Richard S.
Published: April 2021

The Drosophila Enhancer of zeste [E(z)] gene is a member of the Polycomb-group and, as such, is involved in maintaining the transcriptional repression of the homeotic genes of the Antennapedia (ANT-C) and bithorax (BX-C) complexes. It has been proposed that Polycomb-group (Pc- G)-mediated silencing requires the formation of heteromeric protein complexes which modify the chromatin structure of target genes. We describe the in vivo distribution of the E(Z) protein and show it to be ubiquitously present in embryonic and larval nuclei. In salivary gland polytenized nuclei, the identifiable E(Z) chromosome binding sites are a subset of those described for other Polycomb-group proteins, suggesting that E(Z) may also participate in Polycomb-group complexes. E(Z) binds to chromosomes in a DNA sequence-dependent manner, as illustrated by the creation of a new E(Z)-binding site at the location of a P element reporter construct that previously has been shown to contain a Polycomb response element (PRE). We also present the sequences of one null and three temperature-sensitive E(z) alleles, describe the effects these mutations have on the in vivo distribution of E(Z) protein and discuss their implications concerning putative functional domains. Finally, we describe the effect a trithorax mutation has on E(Z) chromosome binding.