bluuebunny/crossref_metadata_2025 huggingface dataset:
hedgehog, wingless and orthodenticle specify adult head development in Drosophila
Authors: Royet Julien , Finkelstein Robert
Published: April 2021

The adult head capsule of Drosophila forms primarily from the eye-antennal imaginal discs. Here, we demonstrate that the head primordium is patterned differently from the discs which give rise to the appendages. We show that the segment polarity genes hedgehog and wingless specify the identities of specific regions of the head capsule. During eye-antennal disc development, hedgehog and wingless expression initially overlap, but subsequently segregate.

This regional segregation is critical to head specification and is regulated by the orthodenticle homeobox gene. We also show that orthodenticle is a candidate hedgehog target gene during early eye-antennal disc development.
Cellular mechanisms of epiboly in leech embryos
Authors: Smith Constance M. , Lans Deborah , Weisblat David A.
Published: April 2021

Gastrulation in leech embryos is dominated by the epibolic movements of two tissues: germinal bands, composed of segmental precursor cells, and an overlying epithelium that is part of a provisional integument. During gastrulation, the germinal bands move over the surface of the embryo and coalesce along the prospective ventral midline. Concurrently, the epithelium spreads to cover the embryo. We have begun to analyze the mechanisms involved in gastrulation in the leech by assessing the independent contributions of the epithelium and the germinal bands to these cell movements. Here we describe cellular events during epiboly in normal embryos and in embryos perturbed by either reducing the number of cells in the epithelium, or by preventing the formation of the germinal bands, or both. These experiments indicate that both the germinal bands and the epithelium are able to undergo epibolic movements independently, although each is required for the other to behave as in control embryos.
Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene
Authors: Uyttendaele Hendrik , Marazzi Giovanna , Wu Guangyu , Yan Qingyou , Sassoon David , Kitajewski Jan
Published: April 2021

The int-3 oncogene was identified as a frequent target in Mouse Mammary Tumor Virus (MMTV)-induced mammary carcinomas and encodes the intracellular domain of a novel mouse Notch gene. To investigate the role of the int-3 proto-oncogene in mouse development and carcinogenesis, we isolated cDNA clones corresponding to the entire coding potential of the int-3 proto-oncogene. We propose to name this gene Notch4 and reserve the int-3 nomenclature for references to the oncogenic form. The deduced amino acid sequence of Notch4 contains conserved motifs found in Notch proteins; however Notch4 has fewer epidermal growth factor (EGF)-like repeats and a shorter intracellular domain than other mouse Notch homologues. Comparison of the coding potential of the int-3 gene to that of Notch4 suggests that loss of the extracellular domain of Notch4 leads to constitutive activation of this murine Notch protein. In situ hybridization revealed that Notch4 transcripts are primarily restricted to endothelial cells in embryonic and adult life. Truncated Notch4 transcripts were detected in post-meiotic male germ cells. The distinct Notch4 protein features and its restricted expression pattern suggests a specific role for Notch4 during development of vertebrate endothelium.
The tumor suppressor gene, lethal(2)giant larvae (l(2)gl), is required for cell shape change of epithelial cells during Drosophila development
Authors: Manfruelli P. , Arquier N. , Hanratty W. P. , Sémériva M.
Published: April 2021

Inactivation of the lethal(2)giant larvae (l(2)gl) gene results in malignant transformation of imaginal disc cells and neuroblasts of the larval brain in Drosophila. Subcellular localization of the l(2)gl gene product, P127, and its biochemical characterization have indicated that it participates in the formation of the cytoskeletal network. In this paper, genetic and phenotypic analyses of a temperaturesensitive mutation (l(2)glts3) that behaves as a hypomorphic allele at restrictive temperature are presented. In experimentally overaged larvae obtained by using mutants in the production of ecdysone, the l(2)glts3 mutation displays a tumorous potential.

This temperature-sensitive allele of the l(2)gl gene has been used to describe the primary function of the gene before tumor progression. A reduced contribution of both maternal and zygotic activities in l(2)glts3 homozygous mutant embryos blocks embryogenesis at the end of germband retraction. The mutant embryos are consequently affected in dorsal closure and head involution and show a hypertrophy of the midgut. These phenotypes are accompanied by an arrest of the cell shape changes normally occurring in lateral epidermis and in epithelial midgut cells. l(2)gl activity is also necessary for larval life and the critical period falls within the third instar larval stage. Finally, l(2)gl activity is required during oogenesis and mutations in the gene disorganize egg chambers and cause abnormalities in the shape of follicle cells, which are eventually internalized within the egg chamber. These results together with the tumoral phenotype of epithelial imaginal disc cells strongly suggest that the l(2)gl product is required in vivo in different types of epithelial cells to control their shape during development.
Central projections of persistent larval sensory neurons prefigure adult sensory pathways in the CNS of Drosophila
Authors: Shepherd David , Smith Simon A.
Published: April 2021

We have used a GAL4 enhancer-trap line driving the expression of a lacZ construct to examine the reorganisation of an identified group of proprioceptive sensory neurons during metamorphosis in Drosophila. The results show that whilst most larval sensory neurons degenerate during the first 24 hours of metamorphosis a segmentally repeated array of 6 neurons per segment persists into the adult stages to become functional adult neurons. These sensory neurons retain their axonal projections in the central nervous system intact and unchanged throughout. The adult sensory neuron axons enter the central nervous system at around 44 hours after puparium formation. Most of these axons grow along the pathways defined by the persistent larval sensory axons. The ordering of the adult sensory projections is, therefore, established upon the larval pattern of projections. The possibility that the larval neurons act as guidance cues for organising the ordered arrays of sensory neurons is discussed.
Roles of cell-autonomous mechanisms for differential expression of regionspecific transcription factors in neuroepithelial cells
Authors: Nakagawa Yasushi , Kaneko Tadahiro , Ogura Toshihiko , Suzuki Taiga , Torii Masaaki , Kaibuchi Kozo , Arai Ken-ichi , Nakamura Shun , Nakafuku Masato
Published: April 2021

Although a number of genes have been found to have restricted expression domains in the embryonic forebrain and midbrain, it remains largely unknown how the expression of these genes is regulated at the cellular level. In this study, we explored the mechanisms for the differential expression of region-specific transcription factors in neuroepithelial cells by using both primary and immortalized neuroepithelial cells from the rat brain at embryonic day 11.5. We found that differential expression patterns of Pax-3, Pax-5, Pax-6, Dlx-1, Dlx-2, Emx2, Otx1 and Dbx observed in vivo were maintained even when the cells were isolated and cultured in vitro, free from environmental influences. Furthermore, in response to Sonic hedgehog, which is a major inductive signal from the environment for regional specification, neuroepithelial cells that maintain distinct regional identities expressed different sets of ventral-specific genes including Islet-1, Nkx-2.1 and Nkx-2.2. These results suggest that certain cell-autonomous mechanisms play important roles in regulating both environmental signal-dependent and -independent expression of region-specific genes. Thus, we propose that use of the in vitro culture systems we describe in this study facilitates the understanding of regulatory mechanisms of region-specific genes in neuroepithelial cells.
Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio
Authors: Hammerschmidt Matthias , Pelegri Francisco , Mullins Mary C. , Kane Donald A. , Brand Michael , Eeden Fredericus J. M. van , Furutani-Seiki Makoto , Granato Michael , Haffter Pascal , Heisenberg Carl-Philipp , Jiang Yun-Jin , Kelsh Robert N. , Odenthal Jörg , Warga Rachel M. , Nüsslein-Volhard Christiane
Published: April 2021

We have identified several genes that are required for various morphogenetic processes during gastrulation and tail formation. Two genes are required in the anterior region of the body axis: one eyed pinhead (oep) and dirty nose (dns). oep mutant embryos are defective in prechordal plate formation and the specification of anterior and ventral structures of the central nervous system. In dns mutants, cells of the prechordal plate, such as the prospective hatching gland cells, fail to specify.

Two genes are required for convergence and extension movements. In mutant trilobite embryos, extension movements on the dorsal side of the embryo are affected, whereas in the formerly described spadetail mutants, for which two new alleles have been isolated, convergent movements of ventrolateral cells to the dorsal side are blocked.

Two genes are required for the development of the posterior end of the body axis. In pipetail mutants, the tailbud fails to move ventrally on the yolk sac after germ ring closure, and the tip of the tail fails to detach from the yolk tube. Mutants in kugelig (kgg) do not form the yolk tube at the posterior side of the yolk sac.
Genes involved in forebrain development in the zebrafish, Danio rerio
Authors: Heisenberg Carl-Philipp , Brand Michael , Jiang Yun-Jin , Warga Rachel M. , Beuchle Dirk , Eeden Fredericus J. M. van , Furutani-Seiki Makoto , Granato Michael , Haffter Pascal , Hammerschmidt Matthias , Kane Donald A. , Kelsh Robert N. , Mullins Mary C. , Odenthal Jörg , Nüsslein-Volhard Christiane
Published: April 2021

We identified four zebrafish mutants with defects in forebrain induction and patterning during embryogenesis. The four mutants define three genes: masterblind (mbl), silberblick (slb), and knollnase (kas). In mbl embryos, the anterior forebrain acquires posterior forebrain characteristics: anterior structures such as the eyes, olfactory placodes and the telencephalon are missing, whereas the epiphysis located in the posterior forebrain is expanded. In slb embryos, the extension of the embryonic axis is initially delayed and eventually followed by a partial fusion of the eyes. Finally, in kas embryos, separation of the telencephalic primordia is incomplete and dorsal midline cells fail to form a differentiated roof plate. Analysis of the mutant phenotypes indicates that we have identified genes essential for the specification of the anterior forebrain (mbl), positioning of the eyes (slb) and differentiation of the roof plate (kas).

In an appendix to this study we list mutants showing alterations in the size of the eyes and abnormal differentiation of the lenses.
Genetic dissection of the retinotectal projection
Authors: Baier Herwig , Klostermann Stefan , Trowe Torsten , Karlstrom Rolf O. , Nüsslein-Volhard Christiane , Bonhoeffer Friedrich
Published: April 2021

A systematic search for mutations affecting the retinotectal projection in zebrafish larvae was performed, as part of the large-scale Tübingen screen for homozygous diploid mutants in embryonic development. 2,746 inbred lines (F2 families) from males mutagenized with ethylnitroso urea were screened. In wild-type larvae, developing retinal axons travel along a stereotyped route to the contralateral optic tectum. Here, their terminals form a highly ordered retinotopic map. To detect deviations from this pattern, an axon tracing assay was developed that permits screening of large numbers of mutagenized fish. Two fluorescent tracer dyes (DiI and DiO) were injected at opposite poles of the eyes of day-5 aldehyde-fixed larvae. 12 hours later, retinal axons were labelled over their entire length, and could be observed through the intact skin. The assay procedure (aldehyde fixation, mounting, injection of dyes, microscopic analysis) took about 1 minute per fish. In total, 125,000 individual fish larvae were processed.

During the screen, 114 mutations in approx. 35 genes were discovered. For the mutants subjected to complementation testing, the number of alleles per locus ranges from 1 to 15. The mutations affect distinct steps in the retinotectal pathway, from pathfinding between eye and tectum to map formation along the dorsal-ventral and the anterior-posterior axis of the tectum. Mutations that disturb axon pathfinding to the tectum for the most part do not disrupt retinotopic mapping, and vice versa. The majority of the mutants display associated defects in other tissues and die before day 10. These mutants provide new tools for studying the formation of neuronal maps. The results of this screen show that a large-scale genetic approach can be applied to relatively late and circum-scribed developmental processes in the vertebrate brain.
Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae
Authors: Arnone Maria I. , Bogarad Leonard D. , Collazo Andres , Kirchhamer Carmen V. , Cameron R. Andrew , Rast Jonathan P. , Gregorians Armenia , Davidson Eric H.
Published: April 2021

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected.
PDP1, a novel Drosophila PAR domain bZIP transcription factor expressed in developing mesoderm, endoderm and ectoderm, is a transcriptional regulator of somatic muscle genes
Authors: Lin Shu-Chun , Lin Meei-Hua , Horváth Péter , Reddy Karen L. , Storti Robert V.
Published: April 2021

In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of MEF2 and the bHLH MyoD family of myogenic factors. We have shown that Drosophila MEF2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic muscles, however, unlike vertebrates, Drosophila MEF2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors or any other known Drosophila transcription factors. We describe here the isolation and characterization of a component of the muscle activator region that we have named PDP1 (PAR domain protein 1). PDP1 is a novel transcription factor that is highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. We show that PDP1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus MEF2 mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger protein/DNA complex that interacts with MEF2 to regulate transcription of Drosophila muscle genes. Furthermore, in addition to being expressed in the mesoderm that gives rise to the somatic muscles, PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also involved in the terminal differentiation of these tissues.
A role for the fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina
Authors: McFarlane S. , Zuber M.E. , Holt C.E.
Published: April 2021

The mature vertebrate retina contains seven major cell types that develop from an apparently homogenous population of precursor cells. Clonal analyses have suggested that environmental influences play a major role in specifying retinal cell identity. Fibroblast growth factor-2 is present in the developing retina and regulates the survival, proliferation and differentiation of developing retinal cells in culture. Here we have tested whether fibroblast growth factor receptor signaling biases retinal cell fate decisions in vivo. Fibroblast growth factor receptors were inhibited in retinal precursors in Xenopus embryos by expressing a dominant negative form of the receptor, XFD. Dorsal animal blastomeres that give rise to the retina were injected with cDNA expression constructs for XFD and a control non-functional mutant receptor, D48, and the cell fates of transgene-expressing cells in the mature retina determined. Fibroblast growth factor receptor blockade results in almost a 50% loss of photoreceptors and amacrine cells, and a concurrent 3.5-fold increase in Muller glia, suggesting a shift towards a Muller cell fate in the absence of a fibroblast growth factor receptor signal. Inhibition of non-fibroblast-growth-factor-mediated receptor signaling with a third mutant receptor, HAVO, alters cell fate in an opposite manner. These results suggest that it is the balance of fibroblast growth factor and non-fibroblast growth factor ligand signals that influences retinal cell genesis.
Bix1, a direct target of Xenopus T-box genes, causes formation of ventral mesoderm and endoderm
Authors: Tada M. , Casey E.S. , Fairclough L. , Smith J.C.
Published: April 2021

Brachyury, a member of the T-box gene family, is required for posterior mesoderm and notochord differentiation in vertebrate development, and mis-expression of Xenopus Brachyury causes ectopic mesoderm formation. Brachyury is a transcription activator, and its ability to activate transcription is essential for its biological function, but Brachyury target genes have proved difficult to identify. Here we employ a hormone-inducible Brachyury construct and subtractive hybridization to search for such targets. Using this approach we have isolated Bix1, a homeobox gene expressed both in the marginal zone of Xenopus and in the vegetal hemisphere. Expression of Bix1 is induced in an immediate-early fashion by mesoderm-inducing factors such as activin as well as by the products of the T-box genes Xbra and VegT (also known as Antipodean, Brat and Xombi). Activation of Bix1 in response to Xbra is direct in the sense that it does not require protein synthesis, and both Xbra and VegT activate expression of a reporter gene driven by the Bix 5′ regulatory region, which contains an Xbra/VegT binding site. Mis-expression of low levels of Bix1 causes formation of ventral mesoderm, while high levels induce endodermal differentiation. These results suggest that Bix1 acts downstream of both VegT and Xbra to induce formation of mesoderm and endoderm.
Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and notochord
Authors: Müller F. , Chang B.-E. , Albert S. , Fischer N. , Tora L. , Strähle U.
Published: April 2021

The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish. We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in intron 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse.

Functional analysis in the zebrafish embryo revealed that the intronic enhancers have a complex organisation. Two activator regions, ar-A and ar-C, were identified in intron 1 and 2, respectively, which mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three regions of 40 bp to be essential for activity. These regions do not contain binding sites for HNF3β? the winged helix transcription factor previously implicated in the regulation of shh expression? indicating the presence of novel regulatory mechanisms. A T-box transcription factor- binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord-deficient mutant embryos.
Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions
Authors: Eickholt Britta J. , Mackenzie Sarah L. , Graham Anthony , Walsh Frank S. , Doherty Patrick
Published: April 2021

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y., Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.
Direct regulatory interaction of the eyeless protein with an eye-specific enhancer in the sine oculis gene during eye induction in Drosophila
Authors: Niimi Teruyuki , Seimiya Makiko , Kloter Urs , Flister Susanne , Gehring Walter J.
Published: April 2021

The Pax-6 gene encodes a transcription factor with two DNA-binding domains, a paired and a homeodomain, and is expressed during eye morphogenesis and development of the nervous system. Pax-6 homologs have been isolated from a wide variety of organisms ranging from flatworms to humans. Since loss-of-function mutants in insects and mammals lead to an eyeless phenotype and Pax-6 orthologs from distantly related species are capable of inducing ectopic eyes in Drosophila, we have proposed that Pax-6 is a universal master control gene for eye morphogenesis. To determine the extent of evolutionary conservation of the eye morphogenetic pathway, we have begun to identify subordinate target genes of Pax-6. Previously we have shown that expression of two genes, sine oculis (so) and eyes absent (eya), is induced by eyeless (ey), the Pax-6 homolog of Drosophila. Here we present evidence from ectopic expression studies in transgenic flies, from transcription activation studies in yeast, and from gel shift assays in vitro that the EY protein activates transcription of sine oculis by direct interaction with an eye-specific enhancer in the long intron of the so gene.
β-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion
Authors: Zhu Alan Jian , Watt Fiona M.
Published: April 2021

We found that cultured human keratinocytes with high proliferative potential, the putative epidermal stem cells, expressed a higher level of noncadherin-associated β- catenin than populations enriched for keratinocytes of lower proliferative potential. To investigate the physiological significance of this, a series of β-catenin constructs was introduced into keratinocytes via retroviral infection. Full-length β-catenin and a mutant containing only nine armadillo repeats had little effect on proliferative potential in culture, the full-length protein being rapidly degraded. However, expression of stabilised, N-terminally truncated β-catenin increased the proportion of putative stem cells to almost 90% of the proliferative population in vitro without inducing malignant transformation, and relieved the differentiation stimulatory effect of overexpressing the E-cadherin cytoplasmic domain. Conversely, β-catenin lacking armadillo repeats acted as a dominant negative mutant and stimulated exit from the stem cell compartment in culture. The positive and negative effects of the β-catenin mutants on proliferative potential were independent of effects on cell-cycle kinetics, overt terminal differentiation or intercellular adhesion, and correlated with stimulation or inhibition of transactivation of a TCF/LEF reporter in basal keratinocytes. We conclude that the elevated level of cytoplasmic β-catenin in those keratinocytes with characteristics of epidermal stem cells contributes to their high proliferative potential.
Drosophila embryonic pattern repair: how embryos respond to cyclin E-induced ectopic division
Authors: Li Qian-Jun , Pazdera Todd M. , Minden Jonathan S.
Published: April 2021

The Drosophila melanogaster embryo ordinarily undergoes thirteen cycles of rapid syncytial division followed by three rounds of cellular division for most cells. Strict regulation of the number of divisions is believed to be essential for normal patterning and development. To determine how the embryo responds to hyperplastic growth, we have examined epidermal development in embryos that experience additional rounds of mitosis as the result of ectopic Cyclin E expression. We observed that the cell density in the epidermis nearly doubled within 1 hour of Cyclin E induction. The spacing and width of the ENGRAILED and wingless stripes was unchanged, but the cell density within the stripes was increased. By 4 hours after Cyclin E induction, the cell density had returned to almost normal values. The embryos developed, albeit more slowly, to produce viable larvae and adults. The excess cells were removed by apoptosis in a reaper-dependent fashion as evidenced by increased reaper expression. Embryos lacking cell death in the abdomen exhibited changes in ENGRAILED expression. In addition, germband retraction and dorsal closure were slower than normal. Ectopic Cyclin E expression in cell-death-deficient embryos exacerbated the germband retraction and ENGRAILED-expression defects.
Opposing roles for neurotrophin-3 in targeting and collateral formation of distinct sets of developing cortical neurons
Authors: Castellani Valérie , Bolz Jürgen
Published: April 2021

Neurotrophin-3 and its receptor TrkC are expressed during the development of the mammalian cerebral cortex. To examine whether neurotrophin-3 might play a role in the elaboration of layer-specific cortical circuits, slices of layer 6 and layers 2/3 neurons were cultured in the presence of exogenously applied neurotrophin-3. Results indicate that neurotrophin-3 promotes axonal branching of layer 6 axons, which target neurotrophin-3-expressing layers in vivo, and that it inhibits branching of layers 2/3 axons, which avoid neurotrophin-3-expressing layers. Such opposing effects of neurotrophin-3 on axonal branching were also observed with embryonic cortical neurons, indicating that the response to neurotrophin-3 is specified at early developmental stages, prior to cell migration. In addition to its effects on fiber branching, axonal guidance assays also indicate that neurotrophin-3 is an attractive signal for layer 6 axons and a repellent guidance cue for layers 2/3 axons. Experiments with specific antibodies to neutralize neurotrophin-3 in cortical membranes revealed that endogenous levels of neurotrophin-3 are sufficient to regulate branching and targeting of cortical axons. These opposing effects of neurotrophin-3 on specific populations of axons demonstrate that it could serve as one of the signals for the elaboration of local cortical circuits.
Inductive regulation of cell fusion in leech
Authors: Isaksen Deborah E. , Liu Nai-Jia L. , Weisblat David A.
Published: April 2021

Cell-cell fusion is a component of many different developmental processes, but little is known about how cell- cell fusion is regulated. Here we investigate the regulation of a stereotyped cell-cell fusion event that occurs among the endodermal precursor cells of the glossiphoniid leech Helobdella robusta. We find that this fusion event is regulated inductively by a cell that does not itself fuse. We also show that biochemical arrest (by microinjection with ricin A chain or ribonuclease A) of the inducer or either of the fusion partners prevents fusion, but only if the arrest is initiated during a critical period long before the time at which fusion normally occurs. If the arrest occurs after this critical period, fusion occurs on schedule. These results suggest that both fusion partners play active roles in the process and that neither the induction nor the fusion itself requires concomitant protein synthesis.
The C. elegans gene lin-36 acts cell autonomously in the lin-35 Rb pathway
Authors: Thomas Jeffrey H. , Horvitz H. Robert
Published: April 2021

The Caenorhabditis elegans gene lin-36 acts to antagonize Ras-mediated vulval induction in a pathway that includes genes with products similar to the mammalian retinoblastoma (Rb) protein and the Rb-binding protein p48. We report that lin-36 encodes a novel protein of 962 amino acids. We demonstrate that lin-36 functions in and is expressed in the vulval precursor cells, establishing that the lin-36 pathway is involved in intercellular signaling. We also report that the lin-36 pathway and/or another pathway that is functionally redundant with the lin-36 pathway antagonize a ligand-independent activity of the receptor tyrosine kinase/Ras vulval induction pathway.
An SCL 3’ enhancer targets developing endothelium together with embryonic and adult haematopoietic progenitors
Authors: Sanchez M.-J. , Gottgens B. , Sinclair A. M. , Stanley M. , Begley C. G. , Hunter S. , Green A. R.
Published: April 2021

The SCL gene encodes a basic helix-loop-helix transcription factor which is expressed in early haematopoietic progenitors throughout ontogeny and is essential for the normal development of blood and blood vessels. Transgenic studies have characterised spatially distinct 5’ enhancers which direct lacZ expression to subdomains of the normal SCL expression pattern, but the same elements failed to produce appropriate haematopoietic expression. We now describe an SCL 3’ enhancer with unique properties. It directed lacZ expression in transgenic mice to extra-embryonic mesoderm and subsequently to both endothelial cells and to a subset of blood cells at multiple sites of embryonic haematopoiesis including the yolk sac, para-aortic splanchnopleura and AGM region. The 3’ enhancer also targeted expression to haematopoietic progenitors in both foetal liver and adult bone marrow. Purified lacZ + cells were highly enriched for clonogenic myeloid and erythroid progenitors as well as day-12 spleen colony forming units (CFU-S). Within the total gated population from bone marrow, 95% of the myeloid and 90% of the erythroid colony-forming cells were contained in the lacZ+ fraction, as were 98% of the CFU-S. Activation of the enhancer did not require SCL protein. On the contrary, transgene expression in yolk sacs was markedly increased in an SCL_/“ background, suggesting that SCL is subject to negative autoregulation. Alternatively the SCL∼I_ environment may alter differentiation of extra-embryonic mesoderm and result in an increased number of cells capable of expressing high levels of the transgene. Our data represents the first description of an enhancer that integrates information necessary for expression in developing endothelium and early haematopoietic progenitors at distinct times and sites throughout ontogeny. This enhancer provides a potent tool for the manipulation of haematopoiesis and vasculogenesis in vivo.
The DNA-binding Polycomb group protein Pleiohomeotic mediates silencing of a Drosophila homeotic gene
Authors: Fritsch Cornelia , Lesley Brown J. , Kassis Judith A. , Müller Jürg
Published: April 2021

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements. Currently, these silencers are ill-defined sequences and it is not known how PcG proteins associate with DNA. Here, we show that the Drosophila PcG protein Pleiohomeotic binds to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers.
Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling
Authors: Sasaki Hiroshi , Nishizaki Yuriko , Hui Chi-chung , Nakafuku Masato , Kondoh Hisato
Published: April 2021

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C- terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3β, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.
The Spineless-Aristapedia and Tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila
Authors: Emmons Richard B. , Duncan Dianne , Estes Patricia A. , Kiefel Paula , Mosher Jack T. , Sonnenfeld Margaret , Ward Mary P. , Duncan Ian , Crews Stephen T.
Published: April 2021

The Drosophila spineless (ss) gene encodes a basic-helix- loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH- PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss− mutations. The results of yeast two- hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus. Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved.
A head-activator binding protein is present in hydra in a soluble and a membrane-anchored form
Authors: Hampe Wolfgang , Urny Jens , Franke Inga , Hoffmeister-Ullerich Sabine A. H. , Herrmann Doris , Petersen Claus M. , Lohmann Jan , Schaller H. Chica
Published: April 2021

The neuropeptide head activator plays an important role for proliferation and determination of stem cells in hydra. By affinity chromatography a 200 kDa head-activator binding protein, HAB, was isolated from the multiheaded mutant of Chlorohydra viridissima. Partial amino acid sequences were used to clone the HAB cDNA which coded for a receptor with a unique alignment of extracellular modules, a transmembrane domain, and a short carboxy-terminal cytoplasmic tail. A mammalian HAB homologue with identical alignment of these modules is expressed early in brain development. Specific antibodies revealed the presence of HAB in hydra as a transmembrane receptor, but also as secreted protein, both capable of binding head activator. Secretion of HAB during regeneration and expression in regions of high determination potential hint at a role for HAB in regulating the concentration and range of action of head activator.
Frizzled and DFrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development
Authors: Bhanot Purnima , Fish Matthew , Jemison Jamileh A. , Nusse Roel , Nathans Jeremy , Cadigan Ken M.
Published: April 2021

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.
Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis
Authors: Kuo Hai-Chien , Chen Ju , Ruiz-Lozano Pilar , Zou Yimin , Nemer Mona , Chien Kenneth R.
Published: April 2021

Although accumulating evidence suggests that the heart develops in a segmental fashion, the molecular mechanisms that control regional specification of cardiomyocytes in the developing heart remain largely unknown. In this study, we have used the mouse cardiac-restricted ankyrin repeat protein (CARP) gene as a model system to study these mechanisms. The CARP gene encodes a nuclear co-regulator for cardiac gene expression, which lies downstream of the cardiac homeobox gene, Nkx 2.5, and is an early marker of the cardiac muscle cell lineage. We have demonstrated that the expression of the gene is developmentally down regulated and dramatically induced as part of the embryonic gene program during cardiac hypertrophy. Using a lacZ/knock-in mouse and three lines of transgenic mouse harboring various CARP promoter/lacZ reporters, we have identified distinct 5′ cis regulatory elements of the gene that can direct heart segment-specific transgene expression, such as atrial versus ventricular and left versus right. Most interestingly, a 213 base pair sequence element of the gene was found to confer conotruncal segment-specific transgene expression. Using the transgene as a conotruncal segment-specific marker, we were able to document the developmental fate of a subset of cardiomyocytes in the conotruncus during cardiogenesis. In addition, we have identified an essential GATA-4 binding site in the proximal upstream regulatory region of the gene and cooperative transcriptional regulation mediated by Nkx2.5 and GATA-4. We have shown that this cooperative regulation is dependent on binding of GATA-4 to its cognate DNA sequence in the promoter, which suggests that Nkx2.5 controls CARP expression, at least in part, through GATA-4.
Inhibition of TGF-β receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass
Authors: Filvaroff Ellen , Erlebacher Adrian , Ye Jian-Qin , Gitelman Stephen E. , Lotz Jeffrey , Heillman Moira , Derynck Rik
Published: April 2021

Transforming growth factor-β (TGF-β) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-β in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-β in transgenic mice by expressing a cytoplasmically truncated type II TGF-β receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-β signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-β acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.
The Drosophila dead ringer gene is required for early embryonic patterning through regulation of argos and buttonhead expression
Authors: Shandala Tatiana , Kortschak R. Daniel , Gregory Stephen , Saint Robert
Published: April 2021

The dead ringer (dri) gene of Drosophila melanogaster is a member of the recently discovered ARID-box family of eukaryotic genes that encode proteins with a conserved DNA binding domain. dri itself is highly conserved, with specific orthologs in the human, mouse, zebrafish and C. elegans genomes. We have generated dri mutant alleles to show that dri is essential for anterior-posterior patterning and for muscle development in the embryo. Consistent with the mutant phenotype and the sequence-specific DNA-binding properties of its product, dri was found to be essential for the normal early embryonic expression pattern of several key regulatory genes. In dri mutant embryos, expression of argos in the terminal domains was severely reduced, accounting for the dri mutant head phenotype. Conversely, buttonhead expression was found to be deregulated in the trunk region, accounting for the appearance of ectopic cephalic furrows. Curiously, dri was found also to be required for maintenance of expression of the ventrolateral region of even-skipped stripe four. This study establishes dri as an essential co-factor in the regulated expression of specific patterning genes during early embryogenesis.