bluuebunny/crossref_metadata_2025 huggingface dataset:
GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene
Authors: Long Qiaoming , Meng Anming , Wang Han , Jessen Jason R. , Farrell Michael J. , Lin Shuo
Published: April 2021

In this study, DNA constructs containing the putative zebrafish promoter sequences of GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos. Erythroid-specific activity of the GATA-1 promoter was observed in living embryos during early development. Fluorescent circulating blood cells were detected in microinjected embryos 24 hours after fertilization and were still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continued to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish were consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies. By generating transgenic fish using constructs containing other zebrafish promoters and green fluorescent protein reporter gene, it should be possible to visualize the origin and migration of any lineage-specific progenitor cells in a living embryo.
Sperm from β1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly
Authors: Lu Qingxian , Shur Barry D.
Published: April 2021

A variety of sperm surface components have been suggested to mediate gamete recognition by binding to glycoside ligands on the egg coat glycoprotein ZP3. The function of each of these candidate receptors is based upon varying degrees of circumstantial and direct evidence; however, the effects on fertilization of targeted mutations in any of these candidate receptors have not yet been reported. In this paper, we describe the effects of targeted mutations in β1,4-galactosyltransferase, the best studied of the candidate receptors for ZP3. Surprisingly, galactosyl-transferase-null (gt−/−) males are fertile; however, sperm from gt−/− males bind less radiolabeled ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either ZP3 or anti-galactosyltransferase anti-bodies, as do wild-type sperm. In contrast, gt−/− sperm undergo the acrosome reaction normally in response to calcium ionophore, which bypasses the requirement for ZP3 binding. The inability of gt−/− sperm to undergo a ZP3-induced acrosome reaction renders them physiologically inferior to wild-type sperm, as assayed by their relative inability to penetrate the egg coat and fertilize the oocyte in vitro. Thus, although ZP3 binding and subsequent induction of the acrosome reaction are dispensable for fer-tilization, they impart a physiological advantage to the fer-tilizing sperm. A second strain of mice was created that is characterized by a loss of of the long galactosyltransferase isoform responsible for ZP3-dependent signal transduc-tion, but which maintains normal levels of Golgi galacto-sylation. Sperm from these mice show that the defective sperm-egg interactions in gt−/− mice are due directly to a loss of the long galactosyltransferase isoform from the sperm surface and are independent of the state of intracel-lular galactosylation during spermatogenesis.
meander tail acts intrinsic to granule cell precursors to disrupt cerebellar development: analysis of meander tail chimeric mice
Authors: Hamre Kristin M , Goldowitz Dan
Published: April 2021

The murine mutation meander tail (gene symbol: mea) causes a near-total depletion of granule cells in the anterior lobe of the cerebellum, as well as aberrantly located Purkinje cells with misoriented dendrites and radial glia with stunted processes. Whether one, two or all three of these cell types is the primary cellular target(s) of the mutant gene is unknown. This issue is addressed by examining cerebella from adult chimeras in which both the genotype and phenotype of individual cells are marked and examined. From this analysis, three novel observations are made. First, genotypically mea/mea Purkinje cells and glial cells exhibit normal morphologies in the cerebella of chimeric mice indicating that the mea gene acts extrinsically to these two cell populations. Second, few genotypically mea/mea granule cells are present in the anterior lobe or, unexpectedly, in the posterior lobe. These findings indicate that the mea gene acts intrinsically to the granule cell or its precursors to perturb their development. Third, there are near-normal numbers of cerebellar granule cells in the chimeric cerebellum. This result suggests that mea/mea cells are out-competed and subsequently replaced by an increased cohort of wild-type granule cells resulting from an upregulation of wild-type granule cells in the chimeric environment. We propose that the wild-type allele of the mea gene is critical for the developmental progression of the early granule cell neuroblast.
muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins
Authors: Begemann Gerrit , Paricio Nuria , Artero Ruben , Kiss Istvan , Pérez-Alonso Manuel , Mlodzik Marek
Published: April 2021

We have isolated the embryonic lethal gene muscleblind (mbl) as a suppressor of the sev-svp2 eye phenotype. Analysis of clones mutant for mbl during eye development shows that it is autonomously required for photoreceptor differentiation. Mutant cells are recruited into developing ommatidia and initiate neural differentiation, but they fail to properly differentiate as photoreceptors. Molecular analysis reveals that the mbl locus is large and complex, giving rise to multiple different proteins with common 5′ sequences but different carboxy termini. Mbl proteins are nuclear and share a Cys 3His zinc-finger motif which is also found in the TIS11/NUP475/TTP family of proteins and is highly conserved in vertebrates and invertebrates. Functional analysis of mbl, the observation that it also dominantly suppresses the sE-Jun Asp gain-of-function phenotype and the phenotypic similarity to mutants in the photoreceptor-specific glass gene suggest that mbl is a general factor required for photoreceptor differentiation.
Only a subset of the binary cell fate decisions mediated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless
Authors: Wang Shuwen , Younger-Shepherd Susan , Jan Lily Y. , Jan Yuh Nung
Published: April 2021

In Drosophila, an adult external sensory organ (bristle) consists of four distinct cells which arise from a sensory organ precursor cell via two rounds of asymmetric divisions. The sensory organ precursor cell first divides to generate two secondary precursor cells, IIa and IIb. The IIa cell then divides to produce the hair cell and the socket cell. Shortly after, the IIb cell divides to generate the neuron and the sheath cell. The membrane-associated protein Numb has been shown to be required for the first two asymmetric divisions. We now report that a new hypo- morphic numb mutant not only displays a double-socket phenotype, due to a hair cell to socket cell transformation, but also a double-sheath phenotype, due to a neuron to sheath cell transformation. This provides direct evidence that numb functions in the neuron/sheath cell lineage as well. Those results, together with our observation from immunofluorescence analysis that Numb forms a crescent in the dividing IIa and IIb cells suggest that asymmetric localization of Numb is important for the cell fate determination in all three asymmetric cell divisions in the sensory organ lineage. Interestingly, we found that in the hair/socket cell lineage but not the neuron/sheath cell lineage, a Suppressor of Hairless mutation acts as a dominant suppressor of numb mutations whereas Hairless mutations act as enhancers of numb. Moreover, epistasis analysis indicates that Suppressor of Hairless acts down- stream of numb, and results from in vitro binding analysis suggest that the genetic interaction between numb and Hairless may occur through direct protein-protein interaction. These studies reveal that Suppressor of Hairless is required for only a subset of the asymmetric divisions that depend on the function of numb and Notch.
Fkh5-deficient mice show dysgenesis in the caudal midbrain and hypothalamic mammillary body
Authors: Wehr Roland , Mansouri Ahmed , Maeyer Tine de , Gruss Peter
Published: April 2021

The murine winged helix gene Fkh5 is specifically expressed in the developing central nervous system (CNS). Early embryonic Fkh5 expression is restricted to the mammillary body region of the caudal hypothalamus, midbrain, hindbrain and spinal cord. Postnatally, signals persist in specific nuclei of the mammillary body and in the midbrain. We generated Fkh5 deficient mice by homolo- gous recombination to assess its in vivo function. At birth, Fkh5-deficient mice are viable and indistinguishable from wild –type and Fkh5 heterozygous littermates. However, about one third die within the first two days and another fifth before weaning. Surviving Fkh5-deficient mice become growth retarded within the first week and remain smaller throughout their whole life span. Fkh5-deficient females on 129Sv ×C57BL/6 genetic background are fertile, but do not nurture their pups. More detailed analysis of Fkh5- deficient brains reveals distinct alterations in the CNS. In the midbrain, mutant mice exhibit reduced inferior colliculi and an overgrown anterior cerebellum. Further- more, the hypothalamic mammillary body of Fkh5- deficient brains lacks the medial mammillary nucleus. These results suggest that Fkh5 plays a major role during CNS development.
The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning
Authors: Kishimoto Yasuyuki , Lee Kyu-Ho , Zon Len , Hammerschmidt Matthias , Schulte-Merker Stefan
Published: April 2021

Early dorsoventral pattern formation in vertebrate embryos is regulated by opposing activities of ventralizing bone morphogenetic proteins (BMPs) and dorsal-specific BMP antagonists such as Chordin, Noggin and Follistatin. Specific defects in early dorsoventral patterning have been recently found in a number of zebrafish mutants, which exhibit either a ventralized or dorsalized phenotype. One of these, the ventralized mutant chordino (originally called dino) is caused by a mutation in the zebrafish chordin homologue and interacts genetically with the dorsalized mutant swirl. In swirl mutant embryos, dorsal structures such as notochord and somites are expanded while ventral structures such as blood and nephros are missing. Here we demonstrate that the swirl phenotype is caused by mutations in the zebrafish bmp2 gene (zbmp2). While injection of mRNAs encoded by the mutant alleles has no ventralizing effect, injection of wild-type zbmp2 mRNA leads to a complete rescue of the swirl mutant phenotype. Fertile adult mutant fish were obtained, showing that development after gastrulation is not dependent on zbmp2 function. In addition zBMP2 has no maternal role in mesoderm induction. Our analysis shows that swirl/BMP2, unlike mouse BMP2 but like mouse BMP4, is required for early dorsoventral patterning of the zebrafish embryo.
Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize BMP4 in avian somite patterning
Authors: Hirsinger Estelle , Duprez Delphine , Jouve Caroline , Malapert Pascale , Cooke Jonathan , Pourquié Olivier
Published: April 2021

In the vertebrate embryo, the lateral compartment of the somite gives rise to muscles of the limb and body wall and is patterned in response to lateral-plate-derived BMP4. Activation of the myogenic program distinctive to the medial somite, i.e. relatively immediate development of the epaxial muscle lineage, requires neutralization of this lateral signal. We have analyzed the properties of molecules likely to play a role in opposing lateral somite specification by BMP4. We propose that the BMP4 antagonist Noggin plays an important role in promoting medial somite patterning in vivo. We demonstrate that Noggin expression in the somite is under the control of a neural-tube-derived factor, whose effect can be mimicked experimentally by Wnt1. Wnt1 is appropriately expressed in the neural tube. Furthermore, we show that Sonic Hedgehog is able to activate ectopic expression of Noggin resulting in the blocking of BMP4 specification of the lateral somite. Our results are consistent with a model in which Noggin activation lies downstream of the SHH and Wnt signaling pathways.
maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA
Authors: Clegg Nigel J. , Frost Deanna M. , Larkin Michele Keller , Subrahmanyan Lakshman , Bryant Zev , Ruohola-Baker Hannele
Published: April 2021

We describe a mutant, maelstrom, that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. Posterior localization of mRNA in stage 3-6 oocytes could therefore be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein that has a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis.
A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing
Authors: Strigini Maura , Cohen Stephen M.
Published: April 2021

The secreted protein Hedgehog (Hh) transmits a signal from posterior to anterior cells that is essential for limb development in insects and vertebrates. In Drosophila, Hh has been thought to act primarily to induce localized expression of Decapentaplegic and Wingless which in turn relay patterning cues at long range. We report here that Hh plays an additional role in patterning the wing. By replacing endogenous Hh activity with that of a membranetethered form of Hh, we show that Hh acts directly to pattern the central region of the wing, in addition to its role as an inducer of Dpp. Comparing the biological activities of secreted and membrane-tethered Hh provides evidence that Hh forms a local concentration gradient and functions as a concentration-dependent morphogen in the fly wing.
Interactions between the EGF receptor and DPP pathways establish distinct cell fates in the tracheal placodes
Authors: Wappner Pablo , Gabay Limor , Shilo Ben-Zion
Published: April 2021

The formation of the tracheal network in Drosophila is driven by stereotyped migration of cells from the tracheal pits. No cell divisions take place during tracheal migration and the number of cells in each branch is fixed. This work examines the basis for the determination of tracheal branch fates, prior to the onset of migration. We show that the EGF receptor pathway is activated by localized processing of the ligand SPITZ in the tracheal placodes and is responsible for the capacity to form the dorsal trunk and visceral branch. The DPP pathway, on the contrary, is induced in the tracheal pit by local presentation of DPP from the adjacent dorsal and ventral ectodermal cells. This pathway patterns the dorsal and lateral branches. Elimination of both pathways blocks migration of all tracheal branches. Antagonistic interactions between the two pathways are demonstrated. The opposing activities of two pathways may refine the final determination of tracheal branch fates.
Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-α in vitro
Authors: Pampfer Serge , Vanderheyden Ivo , McCracken Jane E. , Vesela Jarmila , Hertogh René De
Published: April 2021

The morphogenetic function of the transient phase of cell death that occurs during blastocyst maturation is not known but it is thought that its regulation results from a delicate balance between survival and lethal signals in the uterine milieu. In this paper, we show that blastocysts from diabetic rats have a higher incidence of dead cells than control embryos. Differential lineage staining indicated that increased nuclear fragmentation occurred mainly in the inner cell mass. In addition, terminal transferase-mediated dUTP nick end labeling (TUNEL) demonstrated an increase in the incidence of non-fragmented DNA-damaged nuclei in these blastocysts. Analysis of the expression of clusterin, a gene associated with apoptosis, by quantitative reverse transcription-polymerase chain reaction detected an increase in the steady-state level of its transcripts in blastocysts from diabetic rats. In situ hybridization revealed that about half the cells identified as expressing clusterin mRNA exhibited signs of nuclear fragmentation. In vitro experiments demonstrated that high D-glucose increased nuclear fragmentation, TUNEL labeling and clusterin transcription. Tumor necrosis factor-α(TNFα), a cytokine whose synthesis is up-regulated in the diabetic uterus, did not induce nuclear fragmentation nor clusterin expression but increased the incidence of TUNEL-positive nuclei. The data suggest that excessive cell death in the blastocyst, most probably resulting from the overstimulation of a basal suicidal program by such inducers as glucose and TNF-α, may be a contributing factor of the early embryopathy associated with maternal diabetes.
Segregation of myogenic lineages in Drosophila requires Numb
Authors: Gómez Mar Ruiz , Bate Michael
Published: April 2021

Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that found larval muscles or form precursors of adult muscles. Alternative fates adopted by sibling myoblasts are associated with distinct patterns of gene expression. Genes expressed in the progenitor cell are maintained in one sibling and repressed in the other. These differences depend on an asymmetric segregation of Numb between sibling cells. In numb mutants, muscle fates associated with repression are duplicated and alternative muscles are lost. If numb is overexpressed the reverse transformation occurs. Numb acts to block Notch-mediated repression of genes expressed in muscle progenitor cells. Thus asymmetric cell divisions are essential determinants of muscle fates during myogenesis in Drosophila
Chitin oligosaccharides can induce cortical cell division in roots of Vicia sativa when delivered by ballistic microtargeting
Authors: Schlaman Helmi R. M. , Gisel Andreas A. , Quaedvlieg Nicolette E. M. , Bloemberg Guido V. , Lugtenberg Ben J. J. , Kijne Jan W. , Potrykus Ingo , Spaink Herman P. , Sautter Christof
Published: April 2021

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-chitin oligosaccharide (LCO) signal molecules that can induce nodule organogenesis in the cortex of legume roots in a host-specific way. The multi-unsaturated fatty acyl and the O-acetyl moieties of the LCOs of Rhizobium leguminosarum biovar viciae were shown to be essential for obtaining root nodule induction in Vicia sativa plants. We have used ballistic microtargeting as a novel approach to deliver derivatives of the nodulation signal molecules inside the roots of V. sativa. This method offers the unique ability to introduce soluble compounds into the tissue at a small area. The mitogenic effect of microtargeting of chitin oligosaccharides, including an analysis of the influence of the chain length and modifications, was tested in a qualitative assay. The role of a cell division factor from the root stele, uridine, has also been examined in these experiments. The results show that O-acetylated chitin oligosaccharides can induce root cortical cell divisions when delivered by microtargeting. For this effect it is essential that uridine is co-targeted. The foci of cortical cell division were often similar to root nodule primordia. Anatomical examination also revealed chimeric structures that share characteristics with lateral root and nodule primordia. Our data favour a model in which the oligosaccharide moiety of the rhizobial LCO induces cortical cell division and the fatty acyl moiety plays a role in transport of the LCO into the plant tissue.
Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination
Authors: Hager Jeffrey H. , Cline Thomas W.
Published: April 2021

With a focus on Sex-lethal (Sx1), the master regulator of Drosophila somatic sex determination, we compare the sex determination mechanism that operates in the germline with that in the soma. In both cell types, Sx1 is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sx1 transcription. Once initiated, the active state of Sx1 is maintained by a positive autoregulatory feedback loop in which SXL protein insures its continued synthesis by binding to Sx1 pre-mRNA and thereby imposing the productive (female) splicing mode. The gene splicing-necessary factor (snf), which encodes a component of U1 and U2 snRNPs, participates in this RNA splicing control. Here we show that an increase in the dose of snf+ can trigger the female Sx1 RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sx1 in the soma and qualify snf as a numerator element of the X:A signal for Sx1 in the germline. We also show that female-specific regulation of Sx1 in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chro-mosome dose in the germline may facilitate it. Engagement of the Sx1 splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sx1 feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sx1 is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sx1, snf, ovo and otu which control female-specific processes in the ovary.
A role for netrin-1 in the guidance of cortical efferents
Authors: Métin Christine , Deléglise Delphine , Serafini Tito , Kennedy Timothy E. , Tessier-Lavigne Marc
Published: April 2021

An intermediate target for axons leaving the cerebral cortex in embryonic mammals is the ganglionic eminence (GE), the embryonic precursor of the basal ganglia. The cues that direct these axons over the initial portion of their trajectory are not well understood, but could include both short-range and long-range attractants and repellents. In the present study, we provide evidence that corticofugal axons might be guided at least partly by a diffusible factor or factors originating in the lateral GE and the sulcus between the lateral and medial ridges of the GE (ISS), as well as evidence implicating the axonal chemoattractant netrin-1 in mediating these effects.

Explants of lateral GE and ISS obtained from E12.5 and E13.5 mouse forebrain have a strong effect on both the outgrowth and orientation of corticofugal axons when cultured at a distance with explants of embryonic cortex in collagen gels. Netrin-1 mRNA is detected in these target tissues by in situ hybridization, and both netrin-1 protein and heterologous cells secreting netrin-1 can mimic the outgrowth-promoting effect of these target tissues in vitro. Furthermore, the growth of corticofugal axons is oriented toward an ectopic source of netrin-1 in vitro, and a function blocking anti-netrin-1 antiserum specifically abolishes the cortical axon outgrowth elicited by explants of lateral GE and the ISS in collagen gel cocultures.

Taken together, these results suggest a role for netrin-1 in the attraction at a distance of early cortical axons by the GE. Thus in mammals – as is also observed in nematodes – the development of non-commissural projections in anterior regions of the embryo might be directed by mechanisms similar to those involved in directing the development of commissural projections in more posterior regions of the central nervous system.
Positive and negative DNA sequence elements are required to establish the pattern of Pax3 expression
Authors: Natoli Thomas A. , Ellsworth Mary Kay , Wu Chuanzhen , Gross Kenneth W. , Pruitt Steven C.
Published: April 2021

The transcription start site and DNA sequence elements required for the induction of Pax3 expression in differentiating P19 embryonal carcinoma cells have been localized. These elements consist of a promoter and additional elements located within 1.6 kbp 5′ to the transcription start site. Sequence elements within this 1.6 kbp region are also sufficient to mediate the induction and dorsal restriction of Pax3 in the neural tube and somites of transgenic mice throughout the hindbrain and trunk. Additional elements required for expression anterior to the hindbrain and in migrating myoblasts are located within 14 kbp 5′ to the transcription start site. This region also contains element(s) that repress Pax3 expression in the ventral body wall mesoderm of the tail bud.
The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis
Authors: Wettstein Daniel A. , Turner David L. , Kintner Chris
Published: April 2021

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.
A genetic pathway for regulation of tra-2 translation
Authors: Goodwin Elizabeth B. , Hofstra Karen , Hurney Carol A. , Mango Susan , Kimble Judith
Published: April 2021

In Caenorhabditis elegans, the tra-2 sex-determining gene is regulated at the translational level by two 28 nt direct repeat elements (DREs) located in its 3′ untranslated region (3′UTR). DRF is a factor that binds the DREs and may be a trans-acting translational regulator of tra-2. Here we identify two genes that are required for the normal pattern of translational control. A newly identified gene, called laf-1, is required for translational repression by the tra-2 3′UTR. In addition, the sex-determining gene, tra-3, appears to promote female development by freeing tra-2 from laf-1 repression. Finally, we show that DRF activity correlates with translational repression of tra-2 during development and that tra-3 regulates DRF activity. We suggest that tra-3 may promote female development by releasing tra-2 from translation repression by laf-1 and that translational control is important for proper sex determination – both in the early embryo and during postembryonic development.
Conservation of Pax-6 in a lower chordate, the ascidian Phallusia mammillata
Authors: Glardon Sacha , Callaerts Patrick , Halder Georg , Gehring Walter J.
Published: April 2021

The Pax-6 genes of vertebrates and invertebrates encode transcription factors with both a paired domain and a homeodomain. They are expressed in the developing eye and in the central nervous system. Loss-of-function mutations in mammals and in flies result in a reduction or absence of eyes and targeted expression of the Drosophila and the mouse Pax-6 genes induces ectopic eye structures in Drosophila. These findings lead to the proposal that the morphogenesis of the different types of eyes is controlled by a Pax-6-dependent genetic pathway and that the various eye types are of monophyletic origin. We have isolated a Pax-6 homologous gene from the ascidian Phallusia mammillata, because ascidians occupy an important position in early chordate evolution. Furthermore, the Phallusia larva has a simple photosensitive ocellus. Phallusia Pax-6 shares extensive sequence identity and conserved genomic organization with the known Pax-6 genes of vertebrates and invertebrates. Expression of Phallusia Pax-6 is first detected at late gastrula stages in distinct regions of the developing neural plate. At the tailbud stage, it is expressed in the spinal cord and the brain vesicle, where the sensory organs (ocellus and otolith) form, suggesting an important function in their development. Ectopic expression of the ascidian Pax-6 gene in Drosophila leads to the induction of supernumerary eyes indicating a highly conserved gene regulatory function for Pax-6 genes.
Ftz-F1 is a cofactor in Ftz activation of the Drosophila engrailed gene
Authors: Florence Brian , Guichet Antoine , Ephrussi Anne , Laughon Allen
Published: April 2021

The fushi tarazu pair-rule gene is required for the formation of alternating parasegmental boundaries in the Drosophila embryo. fushi tarazu encodes a homeodomain protein necessary for transcription of the engrailed gene in even-numbered parasegments. Here we report that, within an engrailed enhancer, adjacent and conserved binding sites for the Fushi tarazu protein and a cofactor are each necessary, and together sufficient, for transcriptional activation. Footprinting shows that the cofactor site can be bound specifically by Ftz-F1, a member of the nuclear receptor superfamily. Ftz-F1 and the Fushi tarazu homeodomain bind the sites with 4-to 8-fold cooperativity, suggesting that direct contact between the two proteins may contribute to target recognition. Even parasegmental reporter expression is dependent on Fushi tarazu and maternal Ftz-F1, suggesting that these two proteins are indeed the factors that act upon the two sites in embryos. The two adjacent binding sites are also required for continued activity of the engrailed enhancer after Fushi tarazu protein is no longer detectable, including the period when engrailed, and the enhancer, become dependent upon wingless. We also report the existence of a separate negative regulatory element that apparently responds to odd-skipped.
Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing
Authors: Neumann Carl J. , Cohen Stephen M.
Published: April 2021

Short-range interaction between dorsal and ventral (D and V) cells establishes an organizing center at the DV compartment boundary that controls growth and specifies cell fate along the dorsal-ventral axis of the Drosophila wing. The secreted signaling molecule Wingless (Wg) is expressed by cells at the DV compartment boundary and has been implicated in mediating its long-range patterning activities. Here we show that Wg acts directly, at long range, to define the expression domains of its target genes, Distal-less and vestigial. Expression of the Achaete-scute genes, Distal-less and vestigial at different distances from the DV boundary is controlled by Wg in a concentration-dependent manner. We propose that Wg acts as a morphogen in patterning the D/V axis of the wing.
Taste buds develop autonomously from endoderm without induction by cephalic neural crest or paraxial mesoderm
Authors: Barlow Linda A. , Northcutt R. Glenn
Published: April 2021

Although it had long been believed that embryonic taste buds in vertebrates were induced to differentiate by ingrowing nerve fibers, we and others have recently shown that embryonic taste buds can develop normally in the complete absence of innervation. This leads to the question of which tissues, if any, induce the formation of taste buds in oropharyngeal endoderm. We proposed that taste buds, like many specialized epithelial cells, might arise via an inductive interaction between the endodermal epithelial cells that line the oropharynx and the adjacent mesenchyme that is derived from both cephalic neural crest and paraxial mesoderm. Using complementary grafting and explant culture techniques, however, we have now found that well-differentiated taste buds will develop in tissue completely devoid of neural crest and paraxial mesoderm derivatives. When the presumptive oropharyngeal region was removed from salamander embryos prior to the onset of cephalic neural crest migration, taste buds developed in grafts and explants coincident with their appearance in intact control embryos. Similarly, explants from neurulae in which movement of paraxial mesoderm had not yet begun also developed taste buds after 9-12 days in vitro. We conclude that neither cranial neural crest nor paraxial mesoderm is responsible for the induction of embryonic taste buds. Surprisingly, the ability to develop taste buds late in embryonic development seems to be an intrinsic feature of the oropharyngeal endoderm that is determined by the completion of gastrulation.
Expression and function of clift in the development of somatic gonadal precursors within the Drosophila mesoderm
Authors: Boyle Monica , Bonini Nancy , DiNardo Stephen
Published: April 2021

The gonad forms from cells of two lineages: the germline and soma. The somatic gonadal cells generate the various cell types within the testis or ovary that support gametogenesis. These cells derive from embryonic mesoderm, but how they are specified is unknown. Here, we describe a novel regulator of Drosophila gonadogenesis, clift, mutations in which abolish gonad formation. clift is expressed within somatic gonadal precursors as these cells first form, demonstrating that 9-12 cells are selected as somatic gonadal precursors within each of three posterior parasegments at early stages in gonadogenesis. Despite this early expression, somatic gonadal precursors are specified in the absence of clift function. However, they fail to maintain their fate and, as a consequence, germ cells do not coalesce into a gonad. In addition, using clift as a marker, we show that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wg, coupled with a gene regulatory hierarchy within the mesoderm. While loss of wg abolishes gonadal precursors, ectopic expression expands the population such that most cells within lateral mesoderm adopt gonadal precursor fates. Initial dorsoventral positioning of somatic gonadal precursors relies on a regulatory cascade that establishes dorsal fates within the mesoderm and is subsequently refined through negative regulation by bagpipe, a gene that specifies nearby visceral mesoderm. Thus, these studies identify essential regulators of gonadal precursor specification and differentiation and reveal novel aspects of the general mechanism whereby distinct fates are allocated within the mesoderm.
The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos
Authors: Jen Wui-Chuong , Wettstein Daniel , Turner David , Chitnis Ajay , Kintner Chris
Published: April 2021

Segmentation of the vertebrate embryo begins when the paraxial mesoderm is subdivided into somites, through a process that remains poorly understood. To study this process, we have characterized X-Delta-2, which encodes the second Xenopus homolog of Drosophila Delta. Strikingly, X-Delta-2 is expressed within the presomitic mesoderm in a set of stripes that corresponds to prospective somitic boundaries, suggesting that Notch signaling within this region establishes a segmental prepattern prior to somitogenesis. To test this idea, we introduced antimorphic forms of X-Delta-2 and Xenopus Suppressor of Hairless (X-Su(H)) into embryos, and assayed the effects of these antimorphs on somite formation. In embryos expressing these antimorphs, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment properly. Both antimorphs also disrupted the segmental expression of X-Delta-2 and Hairy2A, a basic helix-loop-helix (bHLH) gene, within the presomitic mesoderm. These observations suggest that X-Delta-2, via X-Notch-1, plays a role in segmentation, by mediating cell-cell interactions that underlie the formation of a segmental prepattern prior to somitogenesis.
rZIP, a RING-leucine zipper protein that regulates cell fate determination during Dictyostelium development
Authors: Balint-Kurti Peter , Ginsburg Gail , Rivero-Lezcano Octavio , Kimmel Alan R.
Published: April 2021

rZIP is an approx. 32 kDa, multi-domain protein of Dictyostelium discoideum whose structural motifs include a RING (zinc-binding) domain, a leucine zipper, a glutamine repeat, an SH3-binding region and a consensus phosphorylation site for MAP kinase. In vitro, rZIP forms homodimers and interacts specifically with the SH3 domain(s) of the Nck adaptor protein. rZIP is expressed maximally during cell differentiation at approximately equivalent levels in all cells. Disruption of the rZIP gene rzpA results in altered cellular aggregation, impaired slug migration, and aberrant patterning of prespore and prestalk cells, the major progenitor classes. In rzpA− strains, presporespecific genes are overexpressed and prestalk expression zones are reduced. Conversely, constitutive overexpression of rzpA markedly decreases prespore-specific gene expression and significantly increases the expression of prestalk-specific genes. Further, induced transdifferentiation of prespore cells into prestalk cells is inhibited in rzpA− slugs. In light of these patterning defects, we suggest that the RING/zipper protein rZIP plays an important role in early cell fate decisions in Dictyostelium, acting as a positive regulator of prestalk differentiation and an inhibitor of prespore differentiation.
Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene
Authors: Ewart J. L. , Cohen M. F. , Meyer R. A. , Huang G. Y. , Wessels A. , Gourdie R. G. , Chin A. J. , Park S. M. J. , Lazatin B. O. , Villabon S. , Lo C. W.
Published: April 2021

Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 trans-genic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.
Concentration-dependent patterning by an ectopic expression domain of the Drosophila gap gene knirps
Authors: Kosman David , Small Stephen
Published: April 2021

The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is critical for striped patterns of pair-rule gene expression in the Drosophila embryo. To test whether these domains function as sources of morpho-genetic activity, the stripe 2 enhancer of the pair-rule gene even-skipped (eve) was used to express kni in an ectopic position. Manipulating the stripe 2-kni expression constructs and examining transgenic lines with different insertion sites led to the establishment of a series of independent lines that displayed consistently different levels and developmental profiles of expression. Individual lines showed specific disruptions in pair-rule patterning that were correlated with the level and timing of ectopic expression. These results suggest that the ectopic domain acts as a source for morphogenetic activity that specifies regions in the embryo where pair-rule genes can be activated or repressed. Evidence is presented that the level and timing of expression, as well as protein diffusion, are important for determining the specific responses of target genes.
Segregating expression domains of two goosecoid genes during the transition from gastrulation to neurulation in chick embryos
Authors: Lemaire Lydia , Roeser Tobias , Izpisúa-Belmonte Juan Carlos , Kessel Michael
Published: April 2021

We report the isolation and characterization of a chicken gene, GSX, containing a homeobox similar to that of the goosecoid gene. The structure of the GSX gene and the deduced GSX protein are highly related to the previously described goosecoid gene. The two homeodomains are 74% identical. In the first few hours of chick embryogenesis, the expression pattern of GSX is similar to GSC, in the posterior margin of the embryo and the young primitive streak. Later during gastrulation, expression of the two genes segregate. GSC is expressed in the anterior part of the primitive streak, then in the node, and finally in the pre-chordal plate. GSX is expressed in the primitive streak excluding the node, and then demarcating the early neural plate around the anterior streak and overlying the pre-chordal plate. We demonstrate that the GSX-positive part of the primitive streak induces gastrulation, while the GSC-expressing part induces neurulation. After full extension of the streak, the fate of cells now characterized by GSX is to undergo neurulation, while those expressing GSC undergo gastrulation. We discuss the effect of a duplicated basic goosecoid identity for the generation of a chordate nervous system in ontogeny and phylogeny.
Mouse Serrate-1 (Jagged-1): expression in the developing tooth is regulated by epithelial-mesenchymal interactions and fibroblast growth factor-4
Authors: Mitsiadis Thimios A. , Henrique Domingos , Thesleff Irma , Lendahl Urban
Published: April 2021

Serrate-like genes encode transmembrane ligands to Notch receptors and control cell fate decisions during development. In this report, we analyse the regulation of the mouse Serrate-1 gene during embryogenesis. The Serrate-1 gene is expressed from embryonic day 7.5 (E7.5) and expression is often observed at sites of epithelial-mesenchymal interactions, including the developing tooth, where Serrate-1 is first (E11.5) expressed in all cells of the dental epithelium, but not in mesenchyme. A transient upregulation in dental mesenchyme (E12.5-15.5) is correlated with down-regulation of Serrate-1 expression in epithelial cells contacting the mesenchyme, i.e. in the cells destined to become ameloblasts. This expression pattern is reproduced in explants of dental epithelium and mesenchyme in vitro: epithelium induces Serrate-1 expression in mesenchyme, while epithelium in close proximity to this mesenchyme does not express detectable levels of Serrate-1 mRNA, suggesting that downregulation of Serrate-1 expression in preameloblasts is caused by mesenchyme-derived signals.

Finally, regulation of Serrate-1 expression differs from that of Notch genes. The Serrate-1 gene is induced in dental mesenchyme by fibroblast growth factor-4, but not by bone morphogenetic proteins, while the converse is true for Notch genes. This indicates that, at least during tooth development, the expression patterns observed for receptors and ligands in the Notch signaling pathway are generated by different induction mechanisms.
The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate
Authors: Micchelli, Craig A. , Rulifson Eric J. , Blair Seth S.
Published: April 2021

We have investigated the role of the Notch and Wingless signaling pathways in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified about the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells are flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. We show first that cut acts to maintain margin wingless expression, providing a potential explanation of the cut mutant phenotype. Next, we examined the regulation of cut expression. Our results indicate that Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; our results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Finally, we show that Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. We propose that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression.