Causality analysis detects the regulatory role of maternal effect genes in the early Drosophila embryo

In animals, early development is dependent on a pool of maternal factors, both RNA and proteins, which are required for basic cellular process and cell differentiation until zygotic genome activation. The role of a majority of these maternally expressed factors is not fully understood. By exploiting the biallelic editing ability of CRISPR-Cas9, we identify and characterize maternal-effect genes in a single generation, using a maternal crispant technique. We validated the ability to generate biallelic mutations in the germline by creating maternal crispants that phenocopied previously characterized maternal-effect genes: motley/birc5b, tmi/prc1l, and aura/mid1ip1. Additionally, by targeting maternally expressed genes of unknown function in zebrafish, we identified two new maternal-effect zebrafish genes, kpna7 and fhdc3. The genetic identity of these maternal crispants was confirmed by sequencing haploid progeny from F0 females, which allowed the analysis of newly induced lesions in the maternal germ line. Our studies show that maternal crispants allow for the effective identification and primary characterization of maternal-effect genes in a single generation, facilitating the reverse genetics analysis of maternal factors that drive embryonic development.

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Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo

Development ◽

10.1242/dev.115.2.607 ◽

1992 ◽

Vol 115 (2) ◽

pp. 607-616 ◽

Cited By ~ 5

Author(s):

R. Chasan ◽

Y. Jin ◽

K.V. Anderson

Keyword(s):

Maternal Effect ◽

Serine Proteases ◽

Drosophila Embryo ◽

Vitelline Membrane ◽

Mutant Form ◽

Wild Type ◽

Zymogen Activation ◽

Embryonic Polarity ◽

Dominant Mutant ◽

Maternal Effect Genes

The product of the Drosophila easter gene, a member of the trypsin family of serine proteases, must be more active ventrally than dorsally to promote normal embryonic polarity. The majority of the easter protein in the embryo is present in the unprocessed zymogen form and appears to be evenly distributed in the extracellular space, indicating that the asymmetric activity of wild-type easter must arise post-translationally. A dominant mutant form of easter that does not require cleavage of the zymogen for activity (ea delta N) is active both dorsally and ventrally. The ea delta N mutant bypasses the requirement for five other maternal effect genes, indicating that these five genes exert their effects on dorsal-ventral patterning solely by controlling the activation of the easter zymogen. We propose that dorsal-ventral asymmetry is initiated by a ventrally-localized molecule in the vitelline membrane that nucleates an easter zymogen activation complex, leading to the production of ventrally active easter enzyme.

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Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen

Development ◽

10.1242/dev.109.4.811 ◽

1990 ◽

Vol 109 (4) ◽

pp. 811-820 ◽

Cited By ~ 16

Author(s):

W. Driever ◽

V. Siegel ◽

C. Nusslein-Volhard

Keyword(s):

Drosophila Embryo ◽

Beta Globin ◽

P Transformation ◽

Early Drosophila Embryo ◽

Maternal Effect Genes ◽

Translational Block ◽

Maternal Gene ◽

Anterior Posterior

A small number of maternal effect genes determine anterior-posterior pattern in the Drosophila embryo. Embryos from females mutant for the maternal gene bicoid lack head and thorax. bcd mRNA becomes localized to the anterior tip of the egg during oogenesis and is the source for the morphogen gradient of bcd protein. Here we show that in vitro transcribed bicoid mRNA that has its own leader sequences substituted by the Xenopus beta-globin 5′ untranslated sequences is translated more efficiently than bicoid mRNA with the natural 5′ mRNA leader when tested in vitro and in Drosophila Schneider cells. When injected into bicoid mutant embryos, only the bcd mRNA with the beta-globin leader sequence, substituted for the natural leader, is able to induce anterior development. We used P-transformation to show that sequences in the 5′ leader are neither necessary for localization of the transcript nor for the translational block of the bcd mRNA during oogenesis. For our injection experiments, we used only one of the identified splicing forms of bcd mRNA. The bcd protein species derived from this mRNA is able to induce anterior development at any position along the anterior-posterior axis. Thus bicoid protein can induce development of head and thorax independent of any other specifically localized morphogenetic factor. Our findings further support the notion that the concentration gradient of bcd protein, and not the existence of different forms of bcd protein, is responsible for specifying subregions of the embryo.

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The Phenotype of mes-2, mes-3, mes-4 and mes-6, Maternal-Effect Genes Required for Survival of the Germline in Caenorhabditis elegans, Is Sensitive to Chromosome Dosage

Genetics ◽

10.1093/genetics/148.1.167 ◽

1998 ◽

Vol 148 (1) ◽

pp. 167-185 ◽

Cited By ~ 6

Author(s):

Carol Garvin ◽

Richard Holdeman ◽

Susan Strome

Keyword(s):

Gene Expression ◽

Germ Cells ◽

Maternal Effect ◽

Early Embryo ◽

Male Offspring ◽

X Chromosomes ◽

Control Of Gene Expression ◽

Maternal Effect Genes ◽

Surprising Finding

AbstractMutations in mes-2, mes-3, mes-4, and mes-6 result in maternal-effect sterility: hermaphrodite offspring of mes/mes mothers are sterile because of underproliferation and death of the germ cells, as well as an absence of gametes. Mutant germ cells do not undergo programmed cell death, but instead undergo a necrotic-type death, and their general poor health apparently prevents surviving germ cells from forming gametes. Male offspring of mes mothers display a significantly less severe germline phenotype than their hermaphrodite siblings, and males are often fertile. This differential response of hermaphrodite and male offspring to the absence of mes+ product is a result of their different X chromosome compositions; regardless of their sexual phenotype, XX worms display a more severe germline phenotype than XO worms, and XXX worms display the most severe phenotype. The sensitivity of the mutant phenotype to chromosome dosage, along with the similarity of two MES proteins to chromatin-associated regulators of gene expression in Drosophila, suggest that the essential role of the mes genes is in control of gene expression in the germline. An additional, nonessential role of the mes genes in the soma is suggested by the surprising finding that mutations in the mes genes, like mutations in dosage compensation genes, feminize animals whose male sexual identity is somewhat ambiguous. We hypothesize that the mes genes encode maternally supplied regulators of chromatin structure and gene expression in the germline and perhaps in somatic cells of the early embryo, and that at least some of their targets are on the X chromosomes.

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The Role of Maternal-Effect Genes in Mammalian Development: Are Mammalian Embryos Really an Exception?

Stem Cell Reviews and Reports ◽

10.1007/s12015-016-9648-6 ◽

2016 ◽

Vol 12 (3) ◽

pp. 276-284 ◽

Cited By ~ 14

Author(s):

Maureen L. Condic

Keyword(s):

Maternal Effect ◽

Mammalian Development ◽

Maternal Effect Genes ◽

Mammalian Embryos

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THE GENETICS OF THE DORSAL-BICAUDAL-D REGION OF DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/113.3.665 ◽

1986 ◽

Vol 113 (3) ◽

pp. 665-678

Author(s):

Ruth Steward ◽

Christiane Nüsslein-Volhard

Keyword(s):

Maternal Effect ◽

Complementation Group ◽

Drosophila Embryo ◽

Temperature Sensitive ◽

X Ray ◽

Dominant Phenotype ◽

Maternal Effect Genes ◽

D Region ◽

Complementation Groups ◽

Anterior Posterior

ABSTRACT The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respectively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of kelch, two previously identified maternal-effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.

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Alternative linker histone permits fast paced nuclear divisions in early Drosophila embryo

Nucleic Acids Research ◽

10.1093/nar/gkaa624 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9007-9018

Author(s):

László Henn ◽

Anikó Szabó ◽

László Imre ◽

Ádám Román ◽

Andrea Ábrahám ◽

...

Keyword(s):

Low Temperature ◽

Early Embryo ◽

Linker Histone ◽

Early Embryogenesis ◽

Drosophila Embryo ◽

Core Histone ◽

Early Embryos ◽

Early Drosophila Embryo ◽

Nucleosome Stability

Abstract In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis.

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Reassessing the Role and Dynamics of Nonmuscle Myosin II during Furrow Formation in Early Drosophila Embryos

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0440 ◽

2004 ◽

Vol 15 (2) ◽

pp. 838-850 ◽

Cited By ~ 181

Author(s):

Anne Royou ◽

Christine Field ◽

John C. Sisson ◽

William Sullivan ◽

Roger Karess

Keyword(s):

Myosin Ii ◽

Colchicine Treatment ◽

Drosophila Embryo ◽

Dependent Manner ◽

Membrane Invagination ◽

Nonmuscle Myosin Ii ◽

Early Drosophila Embryo ◽

Cytoskeleton Rearrangements ◽

Drosophila Embryos

The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction.

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Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo

The Journal of Cell Biology ◽

10.1083/jcb.200810012 ◽

2009 ◽

Vol 184 (5) ◽

pp. 639-646 ◽

Cited By ~ 25

Author(s):

Mark L. McCleland ◽

Jeffrey A. Farrell ◽

Patrick H. O'Farrell

Keyword(s):

Cell Cycle ◽

Drosophila Melanogaster ◽

Drosophila Embryo ◽

Cell Cycle Regulators ◽

Mitotic Progression ◽

Mitotic Entry ◽

Early Drosophila Embryo ◽

Real Time Visualization ◽

The One

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others.

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Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity

Development ◽

10.1242/dev.109.2.425 ◽

1990 ◽

Vol 109 (2) ◽

pp. 425-433 ◽

Cited By ~ 17

Author(s):

B. Hay ◽

L.Y. Jan ◽

Y.N. Jan

Keyword(s):

Germ Cell ◽

Maternal Effect ◽

Polar Granule ◽

Cell Lineage ◽

Posterior Pole ◽

Drosophila Embryo ◽

Polar Granules ◽

Early Drosophila Embryo ◽

Anteroposterior Polarity ◽

Source Of Information

Cytoplasm at the posterior pole of the early Drosophila embryo, known as polar plasm, serves as a source of information necessary for germ cell determination and for specification of the abdominal region. Likely candidates for cytoplasmic elements important in one or both of these processes are polar granules, organelles concentrated in the cortical cytoplasm of the posterior pole. Females homozygous for any one of the maternal-effect mutations, tudor, oskar, staufen, vasa, or valois give rise to embryos that lack localized polar granules, fail to form the germ cell lineage and have abdominal segment deletions. Using antibodies against a polar granule component, the vasa protein, we find that vasa synthesis or localization is affected by these mutations. In vasa mutants, synthesis of vasa protein is absent or severely restricted. In oskar and staufen mutant females, vasa synthesis appears normal, but the vasa protein is not localized. In tudor and valois mutant females, vasa is localized to the posterior pole of oocytes, but this localization is lost following egg activation. In addition to the posterior localized vasa, there is a low level of vasa distributed throughout the embryo. A function for this distributed vasa is postulated based on the observation that embryos from Bicaudal-D mothers, in which abdominal determinants are incorrectly localized to the anterior pole, do not show any ectopic vasa localization, though abdomen development at the anterior end depends on the amount of vasa protein in the embryo.

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