bluuebunny/crossref_metadata_2025 huggingface dataset:
Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex
Authors: Blaschke Anne J. , Staley Kristina , Chun Jerold
Published: April 2021

A key event in the development of the mammalian cerebral cortex is the generation of neuronal populations during embryonic life. Previous studies have revealed many details of cortical neuron development including cell birthdates, migration patterns and lineage relationships. Programmed cell death is a potentially important mechanism that could alter the numbers and types of developing cortical cells during these early embryonic phases. While programmed cell death has been documented in other parts of the embryonic central nervous system, its operation has not been previously reported in the embryonic cortex because of the lack of cell death markers and the difficulty in following the entire population of cortical cells. Here, we have investigated the spatial and temporal distribution of dying cells in the embryonic cortex using an in situ end- labelling technique called ‘ISEL+’ that identifies fragmented nuclear DNA in dying cells with increased sensitivity. The period encompassing murine cerebral cortical neurogenesis was examined, from embryonic days 10 through 18. Dying cells were rare at embryonic day 10, but by embryonic day 14, 70% of cortical cells were found to be dying. This number declined to 50% by embryonic day 18, and few dying cells were observed in the adult cerebral cortex. Surprisingly, while dying cells were observed throughout the cerebral cortical wall, the majority were found within zones of cell proliferation rather than in regions of postmitotic neurons. These observations suggest that multiple mechanisms may regulate programmed cell death in the developing cortex. Moreover, embryonic cell death could be an important factor enabling the selection of appropriate cortical cells before they complete their differentiation in postnatal life.
Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells
Authors: Koshimizu Uichi , Taga Tetsuya , Watanabe Miho , Saito Mikiyoshi , Shirayoshi Yasuaki , Kishimoto Tadamitsu , Nakatsuji Norio
Published: April 2021

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on Sl/Sl4-m220 cells. Interestingly, IL-11 stimu-lated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action on PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.
Hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars
Authors: Tabata Makoto J. , Kim Kenji , Liu Ji-Guang , Yamashita Kazuo , Matsumura Tatsushi , Kato Joji , Iwamoto Masahiro , Wakisaka Satoshi , Matsumoto Kunio , Nakamura Toshikazu , Kumegawa Masayoshi , Kurisu Kojiro
Published: April 2021

The patterns of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met, were revealed in the tooth germ of rat mandibular molars using RT-PCR. In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role played by HGF in tooth development, HGF translation arrest by antisense phosphorothioate oligodeoxynucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell’s system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the HGF mRNA. Both the control and the sense-treated explants showed normal histological structure, as observed in vivo. On the other hand, antisense-treated explants exhibited an abnormal structure in which the enamel organs were surrounded by a thin layer of dentin and dental papilla, appearing ‘inside-out’ compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human HGF combined with antisense ODN showed normal development, indicating that exogenous HGF rescued the explants from the abnormal structure caused by antisense ODN. The findings of a BrdU incorporation experiment suggested that the imbalance between the proliferation activity of the inner enamel epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar.
The homeobox gene GLABRA 2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana
Authors: Masucci James D. , Rerie William G. , Foreman Daphne R. , Zhang Meng , Galway Moira E. , Marks M. David , Schiefelbein John W.
Published: April 2021

The role of the Arabidopsis homeobox gene, GLABRA 2 (GL2), in the development of the root epidermis has been investigated. The wild-type epidermis is composed of two cell types, root-hair cells and hairless cells, which are located at distinct positions within the root, implying that positional cues control cell-type differentiation. During the development of the root epidermis, the differentiating root-hair cells (trichoblasts) and the differentiating hairless cells (atrichoblasts) can be distinguished by their cytoplasmic density, vacuole formation, and extent of elongation. We have determined that mutations in the GL2 gene specifically alter the differentiation of the hairless epidermal cells, causing them to produce root hairs, which indicates that GL2 affects epidermal cell identity. Detailed analyses of these differentiating cells showed that, despite forming root hairs, they are similar to atrichoblasts of the wild type in their cytoplasmic characteristics, timing of vacuolation, and extent of cell elongation. The results of in situ nucleic acid hybridization and GUS reporter gene fusion studies show that the GL2 gene is preferentially expressed in the differentiating hairless cells of the wild type, during a period in which epidermal cell identity is believed to be established. These results indicate that the GL2 homeodomain protein normally regulates a subset of the processes that occur during the differentiation of hairless epidermal cells of the Arabidopsis root. Specifically, GL2 appears to act in a cell-position-dependent manner to suppress hair formation in differentiating hairless cells.
Vegetal egg cytoplasm promotes gastrulation and is responsible for specification of vegetal blastomeres in embryos of the ascidian Halocynthia roretzi
Authors: Nishida Hiroki
Published: April 2021

An animal-vegetal axis exists in the unfertilized eggs of the ascidian Halocynthia roretzi. The first phase of ooplasmic segregation brings the egg cortex to the vegetal pole very soon after fertilization. In the present study, when 5-8% of the egg cytoplasm in the vegetal pole region was removed between the first and second phase of segregation, most embryos exhibited failure of gastrulation, as reported previously in Styela by Bates and Jeffery (Dev. Biol. 124, 65-76, 1987). The embryos that were deficient in vegetal pole cytoplasm (VC-deficient embryos) developed into permanent blastulae. They consisted for the most part of epidermal cells and most lacked the derivatives of vegetal blastomeres, such as endoderm, muscle and notochord. Removal of cytoplasm from other regions did not affect embryogenesis. The cleavage of the VC-deficient embryos not only exhibited radial symmetry along the animal-vegetal axis but the pattern of the cleavage was also identical in the animal and vegetal hemispheres. Examination of the developmental fates of early blastomeres of VC-deficient embryos revealed that the vegetal blastomeres had assumed the fate of animal cells. These results suggested that the VC-deficient embryos had been totally animalized.

When vegetal pole cytoplasm was transplanted to the animal pole or equatorial position of VC-deficient eggs, gastrulation occurred, starting at the site of the transplantation and tissues derived from vegetal blastomeres formed. Therefore, it appears that vegetal pole cytoplasm specifies the site of gastrulation and the cytoplasm is responsible for the specification of vegetal blastomeres. It is suggested that, during the second phase of ooplasmic segregation, cytoplasmic factors responsible for gastrulation spread throughout the entire vegetal hemisphere.
A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation
Authors: Albanesi Cristina , Geremia Raffaele , Giorgio Marco , Dolci Susanna , Sette Claudio , Rossi Pellegrino
Published: April 2021

In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermato-genesis. Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells. These transcripts should encode a truncated version of the c-kit protein, lacking the extra-cellular, the transmembrane and part of the intracellular tyrosine-kinase domains. The 5′ end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene. We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors. Deletion of both these sequences abolishes activity of the promoter in vitro. We have also established that this promoter is functional in vivo, in a tissue- and cell-specific fashion, since intronic sequences drive the expression of the E. coli lacZ reporter gene in transgenic mice specifically in the testis. Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that a cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis. In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa. An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems.
Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males
Authors: Lin Ting-Yi , Viswanathan Sridhar , Wood Cricket , Wilson Patricia G. , Wolf Nurit , Fuller Margaret T.
Published: April 2021

Wild-type function of four Drosophila genes, spermatocyte arrest, cannonball, always early and meiosis I arrest, is required both for cell-cycle progression through the G2/M transition of meiosis I in males and for onset of spermatid differentiation. In males mutant for any one of these meiotic arrest genes, mature primary spermatocytes with partially condensed chromosomes accumulate and post-meiotic cells are lacking. The arrest in cell-cycle progression occurs prior to degradation of cyclin A protein. The block in spermatogenesis in these mutants is not simply a secondary consequence of meiotic cell-cycle arrest, as spermatid differentiation proceeds in males mutant for the cell cycle activating phosphatase twine. Instead, the arrest of both meiosis and spermiogenesis suggests a control point that may serve to coordinate the male meiotic cell cycle with the spermatid differentiation program. The phenotype of the Drosophila meiotic arrest mutants is strikingly similar to the histopathological features of meiosis I maturation arrest infertility in human males, suggesting that the control point may be conserved from flies to man.
lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H)
Authors: Christensen S. , Kodoyianni V. , Bosenberg M. , Friedman L. , Kimble J.
Published: April 2021

The homologous receptors LIN-12 and GLP-1 mediate diverse cell-signaling events during development of the nematode Caenorhabditis elegans. These two receptors appear to be functionally interchangeable and have sequence similarity to Drosophila Notch. Here we focus on a molecular analysis of the lag-1 gene (lin-12 -and glp-1), which plays a central role in LIN-12 and GLP-1-mediated signal transduction. We find that the predicted LAG-1 protein is homologous to two DNA-binding proteins: human C Promoter Binding Factor (CBF1) and Drosophila Suppressor of Hairless (Su(H)). Furthermore, we show that LAG-1 binds specifically to the DNA sequence RTGGGAA, previously identified as a CBF-1/Su(H)-binding site. Finally, we report that the 5′ flanking regions and first introns of the lin-12, glp-1 and lag-1 genes are enriched for potential LAG-1-binding sites. We propose that LAG-1 is a transcriptional regulator that serves as a primary link between the LIN-12 and GLP-1 receptors and downstream target genes in C. elegans. In addition, we propose that LAG-1 may be a key component of a positive feedback loop that amplifies activity of the LIN-12/GLP-1 pathway.
Retinoic acid signaling is required during early chick limb development
Authors: Helms Jill A. , Kim Chang Hee , Eichele Gregor , Thaller Christina
Published: April 2021

In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.
Contrasting effects of protein synthesis inhibition and of cyclic AMP on apoptosis in the developing retina
Authors: Rehen S. K. , Varella M. H. , Freitas F. G. , Moraes M. O. , Linden R.
Published: April 2021

The role of protein synthesis in apoptosis was investigated in the retina of developing rats. In the neonatal retina, a ganglion cell layer, containing neurons with long, centrally projecting axons, is separated from an immature neuro-blastic layer by a plexiform layer. This trilaminar pattern subsequently evolves to five alternating cell and plexiform layers that constitute the mature retina and a wave of programmed neuron death sweeps through the layers. Apoptosis due to axon damage was found in ganglion cells of retinal explants within 2 days in vitro and was prevented by inhibition of protein synthesis. Simultaneously, protein synthesis blockade induced apoptosis among the undamaged cells of the neuroblastic layer, which could be selectively prevented by an increase in intracellular cyclic AMP. Both the prevention and the induction of apoptosis among ganglion cells or neuroblastic cells, respectively, occurred after inhibition of protein synthesis in vivo. The results show the coexistence of two mechanisms of apoptosis within the organized retinal tissue. One mechanism is triggered in ganglion cells by direct damage and depends on the synthesis of proteins acting as positive modulators of apoptosis. A distinct, latent mechanism is found among immature neuroblasts and may be repressed by continuously synthesized negative modulators, or by an increase in intracellular cyclic AMP.
Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis
Authors: Edwards Kevin A. , Kiehart Daniel P.
Published: April 2021

Morphogenesis is characterized by orchestrated changes in the shape and position of individual cells. Many of these movements are thought to be powered by motor proteins. However, in metazoans, it is often difficult to match specific motors with the movements they drive. The nonmuscle myosin II heavy chain (MHC) encoded by zipper is required for cell sheet movements in Drosophila embryos. To determine if myosin II is required for other processes, we examined the phenotypes of strong and weak larval lethal mutations in spaghetti squash (sqh), which encodes the nonmuscle myosin II regulatory light chain (RLC). sqh mutants can be rescued to adulthood by daily induction of a sqh cDNA transgene driven by the hsp70 promoter. By transiently ceasing induction of the cDNA, we depleted RLC at specific times during development. When RLC is transiently depleted in larvae, the resulting adult phenotypes demonstrate that RLC is required in a stage-specific fashion for proper development of eye and leg imaginal discs. When RLC is depleted in adult females, oogenesis is reversibly disrupted. Without RLC induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks, three morphologically and mechanistically distinct types of follicle cell migration, and completion of nurse cell cytoplasm transport (dumping). Finally, we show that in sqh mutant tissues, MHC is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail-binding protein p127. This suggests that sqh mutant phenotypes are chiefly caused by sequestration of myosin into inactive aggregates. These results show that myosin II is responsible for a surprisingly diverse array of cell shape changes throughout development.
Pathways for inflorescence and floral induction in Antirrhinum
Authors: Bradley Desmond , Vincent Coral , Carpenter Rosemary , Coen Enrico
Published: April 2021

The presentation of flowers on a modified stem, the inflorescence, requires the integration of several aspects of meristem behaviour. In Antirrhinum, the inflorescence can be distinguished by its flowers, hairy stem, modified leaves, short internodes and spiral phyllotaxy. We show, by a combination of physiological, genetical and morphological analysis, that the various aspects of the inflorescence are controlled by three pathways. The first pathway, depends on expression of the floricaula gene, and is rapidly and discretely induced by exposure to long daylength. Activation of this pathway occurs in very young axillary meristems, resulting in a floral identity. In addition, the length of subtending leaves and hairiness of the stem are partially modified. The second pathway affects leaf size, internode length, and stem hairiness, but does not confer floral meristem identity. This pathway is induced by long daylength, but not as rapidly or discretely as the floricauladependent pathway. The third pathway controls the switch in phyllotaxy from decussate to spiral and is activated independently of daylength. The coordination of these three programmes ensures that apical and axillary meristem behaviour is integrated.
The mec-8 gene of C. elegans encodes a protein with two RNA recognition motifs and regulates alternative splicing of unc-52 transcripts
Authors: Lundquist Erik A. , Herman Robert K. , Rogalski Teresa M. , Mullen Gregory P. , Moerman Donald G. , Shaw Jocelyn E.
Published: April 2021

Mutations in the mec-8 gene of Caenorhabditis elegans were previously shown to affect the functions of body wall muscle and mechanosensory and chemosensory neurons. Mutations in mec-8 also strongly enhance the mutant phenotype of specific mutations in unc-52, a gene that encodes, via alternative splicing of pre-mRNA, a set of basement membrane proteins, homologs of perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis and cuticle. We have cloned mec-8 and found that it encodes a protein with two RNA recognition motifs, characteristic of RNA binding proteins. We have used reverse transcription-PCR and RNase protection experiments to show that mec-8 regulates the accumulation of a specific subset of alternatively spliced unc-52 transcripts. We have also shown with antibodies to UNC-52 that mec-8 affects the abundance of a subset of UNC-52 isoforms. We propose that mec-8 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and one or more additional genes that affect mechanosensory and chemosensory neuron function.
A functional homologue of goosecoid in Drosophila
Authors: Goriely Anne , Stella Michael , Coffinier Catherine , Kessler Daniel , Mailhos Carolina , Dessain Scott , Desplan Claude
Published: April 2021

We have cloned a Drosophila homologue (D-gsc) of the ver- tebrate homeobox gene goosecoid (gsc). In the Gsc proteins, the pressure for conservation has been imposed on the homeodomain, the functional domain of the protein: sequence homology is limited to the homeodomain (78% identity) and to a short stretch of 7 aminoacids also found in other homeoproteins such as Engrailed. Despite this weak homology, D-gsc is able to mimic gsc function in a Xenopus assay, as shown by its ability to rescue the axis development of a UV-irradiated embryo. Moreover, our data suggest that the position of insect and vertebrate gsc homologues within a regulatory network has also been conserved: D-gsc expression is controlled by decapenta- plegic, orthodenticle, sloppy-paired and tailless whose homo- logues control gsc expression (for BMP4 and Otx-2), or are expressed at the right time and the right place (for XFKH1/Pintallavis and Tlx) to be interacting with gsc during vertebrate development. However, the pattern of D- gsc expression in ectodermal cells of the nervous system and foregut cannot easily be reconciled with that of ver- tebrate gsc mesodermal expression, suggesting that its precise developmental function might have diverged. Still, this comparison of domains of expression and functions among Gsc proteins could shed light on a common origin of gut formation and/or on basic cellular processes. The identification of gsc target genes and/or other genes involved in similar developmental processes will allow the definition of the precise phylogenetic relationship among Gsc proteins.
Alternative RNA splicing that determines agrin activity regulates binding to heparin and α-dystroglycan
Authors: Campanelli James T. , Gayer Gregory G. , Scheller Richard H.
Published: April 2021

Agrin is a component of the extracellular matrix that regulates aspects of neuromuscular junction differentiation. Identification of agrin-binding proteins has lead to the suggestion that α-dystroglycan is a muscle cell surface proteoglycan that mediates agrin activity. To further test this hypothesis, we have compared the ability of differentially active agrin isoforms to interact with a model component of proteoglycans, heparin, as well as with the putative proteoglycan α-dystroglycan. We demonstrate that an alternately spliced exon (encoding the sequence lysine, serine, arginine, lysine: Y site) is necessary for agrin-heparin interactions. We also show that alternate splicing at another site (Z site) dramatically affects inter-action of α-dystroglycan with agrin. We propose a model in which multiple distinct domains of agrin interact with both protein and sugar moieties of α-dystroglycan. The isoform-specific binding of agrin to α-dystroglycan is con-sistent with a functional role for this interaction during synaptogenesis.
Amphiregulin in lung branching morphogenesis: interaction with heparan sulfate proteoglycan modulates cell proliferation
Authors: Schuger Lucia , Johnson Gibbes R. , Gilbride Kevin , Plowman Gregory D. , Mandel Richard
Published: April 2021

Epithelial and mesenchymal cells isolated from mouse embryonic lungs synthesized and responded to amphiregulin (AR) in a different fashion. Mesenchymal cells produced and deposited 3- to 4-fold more AR than epithelial cells, proliferated in the presence of exogenous AR, and their spontaneous growth was blocked by up to 85% by anti-AR antibodies. In contrast, epithelial cells exhibited a broad response to this growth regulator factor depending on whether they were supplemented with extracellular matrix (ECM) and whether this ECM was of epithelial or mesenchymal origin. AR-treated epithelial cells proliferated by up to 3-fold in the presence of mesenchymal-deposited ECM, remained unchanged in the presence of epithelial-deposited ECM, and decreased in their proliferation rate below controls in the absence of ECM supplementation. This effect was abolished by treatment with the glycosaminoglycan-degrading enzymes heparinase and heparitinase suggesting the specific involvement of heparan sulfate proteoglycan (HSPG) in AR-mediated cell proliferation. In whole lung explants, branching morphogenesis was inhibited by antibodies against the AR heparan sulfate binding site and stimulated by exogenous AR. Since during development, epithelial cells are in contact with mesenchymal ECM at the tips of the growing buds and alongside the basement membrane, focal variations in the proportion of epithelial and mesenchymal HSPG will focally affect epithelial proliferation rates. Therefore, AR-HSPG interaction may underlie the process of branching morphogenesis by inducing differential cell proliferation.
An HF-1a/ HF-1b/MEF-2 combinatorial element confers cardiac ventricular specificity and establishes an anterior-posterior gradient of expression
Authors: Ross Robert S. , Navankasattusas Sutip , Harvey Richard P. , Chien Kenneth R.
Published: April 2021

The molecular determinants that direct gene expression to the ventricles of the heart are for the most part unknown. Additionally, little data is available on how the anterior/posterior axis of the heart tube is determined and whether the left and right atrial and ventricular chambers are assigned as part of this process. Utilizing myosin light chain-2 ventricular promoter/β-galactosidase reporter transgenes, we have determined the minimal cis-acting sequences required for ventricular-specific gene expression. In multiple independent transgenic mouse lines, we found that both a 250 base pair myosin light chain-2 ventricular promoter fragment, as well as a dimerized 28 bp sub-element (HF-1) containing binding sites for HF1a and HF1b/MEF2 factors, directed ventricular-specific reporter expression from as early as the endogenous gene, at day 7.5-8.0 post coitum. While the endogenous gene is expressed uniformly throughout both ventricles, the transgenes were expressed in a right ventricular/conotruncal dominant fashion, suggesting that they contain only a subset of the elements which respond to positional information in the developing heart tube. Expression of the transgene was cell autonomous and its temporospatial characteristics not affected by mouse strain/methylation state of the genome. To determine whether ventricular-specific expression of the transgene was dependent upon regulatory genes required for correct ventricular differentiation, the 250 base pair transgene was bred into both retinoid X receptorα and Nkx2-5 null back-grounds. The transgene was expressed in both mutant backgrounds, despite the absence of endogenous myosin light chain-2 ventricular transcript in Nkx2-5 null embryos. Ventricular specification, as judged by transgene expression, appeared to occur normally in both mutants. Thus, the HF-1 element, directs chamber-specific transcription of a transgene reporter independently of retinoid X receptorα and Nkx2-5, and defines a minimal combinatorial pathway for ventricular chamber gene expression. The patterned expression of this transgene may provide a model system in which to investigate the cues that dictate anterior-posterior (right ventricle/left ventricle) gradients during mammalian heart development.
LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis
Authors: Carland Francine M. , McHale Neil A.
Published: April 2021

We have taken a genetic approach to understanding the mechanisms that control vascular patterning in the leaves of higher plants. Here we present the identification and characterization of the lop1 mutant of Arabidopsis which is defective in basipetal transport of IAA. Mutant leaf midveins show disoriented axial growth, and bifurcation into twin veins that are frequently rotated out of the normal dorsal/ventral axis of the leaf. Mutant plants also display abnormal patterns of cell expansion in the midrib cortex and in the epidermis of the elongation zone of lateral roots. Lateral roots show abnormal curvature during initiation, sometimes encircling the primary root prior to growth in a normal downward direction. Mutant seedlings have normal levels of free IAA, and appear normal in auxin perception, suggesting that transport is the primary lesion. The abnormalities in vascular development, lateral root initiation and patterns of cell expansion observed in the lop1 mutant are consistent with a basic disruption in basipetal transport of IAA.
The Drosophila pipsqueak gene encodes a nuclear BTB-domain-containing protein required early in oogenesis
Authors: Horowitz Heidi , Berg Celeste A.
Published: April 2021

Mutations at the pipsqueak locus affect early patterning in the Drosophila egg and embryo. We have cloned pipsqueak and found that it is a large and complex gene, encoding multiple transcripts and protein isoforms. One protein, PsqA, is absent in all of the mutants that we have examined. We show that PsqA is a nuclear protein present in the germ cells and somatically derived follicle cells throughout oogenesis and that it is required prior to stage one of oogenesis. PsqA contains a BTB (POZ) domain at its amino terminus; additionally, we have identified an evolutionarily conserved motif of unknown function present four times in tandem at the C terminus of the protein. PZ pipsqueak mutants produce a putative fusion protein containing the pipsqueak BTB domain fused to sequences resident on the PZ element (H. Horowitz and C. Berg, 1995 Genetics 139, 327-335). We demonstrate here that expression of this fusion protein in wild-type flies has a dominant effect, resulting in infertility and eggshell defects. These dominant phenotypes are discussed in light of current theories on the role of the BTB domain in protein-protein interactions.
In vitro and transgenic analysis of a human HOXD4 retinoid-responsive enhancer
Authors: Morrison Alastair , Moroni Maria Cristina , Ariza-McNaughton Linda , Krumlauf Robb , Mavilio Fulvio
Published: April 2021

Expression of vertebrate Hox genes is regulated by retinoids in cell culture and in early embryonic development. We have identified a 185-bp retinoid-responsive transcriptional enhancer 5′ of the human HOXD4 gene, which regulates inducibility of the gene in embryonal carcinoma cells through a pattern of DNA-protein interaction on at least two distinct elements. One of these elements contains a direct repeat mediating ligand-dependent interaction with retinoic acid receptors, and is necessary though not sufficient for the enhancer function. The HOXD4 enhancer directs expression of a lacZ reporter gene in the neural tube of transgenic mouse embryos in a timeregulated and regionally restricted fashion, reproducing part of the anterior neuroectodermal expression pattern of the endogenous Hoxd-4 gene. Administration of retinoic acid to developing embryos causes alterations in the spatial restriction of the transgene expression domain, indicating that the HOXD4 enhancer is also a retinoid-responsive element in vivo. The timing of the retinoic acid response differs from that seen with more 3′ Hox genes, in that it occurs much later. This shows that the temporal window of competence in the ability to respond to retinoic acid differs between Hox genes and can be linked to specific enhancers. Mutations in the direct repeat or in a second element in the enhancer affect both retinoid response in culture and developmental regulation in embryos, suggesting that co-operative interactions between different factors mediate the enhancer activity. These data provide further support for a role of endogenous retinoids in regulation and spatial restriction of Hox gene expression in the central nervous system.
Position-effect variegation in Drosophila depends on the dose of the gene encoding the E2F transcriptional activator and cell cycle regulator
Authors: Seum Carole , Spierer Anne , Pauli Daniel , Szidonya Janos , Reuter Gunter , Spierer Pierre
Published: April 2021

A dominant mutation due to the insertion of a P-element at 93E on the third chromosome of Drosophila melanogaster enhances position-effect variegation. The corresponding gene was cloned by transposon tagging and the sequence of the transcript revealed that it corresponds to the gene encoding the transcriptional activator and cell cycle regulator dE2F. The transposon-tagged allele is homozygous viable, and the insertion of the transposon in an intron correlates with a strong reduction in the amount of transcript. A homozygous lethal null allele was found to behave as a strong enhancer when heterozygous. Overexpression of the gene in transgenic flies has the opposite effect of suppressing variegation. A link is established here, and discussed, between the dose of a transcriptional activator, which controls the cell cycle, and epigenetic silencing of chromosomal domains in Drosophila.
Mesoderm and endoderm differentiation in animal cap explants: identification of the HNF4-binding site as an activin A responsive element in the Xenopus HNF1α promoter
Authors: Weber Heike , Holewa Beatrix , Jones Elizabeth A. , Ryffel Gerhart U.
Published: April 2021

The gene encoding the tissue-specific transcription factor HNF1α (LFB1) is transcriptionally activated shortly after mid-blastula transition in Xenopus embryos. We have now shown that the HNF1α protein is localized in the nuclei of the liver, gall bladder, gut and pronephros of the developing larvae. In animal cap explants treated with activin A together with retinoic acid, we induced HNF1α in pronephric tubules and epithelial gut cells, i.e. in mesodermal as well as in endodermal tissues. HNF1α can also be induced by activin A, but not by retinoic acid alone. To define the promoter element responding to the activin A signal, we injected various HNF1α promoter luciferase constructs into fertilized eggs and cultured the isolated animal caps in the presence of activin A. From the activity profiles of the promoter mutants used, we identified the HNF4-binding site as an activin-A-responsive element. As HNF4 is a maternal protein in Xenopus and localized in an animal-to-vegetal gradient in the cleaving embryo, we speculate that the activin A signal emanating from the vegetal pole cooperates with the maternal transcription factor HNF4 to define the embryonic regions expressing HNF1α.
Nonradial migration of interneurons can be experimentally altered in spinal cord slice cultures
Authors: Phelps Patricia E. , Barber Robert P. , Vaughn James E.
Published: April 2021

During development, many migrating neurons are thought to guide on radially oriented glia to reach their adult locations. However, members of the ‘U-shaped’ group of cholinergic interneurons in embryonic rat spinal cord appeared to migrate in a direction perpendicular to the orientation of radial glia. This ‘U-shaped’ group of cells was located around the ventral ventricular zone on embryonic day 16 and, during the next two days, the constituent cells dispersed into the dorsal horn or around the central canal. During this period, these cells could be identified with either ChAT immunocytochemistry or NADPHdiaphorase histochemistry and they appeared to be aligned along commissural axons, suggesting that such processes, rather than radial glia, might guide their migration.

An organotypic spinal cord slice preparation was developed and utilized for three different experimental approaches to studying this migration. In the first experiments, slices of embryonic day 16 cervical spinal cord were cultured for one, two or three days, and a relatively histotypic dorsal migration of ‘U-derived’ cells could be inferred from these sequential cultures. A second set of experiments focused on the direct observation of dorsally directed migration in living spinal cord cultures. Embryonic day 16 slices were injected with a lipophilic fluorescent label near the dorsal boundary of the ‘U-shaped’ cell group and the dorsal movement of labeled cells was observed using confocal microscopy. These experiments confirmed the dorsal migratory pattern inferred from sequentially fixed specimens. A third experimental approach was to transect embryonic day 16 slice cultures microsurgically in order to disturb the migration of ‘U-derived’ cells. Depending upon the amount of ventral spinal cord removed, the source of cells was excised and/or their guidance pathway was perturbed. The number and position of ‘U-derived’ cells varied with the amount of ventral cord excised. If more than 400 μm was removed, no ‘U-derived’ diaphoraselabeled cells were present, whereas if only 200-300 μm was removed, the cultures contained such cells. However, in this instance, many of the ‘U-derived’ neurons did not move as far dorsally, nor did they display their characteristic dorsoventral orientation. When results from these three experiments are taken together, they provide strong evidence that nonradial neuronal migration occurs in developing spinal cord and that the ‘U-derived’ neurons utilize such a migration to move from their ventral generation sites to their dorsal adult locations.
Expression domains of a zebrafish homologue of the Drosophila pair-rule gene hairy correspond to primordia of alternating somites
Authors: Müller Marcus , Weizsäcker Elisabeth v. , Campos-Ortega José A.
Published: April 2021

her1 is a zebrafish cDNA encoding a bHLH protein with all features characteristic of members of the Drosophila HAIRY-E(SPL) family. During late gastrulation stages, her1 is expressed in the epibolic margin and in two distinct transverse bands of hypoblastic cells behind the epibolic front. After completion of epiboly, this pattern persists essentially unchanged through postgastrulation stages; the marginal domain is incorporated in the tail bud and, depending on the time point, either two or three paired bands of expressing cells are present within the paraxial presomitic mesoderm separated by regions devoid of transcripts. Labelling of cells within the her1 expression domains with fluorescein-dextran shows that the cells in the epibolic margin and the tail bud are not allocated to particular somites. However, allocation of cells to somites occurs between the marginal expression domain and the first expression band, anterior to it. Moreover, the her1 bands, and the intervening non-expressing zones, each represents the primordium of a somite. This expression pattern is highly reminiscent of that of Drosophila pair-rule genes. A possible participation of her1 in functions related to somite formation is discussed.
Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons
Authors: Reissmann E. , Ernsberger U. , Francis-West P. H. , Rueger D. , Brickell P. M. , Rohrer H.
Published: April 2021

The neurotransmitter phenotype of sympathetic neurons is specified by interactions with the surrounding embryonic tissues. Adrenergic differentiation is elicited early during development in the vicinity of notochord and dorsal aorta and the importance of axial midline tissues for adrenergic differentiation has been well documented. We now provide evidence that bone morphogenetic proteins, BMP-4 and BMP-7 are signals produced by the dorsal aorta that direct sympathetic neuron differentiation. BMP-4 and BMP-7 are expressed in the dorsal aorta at critical times during sympathetic neuron differentiation and have the ability to enhance the formation of adrenergic sympathetic neurons both in cultures of neural crest cells and when ectopically expressed in the developing embryo.
Profilin is required for posterior patterning of the Drosophila oocyte
Authors: Manseau Lynn , Calley John , Phan Huy
Published: April 2021

We have investigated the role of the actin cytoskeleton during mid-oogenesis and have found that disrupting the actin cytoskeleton with cytochalasin D induces microtubule bundling and microtubule-based cytoplasmic streaming within the oocyte, similar to that which occurs prematurely in cappuccino and spire mutant oocytes. After examining a number of mutants that affect the actin cytoskeleton, we have found that chickadee, which encodes the actin-binding protein, profilin, shares this phenotype. In addition to the microtubule misregulation, mutants in chickadee resemble cappuccino in that they fail to localize STAUFEN and oskar mRNA to the posterior pole of the developing oocyte. Also, a strong allele of cappuccino has multinucleate nurse cells, similar to those previously described in chickadee. In an independent line of experiments, we have identified profilin as a CAPPUCCINO interactor in a two-hybrid screen for proteins that bind to CAPPUCCINO. This, together with the similarity of mutant phenotypes, suggests that profilin and CAPPUCCINO may interact during development.
Symmetry breakage in the development of one-armed gonads in nematodes
Authors: Félix Marie-Anne , Sternberg Paul W.
Published: April 2021

Whereas the hermaphrodite gonad of Caenorhabditis elegans has two symmetric arms (didelphy), the female/hermaphrodite gonad of many nematode species features a single anterior arm (monodelphy). We examined how gonadal cell lineages and intercellular signalling evolve to generate these diverse structures. In C. elegans, the two arms develop symmetrically from two somatic precursor cells, Z1 (anterior) and Z4 (posterior). Each first gives rise to one distal tip cell (which promotes arm growth and germ line proliferation), two ovary precursors and three uterine precursors in the center of the developing gonad. In monodelphic species, Z1 and Z4 have different fates. The first visible asymmetry between them is in the relative timing of their divisions, followed by asymmetric cell movements. The putative posterior distal tip cell is then eliminated in all but one species by programmed cell death. In some species the posterior ovary precursors form a small vestigial posterior arm, the post-vulval sac; in other species, they stay undivided, or die. In Cephalobus sp. PS1197, the specific fate of Z4 progeny is induced by Z1 (or its daughters). In the uterus in C. elegans, symmetric lateral signalling between Z1.ppp and Z4.aaa renders them equally likely to become the anchor cell, which links the uterus to the vulva. In the different monodelphic species, anchor cell specification is biased, or fully fixed, to a descendant of either Z1 or Z4. Replacement regulation upon anchor cell ablation is conserved in some species, but lost in others, leading to a mosaic-type development. Differentiation between Z1 and Z4 is thus manifested at this later stage in the breakage of symmetry of cell interactions in the ventral uterus.
Mad acts downstream of Dpp receptors, revealing a differential requirement for dpp signaling in initiation and propagation of morphogenesis in the Drosophila eye
Authors: Wiersdorff Volker , Lecuit Thomas , Cohen Stephen M. , Mlodzik Marek
Published: April 2021

Decapentaplegic (Dpp), a member of the TGF-β family of cytokines, has been implicated in many patterning processes in Drosophila, including the initial steps of pattern formation in the developing eye. We show that the Mothers against dpp (Mad) gene is required for dpp signaling during eye development. Clonal analysis demonstrates a cell-autonomous function for Mad and genetic interactions indicate that Mad is an essential component of the signal transduction pathway downstream of the Dpp receptors in responding cells. Mad-mediated dpp signaling is absolutely required for the initiation of the morphogenetic furrow in the eye, but has only a minor role in its subsequent propagation across the eye disc. We also present evidence for the repression of wingless transcription by dpp signaling.