Thesis
Reference
The role of bucky ball in germ plasm assembly in zebrafish
BONTEMS, Franck
Abstract
La formation des gamètes est spécifiée par un ensemble de déterminants cytoplasmiques appelé plasma germinatif. Assemblé durant l'ovogenèse, il est essentiel pour la fertilité du futur adulte. Les régulateurs clés de cet assemblage sont inconnus chez les vertébrés. Seul oskar, chez la Drosophile, est le gène connu comme régulateur de l'assemblage du plasma germinatif. Cependant, il n'existe que chez les diptères. Chez le mutant bucky ball (buc) les femelles zebrafishs présentent une perturbation lors de l'assemblage du plasma germinatif.
Nous démontrons d'une part que l'ARNm buc code pour une nouvelle protéine exprimée exclusivement dans l'ovaire, d'autre part que sa surexpression, et celle d'oskar, chez le zebrafish, génèrent des cellules germinales supplémentaires. Nous prouvons donc que buc, comme oskar, est suffisant pour recruter les composants du plasma germinatif. Ainsi tous deux montrent la même fonction chez les vertébrés et les invertébrés : l'organisation du plasma germinatif
BONTEMS, Franck. The role of bucky ball in germ plasm assembly in zebrafish. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4148
URN : urn:nbn:ch:unige-47036
DOI : 10.13097/archive-ouverte/unige:4703
Available at:
http://archive-ouverte.unige.ch/unige:4703
Disclaimer: layout of this document may differ from the published version.
UNIVERSITÉ DE GENÈVE
Département de zoologie et
Biologie animale FACULTE DES SCIENCES
Professeur Denis Duboule Docteur Roland Dosch
The Role of Bucky Ball in Germ Plasm Assembly in Zebrafish
THÈSE
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
Par
Franck BONTEMS de
Reignier (France)
Thèse nº 4148
Genève
Atelier d’impression ReproMail 2009
REMERCIMENTS
Je tiens dans cette partie à rendre mérite à ceux qui à un moment ont rendu possible cette thèse. En effet, chaque personne citée ici mérite une attention particulière. Mes prouesses littéraires étant limitées, je les remercierai simplement.
Je tiens donc à remercier tout d’abord, le Dr Roland Dosch, de m’avoir fait confiance en m'accueillant dans son laboratoire. Mes connaissances en génétique étaient très limitées et mon Anglais très approximatif, il lui a donc fallu patience et disponibilité pour me transmettre ses conseils scientifiques.
Je remercie le Dr Anne Ephrussi et le Pr Denis Duboule de l’honneur qu’ils me font en acceptant d'être membre du jury et d'avoir pris sur leur temps pour consulter ce travail.
J’aimerais aussi remercier Claudia, Sabine, Michèle, Christine, Olivier, Patrick, Joost, Maximilian, Irinka, qui ont participé au bien être des poissons en partageant leur temps pour le nourrissage.
Je remercie Amandine et Susanne pour leur bonne humeur, ainsi que toutes les personnes avec lesquelles j’ai collaboré au sein du laboratoire au cours de ces années.
Je tiens à adresser mes vifs remerciements à
Carole Seum pour ses nombreux et précieux conseils notamment en matière de clonage.
Richard Fish et l'équipe de Juan Montoya pour les discussions que nous avons pu avoir, mais aussi Bozena Polok pour les commentaires et corrections de cet écrit.
Je remercie également l’équipe du laboratoire du Pr Brigitte Galliot et Dr Marie Gomez qui, par leurs échanges scientifiques le mercredi matin, m’ont permit de progresser activement dans mon travail.
Je remercie Lisbeth, Loic, Fabien, Tomohiko, Nicole, Julien, Ludivine pour les bons moments que nous avons pu passer ensemble pendant les repas.
Je remercie également ma famille et belle famille pour leur soutien et leur attention, tout particulièrement ma bête des Vosges qui m’a beaucoup déchargé en prenant soin de nous (notre petite Lune et moi).
Je réserve enfin, une reconnaissance particulière et chaleureuse aux deux personnes sans qui cette thèse n’aurait pas été la même, Jacqueline Lyautey et André Solaro qui, en plus de leur soutien technique et scientifique de chaque instant, ont toujours supportés avec le sourire, mes blagues de chaque jour. C’est pourquoi avec toute l’affection et le respect que j’ai à leur égard, je leur dédie cette thèse.
A Jacqueline et André
INDEX
I. RESUME... 9
II. ABSTRACT ... 11
III. INTRODUCTION ... 13
III.1. Germ plasm history... 14
III.2. Germ plasm specifies gamete development ... 18
III.2.1. Molecular mechanisms of germ cell specification ... 19
III.2.2. Germ plasm synonyms ... 20
III.3. Germ plasm composition ... 21
III.3.1. Vasa... 21
III.3.2. Nanos... 23
III.3.3. Dazl. ... 23
III.4. Germ plasm assembly ... 24
III.4.1. Germ plasm and the Balbiani body... 25
III.4.2. Germ plasm assembly in Drosophila... 29
III.5. Zebrafish as a vertebrate model to study maternal factors... 33
III.6. Germ line development in zebrafish... 35
III.6.1. Germ plasm localization during embryogenesis in zebrafish... 36
III.6.2. Germ plasm localization during oogenesis in zebrafish ... 38
III.7. Transition between maternal and zygotic gene expression in zebrafish... 41
III.7.1. Maternal-effect mutant... 44
III.8. The isolation of maternal factors in vertebrates ... 45
III.8.1. Description of the polarity phenotype of the buc mutant... 47
IV. RESULTS ... 51
IV.1. Buc is required for germ plasm assembly in early zebrafish oocytes ... 51
IV.2. Buc encodes a novel vertebrate specific protein. ... 56
IV.3. buc mRNA expression ... 66
IV.3.1. buc mRNA expression during oogenesis ... 66
IV.3.2. buc mRNA expression during embryogenesis ... 66
IV.4. Bucky ball localization during oogenesis ... 68
IV.5. Buc mRNA translation is required for germ plasm formation... 73
IV.5.1. Buc protein function ... 75
IV.5.2. Buc protein localization with germ plasm in oocytes... 78
IV.6. Buc is localized to the germ plasm in embryos... 80
IV.7. Buc overexpression induces ectopic germ cell formation in zebrafish embryos 84 IV.7.1. Quantitative expression of germ plasm components after buc overexpression... 86
IV.8. Oskar and Bucky ball protein alignment. ... 91
IV.8.1. Oskar transgenic zebrafish ... 93
IV.8.2. Drosophila oskar mRNA overexpression in zebrafish. ... 95
IV.8.3. Germ cell induction assay with osk mRNA in zebrafish ... 96
V. DISCUSSION ... 99
V.1. Buc localization ... 99
V.2. mRNA localization during oogenesis ... 99
V.3. Early polarity is controlled by bucky ball... 100
V.4. Germ plasm assembly is controlled by bucky ball... 102
V.4.1. Differential buc localization during maternal period ... 102
V.5. Buc protein domains ... 103
V.5.1. Buc recruits germ plasm... 104
V.6. Phenotypic differences between oskar and bucky ball... 105
V.6.1. Evolutionary comparison of germ plasm localization ... 106
V.7. Similarity between Bucky ball and Oskar... 107
V.8. Functional similarity between Bucky ball and Oskar ... 108
V.9. Polarity in mammals. ... 109
VI. PERSPECTIVES ... 111
VI.1. Validate Buc and Osk homology through biochemical interactions ... 111
VI.2. Differentiation of additional PGCs into functional gametes... 112
VI.2.1. Changing the sex ration with additional PGCs... 112
VI.2.2. Rescue of sterility with Buc induced PGCs ... 112
VII. MATERIALS AND METHODS... 115
VII.1. Plasmid DNA purification ... 115
VII.1.1. Electro-transformation... 115
VII.1.2. Heat shock bacteria... 116
VII.1.3. Control of the transformation... 117
VII.1.4. Plasmid DNA amplification and extraction... 117
VII.1.5. Enzymatic treatment of plasmid DNA ... 118
VII.1.6. In vitro transcription of RNA... 118
VII.2. Meiotic mapping and genotyping... 120
VII.2.1. PCRs ... 121
VII.3. Site directed mutagenesis ... 123
VII.4. Quantitative real-time PCR... 123
VII.5. Staining... 124
VII.5.1. Antibody staining of zebrafish embryos and ovaries ... 124
VII.5.2. RNA staining... 124
VII.5.3. DiOC6 staining ... 127
VII.6. Phylogenetic and synteny analysis... 128
VII.7. Embryonic or early stage oocytes injection... 129
VII.7.1. Nucleotides materiel ... 129
VII.7.2. 1-cell injection... 130
VII.7.3. 16-cell injections ... 130
VII.7.4. Oocyte injection and culture ... 131
VII.8. Statistics... 134
VIII. ABBREVIATIONS... 135
IX. ANNEXE ... 137
X. REFERENCE ... 147
I. RESUME
Dans de nombreux organismes, la formation des gamètes pendant l’embryogenèse est spécifiée par des déterminants cytoplasmiques réunis sous le terme de plasma germinatif.
Le plasma germinatif est assemblé au cours de l'ovogenèse formant une structure cellulaire distincte, comme le plasma polaire chez la drosophile ou le corps de Balbiani, retrouvé dans les ovocytes de nombreux animaux, dont les humains. Bien que le plasma germinatif soit essentiel pour la fertilité pendant le développement embryonnaire, les régulateurs clés de l'assemblage du corps de Balbiani sont inconnus chez les vertèbres.
Seul, le gène oskar chez la Drosophile est connu comme un régulateur de l’assemblage du plasma germinatif, mais celui-ci existe uniquement chez les diptères.
Chez le mutant bucky ball (buc) les femelles zebrafish mutantes présentent une perturbation lors de la formation du corps de Balbiani. Nous démontrons ainsi que Buc est essentiel pour l’assemblage du plasma germinatif chez les vertébrés. Afin d’identifier le gène Buc, nous avons positionné sur le génome la mutation de Buc. Buc code pour une nouvelle protéine dont l’ARNm est exprimée exclusivement dans l'ovaire, montrant une nouvelle dynamique de localisation d’ARNm, initialement localisée dans le corps de Balbiani. Pour caractériser la fonction de Buc, nous avons développé une nouvelle technique d'injection dans les ovocytes de zebrafish. En utilisant ce nouveau procédé, nous avons montré que l'activité de Buc dans l'ovocyte est suffisante, et que Buc doit être traduit pour assembler le plasma germinatif. Ces résultats sont cohérents avec les résultats découverts lors de l'injection de Buc en fusion avec GFP. En effet Buc-GFP se localise dans le corps de Balbiani et après la fécondation, dans le plasma germinatif de
des cellules germinales supplémentaires, prouvant que Buc est suffisant pour recruter les composants du plasma germinatif. Depuis, nous avons découvert de nombreux homologues de Buc dans le génome des vertébrés, y compris celui des mammifères. Nos résultats suggèrent ainsi que Buc représente le premier régulateur commun dans la formation du plasma germinatif chez les vertébrés. Afin de mieux comprendre le rôle de Buc dans la localisation du corps de Balbiani, nous avons établi une lignée transgénique Buc-GFP. Cette lignée vient au secours du phénotype mutant indiquant que le transgène reflète la majorité de l’activité endogène de Buc. En outre, la fluorescence de Buc-GFP montre, in vivo, la localisation des protéines dans le corps Balbiani mais aussi, au cours de l'ovogenèse, au cortex des ovocytes. Dés les premiers stades embryonnaires, Buc-GFP est localisé avec le plasma germinatif dans les premiers sillons de clivages et plus tard dans les quatres cellules germinales primordiales jusqu'au stade oblong. Cette lignée transgénique offre ainsi l’opportunité d’avoir un rapporteur in vivo du plasma germinatif de l'ovocyte jusqu’à l'embryon précoce. En outre, cette lignée sera dans le futur, un outil pour étudier le rôle de Buc dans sa fonction biochimique et de biologie cellulaire, fournissant ainsi un point d'entrée pour disséquer moléculairement la structure du corps de Balbiani dans l'ovocyte des vertébrés.
De plus, lorsque l’ARNm oskar est exprimé dans des embryons de zebrafish, il génère, tout comme bucky ball, des cellules germinales primordiales additionnelle, ce qui nous démontre ainsi que bucky ball et oskar ont une fonction commune dans l’organisation du plasma germinatif.
II. ABSTRACT
In many organisms the formation of gametes during embryogenesis is specified by maternal cytoplasmic determinants termed germ plasm. Germ plasm is assembled during oogenesis forming a distinct cellular structure, such as pole plasm in Drosophila or the Balbiani body, which has been found in oocytes of many animals including humans.
Although germ plasm is critical for the fertility of the developing embryo, the key regulators of Balbiani body assembly in vertebrates are unknown.
Only the oskar gene in Drosophila is known as a regulator of germ plasm assembly, but exists only in Dipterans. In bucky ball (buc) mutant zebrafish females the formation of the Balbiani body is disrupted demonstrating that Buc is essential for germ plasm assembly in vertebrates. To molecularly identify the buc gene, we positionally cloned the buc mutation. Buc encodes a novel protein and its mRNA is exclusively expressed in the ovary displaying a novel, dynamic mRNA localization pattern initiating in the Balbiani body. To characterize the function of Buc, we developed an oocyte injection assay in zebrafish. Using this assay we show that Buc activity in the oocyte is sufficient and that Buc needs to be translated to aggregate germ plasm. Consistent, with these results we discovered that an injected Buc-GFP fusion localizes to the Balbiani body and after fertilization in the germ plasm of the early embryo. Interestingly, overexpression of buc generates additional germ cells in the zebrafish embryo providing evidence that buc is sufficient to recruit germ plasm components. Since we discovered buc homologs in many vertebrate genomes including mammals our results suggest that Buc represents the first common regulator of germ plasm formation in vertebrates.
transgenic Buc-GFP line. This line fully rescues the mutant phenotype indicating that the transgene reflects most of the endogenous Buc activity. In addition the GFP fluorescence reports Buc protein localization in vivo, which we discover in the Balbiani body and later during oogenesis at the cortex of the oocyte. At the early embryonic stages Buc-GFP is localized to the germ plasm at the first cleavage furrows and later in the four primordial germ cells until oblong stage. This transgenic line provides an in vivo reporter for the germ plasm in the oocyte and the early embryo. Moreover this line will be a good tool to study the biochemical function and cell biological role of Buc and thus provides an entry point to molecularly dissect the oocyte Balbiani body structure in vertebrates.
Additionally, when oskar mRNA is expressed in zebrafish embryos, it generates additional primordial germ cells like bucky ball demonstrating that bucky ball and oskar have a similar function in germ plasm organization.
III. INTRODUCTION
One of the most important processes in the animal kingdom is sexual reproduction. For example, sexual reproduction controls the behavior of individuals inside or outside an animal group. The sensory organs, like olfaction and sight, play an important role in the sexual life of animals and often the behavior is different between male and female.
Moreover, reproduction is associated with evolution in terms of genetic transmission of character. A geographic separation (ecological niche separation) of a species and thus, reproductive separation drives speciation generating new genetic characters and new ways of reproduction.
The newly created species have sometimes small and unperceivable differences, but the morphological or genomic differences can create progeny with difficulties to reproduce with each other. A genetic cross between horse and donkey is a well known example:
hinny or mule are often sterile (Zhao et al., 2006). This sterility is caused by difference in the number of chromosomes of the parents: 64 for the horse and 62 for the donkey (Taylor and Short, 1973).
Sexual reproduction represents a unique step, when part of the genome of two different organisms fuses to give a new organism. Moreover, the cytoplasm of the two gametes also fuses. Already Weismann and Boveri hypothesized that constituents of the egg control the first steps of gamete development and therefore are called “germ plasm”.
To understand the process, that controls the formation of germ plasm and its localization, it is important to describe what is germ plasm, its function and where it is situated in
organism, so I will follow chronologically the discoveries of the scientists in germ plasm research.
III.1. Germ plasm history
In 1882, August Weismann proposed that organisms are subdivided into a somatic part, representing the majority of our body, and the germline part, which transmits the hereditary information to the next generation (Figure 1) (Weismann, 1885). To test this hypothesis he amputated the tails of mice for 60 generations. In each subsequent generation the tails of the offspring were not shortened and were of normal length, as those of their parents prior to the treatment. His conclusion was, that in multicellular organisms, only the gametes (eggs and sperm) transmit the hereditary material to the other cells (somatic cells) of the next generation, because experiments carried out on somatic cells do not affect the transmitted information.
Figure 1. Germline theory after Weismann showing the germ cell and somatic cell lineage. Germ cells (gametes) generate somatic cells and additional germ cells and are the only cells transmitting hereditary information to the next generation.
(from:http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GermlineVsSoma.html).
To explain how the germline becomes different from the somatic cells, Weismann proposed the "mosaic model". In this model the nucleus play a pivotal role in specifying the somatic cells by unequal distribution of determinants into daughter cells. The inheritance of these determinants will then control somatic differentiation (Figure 2).
Only the germline will keep all of the determinants in the nucleus. Thus, in the mosaic model of Weismann the germ cell inducing determinants are localized in the nucleus.
Figure 2. Weismann’s theory of nuclear determination. Weismann assumed that there were factors in the nucleus that were distributed asymmetrically to daughter cells during cleavage and directed their future development. (Figure from Principles of development, 3rd edition, Wolpert.
L)
In 1910, Theodor Boveri proposed that a specific cytoplasm (germ plasm) induces the formation of germ cells (Boveri, 1910). His previous observation showed (Figure 3A) that in the embryo of the nematode Parascaris the first division of the egg is equatorial.
Two cells are formed and the chromosomes in the “south pole” cell (vegetal part refers to the lower hemisphere in Figure 3A) stay unfragmented. Later during development, this cell will become the origin of the germline. The other cells develop into somatic cells and will undergo a phenomenon called chromosome diminution (Figure 3A). Chromosome diminution means that the chromosomes of the somatic cells tend to fragment, with only a few remaining intact, whereas the integrity of the germ-cell chromosomes is maintained. To test his cytoplasmic determinant hypothesis, he centrifuged, fertilized eggs, which reoriented the mitotic spindle and expanded the vegetal cytoplasm into two cells instead of only one under normal conditions (Figure 3B). As a consequence, the chromosomes in the nuclei of both cells stayed intact. After a second cleavage, two of four blastomeres keep unfragmented chromosomes. Boveri concluded that the cytoplasm
located at the vegetal part of the first cell protects the chromosomes and for the first time provided experimental evidence for the existence of germ plasm.
At the same time, he corrected the hypothesis of Weismann, who thought that the chromosomes inside nucleus control the fate of cells that will become the germline. More specifically, Boveri proved that any cytoplasmic factor (called germ plasm in pink on Figure 3) on the vegetal pole protects nuclei from the chromosomal diminution. This cytoplasmic factor, the germ plasm, will determine later on the cells, which are at the origin of the germline and develop into gametes.
Figure 3. Boveri's experiment to demonstrate the presence of germ plasm in the Parascaris embryo. Distribution of germ plasm (pink) during the first two cleavages. (A) During normal development the first cleavage is equatorial. So, only one cell keeps the germ plasm preventing chromosome diminution and thus induces germ cells. (B) After centrifugation, the spindle reorients and the vegetal germ plasm is segregated into two cells. So two cells keep the chromosomes intact. (Figure from Developmental Biology, 6th Edition Scott F Gilbert) Adapted from (Satzinger, 2008)
In 1934 Bounoure visualized for the first time in vertebrates a small island of cytoplasm by staining of frog embryos with Altmann’s acid fuchsin (Bounoure, 1934). He called this cytoplasm “germinal plasm” according to its localization close to the vegetal cortex of the eggs. During the first cleavages, this cytoplasm is distributed into the first cells and later during development, is localized in the cells that migrate into the genital ridges, the primordium of the gonads. In summary, scientists observed in the egg of some animals including vertebrates a localized cytoplasm, which follows the development of the germ cells in the embryo.
III.2. Germ plasm specifies gamete development
Using the information, that the germ plasm localizes to the vegetal pole, Smith irradiated fertilized frog eggs with UV light (Smith, 1966). The offspring are normal but unfertile.
A more precise analysis showed that these larvae completely lacked the primordial germ cells (PGCs), which are the progenitors of the gametes and the cells at the origin of the whole germline. Smith confirmed the accepted theory concerning invertebrates that specific cytoplasmic determinants for germ cells are present in the vegetal hemisphere prior to the first division (Geigy, 1931). In an inverse experiment, Wakahara injected vegetal pole cytoplasm into Xenopus embryos and induced supernumerary PGCs (Smith, 1966; Wakahara, 1978). In summary, these classical loss- and gain-of-function experiments in vertebrates showed that the vegetal pole cytoplasm acts as germ cell determinant.
Since localization occurs in the egg, the mother organizes the germ plasm in the oocyte prior to fertilization i.e. without contribution by the sperm. In general, in mature oocytes cytoplasmic determinants play a central role at the beginning of embryonic development, as they provide the foundation for embryogenesis. Indeed, after several divisions every embryonic cell will have the same genome. Thus, the ability to form several tissues with different functions is not caused by the presence or absence of genes, as Weismann proposed, but by the gene expression level inside the cell. It means that in almost all animals cytoplasmic determinants are already distributed unequally between the two first cells of the embryo. This unequal cytoplasmic distribution activates or inhibits the genes responsible for embryonic patterning. For example, in many animals the germ cells are among the first cells, which are specified at the beginning of embryogenesis by maternal, cytoplasmic determinants, but are the last cells that complete their differentiation program during puberty, when the animal becomes a sexually mature adult. In summary, the formation of gametes is controlled by germ plasm formed by the mother in the egg.
and germinal granules are a constitutive marker for germ cells in an organism (Heasman et al., 1984).
III.2.1. Molecular mechanisms of germ cell specification
There is not much known about molecular regulation of PGC development between the germ plasm phase and germ cell specification. Some particular mRNAs and proteins, present in the germ plasm, are selectively incorporated into germ cells and induce the germline development program (Seydoux and Braun, 2006). In addition, the molecular composition of the germ plasm changes during germline development suggesting
different molecular functions at specific stages. Some of these germ plasm components encode repressors of somatic gene expression. For example, the genes PIE-1 in the worm or Polar granule component (Pgc) in the fly repress transcription in the germline by inhibition of RNA Polymerase II (Pol II) indicating that reduced transcription is a general feature of germ cells (Ghosh and Seydoux, 2008; Hanyu-Nakamura et al., 2008; Strome and Lehmann, 2007). Other germ plasm components are members of the piRNA pathway such as the Piwi protein. piRNAs represent a class of small RNAs in the gonad, which are involved in posttranscriptional regulation. The best-characterized molecular function of piRNAs is the silencing of transposable elements, but how this process contributes to germ cell specification is unclear (Cinalli et al., 2008). In summary, several molecular processes start to emerge to regulate PGCs determination by germ plasm, but it is also clear that they are not sufficient to explain the molecular program of germ cell line development.
III.2.2. Germ plasm synonyms
Germ plasm designates cytoplasmic determinants present in eggs and embryos of many species, which upon inheritance by a few cells will specify their differentiation into germ- cells (Wylie, 2000). Germ plasm has different names according to the species: “pole plasm” in Drosophila or “germinal plasm” in Xenopus, but here I will use the term "germ plasm". Germ plasm shows under electron-microscope observation high-density cytoplasmic inclusions without envelope. These components are called “polar granules”, in Drosophila or “P-granules” in C. elegans, and will constitute the “pole cell” precursors of the germline. Another synonym frequently used for germ plasm is "nuage", which now
designates perinuclear electron-dense material (Bilinski et al., 2004; Kloc et al., 2004).
Nuage consists of the ribonucleoproteins (RNPs) exported from the nucleus and is the precursor of the germ plasm.
III.3. Germ plasm composition
When I describe the composition of the germ plasm, I will focus for clarity on selected genes, but omit for example some of the organelles such as mitochondria, which are frequently associated with the germ plasm. The majority of genes present in the germ plasm are highly conserved during evolution between vertebrates and invertebrates. I will describe some germ plasm components existing in zebrafish and for these components, the similarity with Drosophila melanogaster, the organism in which many of these genes and their function were discovered and characterized.
III.3.1. Vasa.
Vasa was found in a genetic screen together with other maternal-effect mutants in Drosophila, alternating embryonic polarity (Schupbach and Wieschaus, 1986). The embryos from females producing a mutant Vasa protein fail to assemble germ plasm and do not form germ cells. Vasa is the most notable example for highly conserved genes.
vasa homologs were found in almost every animal, for example C. elegans, Xenopus (Xcat3), zebrafish, marsupials, mice and primates (Noce et al., 2001; Raz, 2000). GFP mRNA fused with the zebafish vasa 3’utr was injected into Xenopus and the fluorescence was located to the germ plasm suggesting that the mechanism of vasa RNA localization
is conserved between Xenopus and zebrafish (Knaut et al., 2002). Vasa is specifically expressed in the germ line and is used in almost every animal as a marker for germ cells.
Vasa encodes a RNA binding protein or more precisely a RNA helicase of the DEAD box family, whose biochemical function is the modulation of RNA structure by unwinding. This process is important for the post-transcriptional regulation of RNA such as splicing and translation in primordial germ cells.
In Drosophila, vasa is essential in the oocyte for the perinuclear nuage assembly, which gives rise to the pole plasm, the fly germ plasm. Additionally, a two hybrid screen in Drosophila shows that Vasa protein interacts directly with Oskar protein to assemble the polar granules at the posterior pole and to form germ cells (Breitwieser et al., 1996).
Oskar alone can localize to the posterior pole, but it requires Vasa to assemble a functional pole plasm. In conclusion, vasa represents an essential factor during germ line development for pole plasm assembly.
During zebrafish development vasa mRNA is found at the two first cleavage furrows after fertilization giving rise to four spots of localization (Yoon et al., 1997). Later during embryogenesis it is localized to four cells at the blastula stage, which will develop into the primordial germ cells (see paragraph: “the germ plasm localization during embryogenesis in zebrafish” and Figure 7). More recent data show that vasa mRNA is also localized in the cytoplasm of the oocyte, in a structure called the Balbiani body, close to the vegetal cortex (Kosaka et al., 2007). Thus, vasa mRNA serves as a molecular marker to study the phenotype of genes responsible for germ plasm localization in zebrafish (Pelegri et al., 1999).
III.3.2. Nanos
Nanos RNA encodes an RNA binding zinc finger protein (Mosquera et al., 1993). Nanos appears to regulate PGC survival in several organisms (Subramaniam and Seydoux, 1999). In Drosophila, nanos accumulates in pole cells and is present in the germline until PGCs join the gonads. The Drosophila nanos mutant undergoes premature mitosis before proper migration of PGCs into the gonad (Asaoka-Taguchi et al., 1999). Thus, when Nanos protein is defective, the PGCs do not develop properly. Indeed, inhibition of nanos in zebrafish by morpholino injection (antisense oligonucleotide inhibiting mRNA translation) shows that Nanos is also essential for proper migration and survival of PGCs (Koprunner et al., 2001). The ovaries of adult female zebrafish mutant for nanos show almost only late stage oocytes. Thus like in Drosophila, nanos is required to maintain oocyte production in adult ovaries (Draper et al., 2007).
III.3.3. Dazl.
Dazl (Deleted in azoospermia-like) is a gene, which encodes another RNA binding protein. Dazl and its Drosophila homolog boule are members of the Daz family, which comprises three members (Eberhart et al., 1996). The first gene discovered of this family is DAZ in humans, which is located on the Y chromosome and whose deletion causes male infertility. Dazl is a daz orthologous gene, required for germline development in several organisms (Xu et al., 2001). For example, in Drosophila, a mutation in the Daz- family member boule also shows a defect in male fertility, since it blocks spermatogenesis during meiosis.
dazl RNA is expressed specifically in germ cells and its protein plays a role in meiosis entry (Castrillon et al., 1993). The homology between dazl and boule was demonstrated by the rescue of the Drosophila boule mutant with Xenopus dazl (Houston et al., 1998).
In Xenopus, Xdazl mRNA is present during several stages of development: In the germ plasm and primordial germ cells of the early embryo, in spermatogonia and spermatocytes and during oogenesis in the Balbiani body at the vegetal pole. When dazl is depleted in oocytes, the number of PGCs in tadpoles seems to be reduced and they fail to migrate properly (Houston and King, 2000). More practically, dazl RNA is the most specific marker to highlight the germ plasm by in situ hybridization in zebrafish and will be used in this study to label the Balbiani body, the germ plasm during oogenesis (Howley and Ho, 2000; Kosaka et al., 2007; Maegawa et al., 1999).
In summary, many of the conserved germ plasm components are RNA binding proteins and are found in almost every organism.
III.4. Germ plasm assembly
Although many components of the germ plasm have been identified, they do not explain the biochemical functions of the germ plasm and the mechanisms of germ cell induction.
The assembly of the germ plasm is even less well understood. So I will first give a short historical overview about the formation of the Balbiani body, an evolutionary conserved structure aggregating germ plasm at the beginning of oogenesis. Then I will describe germ plasm formation in the Drosophila oocyte, where the molecular characterization of this process is more advanced then in other animals.
III.4.1. Germ plasm and the Balbiani body.
After the experiments of Bonoure, for about 50 years the origin of the germ plasm at the vegetal pole of the frog embryo was unknown. Then, observing the migration of mitochondria in vivo revealed a connection between a structure called the mitochondrial cloud or the Balbiani body and the vegetal germ plasm in Xenopus (Heasman et al., 1984).
The Balbiani body has previously been described in the oocytes of several organisms.
Von Wittich first reported a round structure that he called “vitelline nucleus” in spider oocytes (von Wittich, 1845). Several years later Balbiani described this structure inside the early oocyte in more detail (Figure 4) and since then a large number of publications refer to this structure as the “Balbiani Body” (Bb) (Balbiani, 1864; Kloc et al., 2004).
During a long time, scientists thought that the Bb is the area, where the yolk was concentrated. In organism like Xenopus, the Bb is formed at the beginning of oogenesis (previtellogenic stage) exactly when the oocytes start to grow significantly by yolk endocytosis.
A careful study observed that the Bb accumulates about half a million of mitochondria and thus forms a spherical mass called the “mitochondria mass” or "mitochondrial cloud"
suggesting that Bb is a replication site for mitochondria (Marinos and Billett, 1981).
Figure 4. Section through the center of a Xenopus laevis oocyte with a diameter of around 200µm, stained with diaminobenzidine. The mitochondrial cloud (MC) is compact and adjacent to the light gray germinal vesicle, the nucleus of the oocyte, from (Marinos and Billett, 1981).
Different studies suggest that maternal components are stored in the Bb, like mitochondria, which are needed for embryogenesis. Fluorescent, microscopic time lapse movies and ultrastructural studies report another role of the Bb like the source of germinal granule material (Heasman et al., 1984). Mitochondria and granulofibrillar materials accumulate during previtellogenic stages in the Bb, which then breaks down into islands and finally moves in a cone-shaped form, towards the vegetal pole (Figue5).
Figure 5. Diagram of mitochondrial cloud formation and dispersion in Xenopus oogenesis and embryogenesis. (1) In prestage I and early stage I oocytes, nuage material (marked in pink) leaves the nucleus and comes into close contact with mitochondria (green), thus becoming the mitochondrial cement within the primary mitochondrial cloud (MC). (2) In stage I oocytes, a round MC is located in the vicinity of the oocyte nucleus. Germinal granules are concentrated in the MC. (3) Between stages II and IV of oogenesis, the MC fragments into islands that move toward the vegetal cortex. (4) Between stages IV and VI of oogenesis, the germ plasm islands (remnants of the MC) anchor in the vegetal cortex of the oocyte. From (Kloc et al., 2004)
The Bb is a cellular structure, which was found in developing oocytes of every species examined with the exception of C. elegans: In the fly Drosophila (Cox and Spradling, 2003) the mouse (Pepling et al., 2007) and humans (Albamonte et al., 2008). Although the Balbiani body is such a widespread structure in oocytes of many species, its connection with the germ plasm in the early oocyte has been only shown in Xenopus and
Drosophila (Cox and Spradling, 2003; Heasman et al., 1984). So it might not harbor the germ plasm in all organisms, where it has been observed.
A lack of molecular markers prevents in many organisms to make the connection between the Balbiani body and the germ plasm. In Xenopus several RNAs such as vasa, nanos (Xcat2 in Xenopus) or Xdazl localize to the germ plasm, but this localization is not conserved in other organisms. For example the vasa gene is expressed in germ cells of all organisms, but the localization of its mRNA to the germ plasm has not been reported in other animals like in Xenopus (Noce et al., 2001; Raz, 2000).
In summary, the Balbiani body at the beginning of oogenesis is present in many species, but only in few species has been shown to represent a precursor of the germ plasm of the mature egg.
As already mentioned, many genes expressed in vertebrate germ cells were originally discovered in Drosophila, since many components like vasa, nanos and dazl are conserved during animal evolution. These genes are not only conserved on the sequence level, but frequently show a similar expression pattern during germ line development or even can replace each other, like the example of the Drosophila boule mutant rescued with Xenopus dazl described previously. Since gamete formation and reproduction are fundamental processes for the survival of a species in Metazoans, it is not surprising that these key genes are evolutionary conserved (Extavour and Akam, 2003).
Surprisingly, oskar is found only in the genomes of Dipterans (Ephrussi et al., 1991; Juhn and James, 2006; Kim-Ha et al., 1991; Webster et al., 1994), although it is at the top of
the hierarchy in germ plasm formation, since it controls nanos and vasa mRNA localization (Thomson and Lasko, 2005), Oskar is the only gene sufficient to induce germ plasm formation. A gene with this activity has never been described in vertebrates.
The similarity between Xenopus germ plasm and Drosophila pole plasm suggests that a homologous mechanisms exists for localizing germ cell-specifying molecules (Wylie, 1999). The only other gene with similar activity is Xpat in Xenopus, which has not been found in the genomes of other organisms. Indeed, when Xpat is injected in the oocyte, ectopic granules are formed similar to germ plasm in the oocyte (Machado et al., 2005), but whether Xpat is sufficient to form ectopic germ cells in the embryo was not tested.
In summary, whereas the players of germ cell development such as vasa are conserved during animal evolution, the key regulators of germ plasm formation such as oskar were not discovered in other animals.
III.4.2. Germ plasm assembly in Drosophila
Oskar is the only gene, which is able to organize germ plasm components and it was found only in flies. To understand how oskar organizes germ plasm in Drosophila, this chapter describes pole plasm and pole cell formation in Drosophila.
After fertilization, the first nuclear divisions are initiated without cytokinesis. Thus, the nuclei form a syncytium not separated by a plasma membrane (Figure 6). A group of nuclei, around 10, migrate to the posterior end (Sullivan et al., 1993), where the germ plasm is localized (red in Figure 6). At syncytial blastoderm stage, the vast majority of
nuclei migrates to the embryonic cortex and will become separated by a plasma membrane, so that the embryo is cellularized only during the fourteenth cell cycle.
Figure 6. Drosophila germ line development from fertilization to the blastoderm stage.
Drosophila pole plasm is localized at the posterior end of the mature egg (red, left panel). The early embryo undergoes synchronous nuclear divisions without cytokinesis (middle panel).
Nuclei that arrive at the posterior end of the embryo are the first to cellularize, thus forming the pole cells which contain the pole plasm (right panel) (adapted from (Mahowald, 2001; Matova and Cooley, 2001).
III.4.2.1. Pole plasm assembly in Drosophila.
During development, the polar granules are localized at posterior pole, close to the cortex (red in Figure 6). Since the pole cells always form at the posterior pole, defects in anterior/posterior patterning also interfere with pole cell formation. For example, genes like cappuccino or spire are important for anterior-posterior polarity of the oocyte and a defect in their function disrupts pole cell formation (Manseau and Schupbach, 1989) . The process of germ plasm localization at the posterior pole is separated in several phases: the transport of germ plasm components from the nuage in the nurse cells and the assembly of functional pole plasm at the posterior pole of the oocyte. Ectopic transplantation of pole plasm into the embryo creates additional pole cells demonstrating
that in Drosophila germ plasm is sufficient to establish the germ cell linage (Illmensee and Mahowald, 1974).
III.4.2.2. Oskar the orchestral leader of the germ cell symphony.
One of the most important genes for germ plasm assembly is oskar. It acts like a master gene that directs the other genes. oskar is required for pole plasm formation since in oskar mutants this process does not take place. In addition, ectopic localization of oskar at the anterior pole can be induced by replacement of its mRNA localization sequence (3’UTR of oskar) by the 3’UTR of bicoid. This additional anterior localization of oskar mRNA results in the formation of additional pole cells at the anterior pole mimicking the transplantation of germ plasm (Ephrussi et al., 1991; Ephrussi and Lehmann, 1992; Smith et al., 1992).
Taken together, this means that oskar is not only required but also sufficient for pole cells formation and represents the only gene in the animal kingdom, which can induce functional pole cells (Lehmann and Ephrussi, 1994).
The easiest way to understand and remember the function of the different genes is to compare each of them to the musicians of a big symphony orchestra, where oskar plays the role of a conductor. Several nuclear shuttling proteins (Kinesin I, Mago Nashi, Y14, Staufen) are required for oskar mRNA localization (Hachet and Ephrussi, 2001). In fact, they take care of oskar like a chauffeur. Oskar mRNA comes from the close country (the nurse cells). It is brought to its final destination, the city, where there is an auditorium (the oocyte). The road, which it follows, is constituted by polarized microtubules.
The conductor is not yet ready to manage the group (not yet translated in protein). He will be ready only after arrival at the auditorium (posterior pole). Thus oskar mRNA should be “active” only when its body guard (Bruno) is alright, (permits translation in Oskar protein). The auditorium, where the group will play, is localized at the posterior pole of the oocyte. But, as explained above in the text, its road map is its 3’UTR, which means, that if oskar has another map (3’UTR of bicoid see part before) it goes in the wrong direction, and the music will be played elsewhere. To conclude Oskar is probably the most important actor in the germ plasm symphony. Even if it needs other genes, where Oskar goes, the music will be played. Every organism has to play music to generate germ cells for the species to survive. Moreover, every organism has almost the same music players but surprisingly, it seems that only Drosophila has the music conductor!
III.4.2.3. Germ plasm cellularization. – Pole cell formation.
The group of 10 nuclei will be the first nuclei surrounded by a plasma membrane and contain the germ plasm localized at the posterior pole. (red in Figure 6). In the embryo, all the germ plasm components are fundamentally important, and it is the priority of the embryo to quickly protect them by a membrane.
Thus, the cellularized germ plasm will give rise to the first embryonic cells, separated from the rest of the embryo and called, pole cells or primordial germ cells (PGCs) (Huettner, 1923). So, the pole cells will give rise to the future gametes in the adult fly. A modification of germ plasm quantity will modify the number of gametes. For example, the oskar gene, expressed during oogenesis and localized in posterior pole, regulates in a
concentration-dependent manner the number of pole cells (Ephrussi and Lehmann, 1992).
Moreover, other genes such as mtlr (mitochondrial RNA) restore after injection the ability of cellularization when the embryo was irradiated, but in contrast to oskar, mtlr does not give rise to extra germ cells when its RNA is localized to the anterior cortical region (Okada, 1998). This means that oskar is this only gene sufficient to initiate the whole program of germ cell formation.
III.5. Zebrafish as a vertebrate model to study maternal factors.
To study the genes or more precisely the maternal factors, which control the first steps of development, it is important to select laboratory animals with suitable characteristics.
Zebrafish combines several properties useful for research: The propagation of zebrafish lines does not require extensive training or expertise. Although the mouse genome is much more similar to humans, the zebrafish is a vertebrate and thus, its genome is also comparable to humans. More and more researchers work with this model organism and contribute to complete the sequencing of the zebrafish genome. These different efforts facilitate the identification of mutant genes.
The major strength of zebrafish is its suitability for forward genetics in systematic mutagenesis screens. Zebrafish has a small body size, but it is extremely productive. A zebrafish couple produces around 100 eggs by week. The zebrafish has also a relatively short generation time for a vertebrate, since it will develop into an adult fish in three months, which is then able to reproduce. Like in Drosophila this high reproductive rate is helpful for forward genetics.
Additionally, the zebrafish embryo is transparent during the first days of its life. The transparency allows us to study, in vivo, the first steps of development and the biological processes like organogenesis. One can also investigate protein localization, when the gene of interest is fused to a reporter gene like GFP (green fluorescent protein), which allows following its dynamics under the fluorescent microscope. So the transparency of the embryo allows detailed investigation of cell migration and organogenesis and the feasibility of observing these processes in the living animal has provided novel insights in these areas of vertebrate embryogenesis in vertebrates.
The microinjection into the 1-cell stage embryo is frequently used in zebrafish. External fertilization easily permits the microinjection with several molecules. Moreover, the relative big size of the 1-cell embryo, around 0.8 mm, is another principal advantage for microinjection. For all these reasons, it is quite simple to obtain a transgenic line in zebrafish, but also to study the effect of antisense oligonucleotides (morpholinos), to microinject mRNAs or chemical molecules. Furthermore, transplantation techniques are also possible in zebrafish and allow us to perform mosaic analysis i.e. to study mutant or transgenic cells in a wild-type background or vice versa.
In addition, zebrafish live in water, so it is easy to adjust the concentration of chemical products added to their natural medium. Consequently, we can easily control the precise concentration of pharmaceutical drugs, pollutants or N-ethyl-N-nitrosourea (ENU) used for mutagenesis treatment.
However, some genetic tools are not yet developed in zebrafish like in Drosophila and mice for example to obtain a knock-out animal. As an alternative to knock-out genes,
tilling and zinc finger nucleases have been successfully applied to zebrafish (Ekker, 2008; Maeder et al., 2009).
All together these properties and the available methods distinguish zebrafish as a good model organism to study genes implicated in maternally controlled processes during embryogenesis such as germ plasm localization in vertebrates.
III.6. Germ line development in zebrafish
During zebrafish development the localization of germ cells inside the organism and of germ plasm inside the germ cells is very dynamic (Figure 7). Because germ cells are immortal and constantly transmitted from one generation to the next, there is no beginning and no end of their development. So unlike other cell types in the organism germ cell development represents a cycle. In this part, I arbitrarily chose to start the description of germ line development at the moment of fertilization.
III.6.1. Germ plasm localization during embryogenesis in zebrafish.
Figure 7. Germ line development in zebrafish. In each small pictogram, the red granules and red area represent the germ plasm, which segregates from the rest of the embryo. Stages written in blue indicate a lateral view, and black writing an animal view. The colored arrows represent the transition between developmental stages: red arrows, when the development is under control of maternal factors (j-e). Blue arrows, when the zygotic genome is activated (f-j). The transition between maternal and zygotic control is symbolized by a purple arrow (e-f). (Figure adapted from (Pelegri, 2003; Raz, 2003))
(a) At the time of fertilization the germ plasm, represented here by vasa mRNA in red, is localized at the base of the blastodisc (blue) close to the yolk (yellow). The blastodisc
represents the first cell and will give rise to all cells of the embryo. (b) After 45 min, the first division is established and the germ plasm is recruited to two domains of the first cleavage furrow. (c) The development continues and gives rise to the 4-cell embryo after a second cleavage. The germ plasm is now also localized to the second cleavage furrow forming four domains in the embryo. At this stage, the only protein known to be localized to the germ plasm at the cleavage furrow is Ziwi, the zebrafish Piwi homolog (Houwing et al., 2007). Depletion of the germ plasm at the cleavage furrows generates sterile zebrafish demonstrating that it is required for germ cell formation (Hashimoto et al., 2004). (d-e) During the next stages of development until 3-4 hours post fertilization, only four cells will keep the germ plasm due to asymmetric cell division. The insert in (e) magnifies a dividing germ cell, in which the two arrowheads symbolize the spindle. The germ plasm (red) segregates with one of the centrosomes of the spindle and eventually is localized to only one of the two daughter cells. (f) At sphere stage the embryonic genome is activated and the germ plasm is now distributed symmetrically in the two daughter cells of the PGCs (see insert (f)). After this symmetric division, the germ plasm will be distributed into four cell clusters. (g-h) During the next period of development, the primordial germ cells migrate in a dorsal direction (arrows) to the shield, the zebrafish Spemann organizer (indicated with a blue semi-circle on the right side of the embryo in g). (i) At 1 day post fertilization the PGCs arrive at the future gonads. (j) In the adult the primordial germ cells will differentiate into sperm in males and into oocytes in females.
The insert represents an oocyte at stage I with germ plasm inside the Balbiani body (red dots).
III.6.2. Germ plasm localization during oogenesis in zebrafish
Figure 8. Schematic representation of germ plasm localization during the five stages of oogenesis in zebrafish. The center of the schema points to the vegetal pole; the germ plasm in red, localized in the vegetal part, is always orientated towards the centre of the figure; the animal marker, in green, is always localized at the exterior. Note that that in an ovary all stages are intermingled and the oocytes in this scheme are not drawn to scale. The diameter of oocytes at specific stages is indicated. (Figure adapted from (Bally-Cuif et al., 1998; Clelland et al., 2007; Selman et al., 1993)).
a) Stage Ia: oocytes form a nest of connected oocytes.
b) Stage Ib: the oocyte is surrounded and individualized by a follicle cell layer. In this stage the germ plasm aggregates to form the Balbiani body in the oocyte (red in Figure 8). At stage I the oocyte begins to grow. In fact, the Balbiani body was believed to control yolk accumulation, since it forms at the beginning of vitellogenesis (see paragraph, Germ plasm and the Balbiani body). At the end of stage I the oocyte is still transparent, because it does not contain a lot of yolk. The transparency allows to use living markers like DIOC6 to visualize mitochondria and the endoplasmic reticulum, which are components of the Balbiani body. Moreover, in situ hybridization shows that the germ plasm mRNAs vasa, nanos and dazl are also localized to the Balbiani body (Kosaka et al., 2007). During stage I, the axis of Balbiani body and nucleus (germinal vesicle, grey) indicates for the first time the animal-vegetal polarity of the oocyte, which later develops into the anterior-posterior axis of the embryo.
c) Stage II is also called cortical alveolus stage, due to the size variability of cortical granules (light vesicles) surrounding the germinal vesicle. The vitelline envelope or chorion, an acellular membrane surrounding the oocyte, becomes also more prominent.
Moreover, germ plasm mRNAs spread towards vegetal pole (red, arrows indicate direction of germ plasm movement). CyclinB1 mRNA (green) is localized to the cortex of the animal pole until embryogenesis and represents the earliest molecular marker for the animal pole (Howley and Ho, 2000).
d) Stage III: The oocyte grows massively as a result of vitellogenesis; yolk protein accumulates to such a high concentration that it forms crystalline structures within yolk bodies (brown vesicles). The oocyte is ready to react to steroid hormone, which triggers
oocyte maturation. For the first time morphological animal/vegetal asymmetries become visible: the formation of the micropylar cell (unique red spot in the follicle cells layer at the animal pole). The mRNAs localized at the animal and vegetal pole are more spread along the oocyte membrane and will to stay at the cortex until embryogenesis
e) Stage IV: Oocyte maturation occurs including germinal vesicle migration towards the animal pole and subsequent germinal vesicle breakdown (GVBD) as a hallmark of the completion of the first meiotic cell cycle. During oocyte maturation the yolk proteins are cleaved proteolytically, which makes the oocyte transparent.
f) Stage V: The oocyte is now mature and called an egg. During ovulation the oocyte is released from the follicle cell layer into the oviduct and is able to be fertilized. In fact, the micropylar cell forms the micopyle, an actin canal, which allows sperm entry through the chorion.
g) 1-cell embryo: Fertilization represents the moment when many events happen at the same time. It is the transition between oogenesis and embryogenesis, when the blastodisc forms and the egg is activated. Cytoplasm segregates from the yolk and starts to migrate towards the animal pole to form the blastodisc. During this time, mRNAs are redistributed: the germ plasm mRNAs migrate towards the animal pole (red arrows indicate direction of germ plasm movement). vasa, dnd and nos1 mRNAs were reported to be enriched at the base of the blastodisc, where the rearrangement of the cytoskeleton (f-actin and microtubules) organizes the germ plasm mRNAs. Thus, the germ plasm will become enriched in the cytokinetic ring (red between blastodisc in green and yolk in yellow). daz-like mRNA is recruited a little bit later to the germ plasm suggesting a separate route of RNA transport. Moreover, dazl mRNA localization in the embryo does
not completely overlap with vasa and nanos mRNA indicating compartmentalization of the germ plasm (Theusch et al., 2006). Recently, three elements in the dazl 3’UTR have been mapped, which seem to be important for its localization during oogenesis and embryogenesis (Kosaka et al., 2007), but the significance of the different RNA localization domains within the germ plasm is still unresolved. During the next stages of embryogenesis the localization of germ plasm mRNAs is segregated asymmetrically during cell divisions until the activation of the zygotic genome (see Figure 7f).
III.7. Transition between maternal and zygotic gene expression in zebrafish.
The egg is full of mRNAs and protein molecules, which are produced and deposited by the mother inside the oocyte. These components are important for the early steps of embryonic development and control the first hours of embryogenesis before the activation of the embryonic genome, called zygotic activation. One of the genes expressed during this genome activation in zebrafish encodes micro RNA-430.
Figure 9. During embryonic development, micro RNA-430 expression is activated during the transition from maternal (red) to zygotic period (blue). In the absence of micro RNA-430, maternal mRNAs persist longer and interfere with normal embryonic development. Figure from (Cohen and Brennecke, 2006; Weigel and Izaurralde, 2006)
Micro RNA-430 promotes the maternal mRNA turnover by targeting 40% of several hundred maternal mRNAs for degradation (Giraldez et al., 2005). Micro RNA-430 targets mRNAs by specific sequences in the 3’UTR leading to deadenylation, which causes rapid mRNA decay. The poly-A tail is known to stabilize the RNA and its translation. A well-characterized example in germ cells is the interaction between miRNA and nanos mRNA involving Dead end (DND) protein to regulate nanos expression
Dead end (DND) protein is an activator of nanos mRNA translation. When dead end morpholino is injected, the protein Dead end is not translated and thus cannot block the
binding of micro RNA-430, which in a wild type situation inhibits the translation of nanos mRNA (Figure 10). So, translation inhibition of dead end inhibits the translation of nanos, which is required for primordial germ cell migration and survival (Kedde et al., 2007). After the loss of germ cells, every fish becomes a male and is unfertile (Weidinger et al., 2003). So Nanos is an important player in PGC survival.
Figure 10. Schematic model depicting the mechanism of Dnd1 action. The miRNA-RISC complex loaded with miRNAs targeting a 3′UTR inhibits mRNA translation (upper panel). By binding U-rich regions in the 3′UTR, Dnd1 prevents miRNAs from binding to and inhibiting translation, thereby allowing mRNA translation (lower panel). CR: coding region. (RISC): RNA- Induced Silencing Complex. Figure from (Kedde et al., 2007)
III.7.1. Maternal-effect mutant.
In zebrafish, the maternal/zygotic transition takes place 3-4 hours after fertilization. At this time, the embryo has around one thousand cells. Before this time all the development is under control of the maternal genome and consequently, a zygotic mutation does not show a phenotype. At the contrary, if the mutation affects a maternal gene, it will appear, if the mother is homozygous, even if the embryo is not homozygous for the mutation.
Figure 11. Difference between recessive zygotic and maternal mutant. The colors represent the genotype; the full red for homozygous mutant and red stripes for heterozygous carriers. The shape of the embryo represents the phenotype: a totally round embryo corresponds to a mutant phenotype (every embryo in B), whereas the yellow blastodisc indicates a wild-type embryo.
(A) A zygotic mutation gives rise to a mutant phenotype after the embryonic genome is activated in zebrafish 3 hours post fertilization. So, only the red embryos (25%) have a mutant phenotype.
(B) All the offspring of a maternal mutant female show a mutant phenotype (100%) even if the genotype is heterozygous. During the first part of development, the embryo is under
control of the genotype of its mother, since the zygotic genome is not yet activated.
Therefore zygotic mutations affecting the one-cell embryo before 3-4 hours post fertilization (hpf) like the hypothetical (Figure 11 A) do not exist. In a maternal-effect mutant the genotype of the male has no influence on the phenotype of the embryos. Since germ plasm localization occurs before activation of the embryonic genome, this process is controlled by maternal factors (Figure 12).
III.8. The isolation of maternal factors in vertebrates
The most successful approach to discover maternal regulators are systematic mutagenesis screens in invertebrates. To study processes controlled by maternal factors in vertebrates and to identify key genes mediating these processes, a systematic, recessive maternal- effect mutant screen in the zebrafish was conducted (Dosch et al., 2004). 68 maternal- effect mutants were identified, about half of which displayed specific abnormalities. 1 of the 15 mutations that affect embryogenesis prior to the onset of zygotic transcription shows a defect in animal-vegetal polarity and thus, is called bucky ball (buc). The bucky ball mutant name comes from a completely round chemical structure, the Buckminster Fullerenes, which show no polarity similar to the mutant phenotype.
Figure 12. Bucky ball: sphere molecule composed entirely of carbon, which does not show polarity like the mutant embryo in Figure 13 B.
Although it is the homozygous female, which is the actual homozygous mutant, I will refer to the embryo showing the mutant phenotype, when I use the term "mutant".
III.8.1. Description of the polarity phenotype of the buc mutant
Figure 13. Morphological phenotype of the bucky ball mutant. Living embryos 30 minutes post fertilization, lateral views, animal pole to the top. The polarity of cytoplasmic streaming causes the blastodisc to form specifically at the animal pole in wild type (A). In the bucky ball mutant (bucp106re) the cytoplasm segregates radially around the circumference of the yolk and subsequent cellular cleavages do not occur (B). In the bucky ball mutant, cytoplasmic streaming was evident in multiple orientations, rather than in a single orientation toward the animal pole as in wild type, suggesting a defect in animal-vegetal polarity of the egg (Dosch et al., 2004).
To investigate the animal-vegetal polarity phenotype of bucky ball mutant embryos, two localized mRNAs were examined as molecular markers for embryonic polarity: cyclinB mRNA (Kondo et al., 1997) and bruno-like mRNA (Howley and Ho, 2000; Suzuki et al., 2000).
Figure 14. Molecular markers showing embryonic polarity defect in bucky ball. Whole-mount in situ hybridizations of wild-type and mutant embryos, 30 minutes post fertilization, lateral views, animal pole to the top. The cyclinB mRNA, which is normally localized to the animal pole of the egg (A), extends around the circumference of the mutant (B) The bruno-like mRNA, which localizes to the animal and vegetal poles following egg activation (C), is not localized in the mutant (D) Since mRNA at both the animal and the vegetal pole is unlocalized, the animal- vegetal axis appears disrupted, rather than an animal- or vegetal-specific RNA localization mechanism. (Dosch et al., 2004)
In summary, the phenotype and mislocalization of mRNA in the bucky ball mutant suggest a defect in embryonic polarity along the animal-vegetal axis.
To better understand the biological role of the bucky ball gene this thesis has the following goals:
1. Study the bucky ball mutant phenotype.
2. Identify the bucky ball gene in the zebrafish genome.
3. Characterize the bucky ball gene.
