Roland Dosch

The zebrafish germline is specified during early embryogenesis by inherited maternal RNAs and proteins collectively called germ plasm. Only the cells containing germ plasm will become part of the germline, whereas the other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is key for germ cell specification and its removal is crucial for the development of the soma. The molecular mechanism underlying this process in vertebrates is largely unknown. Here, we show that germ plasm localization in zebrafish is similar to that in Xenopus but distinct from Drosophila. We identified non muscle myosin II (NMII) and tight junction (TJ) components, such as ZO2 and claudin-d (Cldn-d) as interaction candidates of Bucky ball (Buc), which is the germ plasm organizer in zebrafish. Remarkably, we also found that TJ protein ZO1 colocalizes with germ plasm, and electron microscopy of zebrafish embryos uncovered TJ-like structures at the cleavage furrows where the germ plasm is anchored. In addition, injection of the TJ receptor Cldn-d produced extra germ plasm aggregates, whereas expression of a dominant-negative version inhibited germ plasm aggregate formation. Our findings support for the first time a role for TJs in germ plasm localization.

The zebrafish germline is specified during early embryogenesis by inherited maternal RNAs and proteins collectively called germ plasm. Only the cells containing germ plasm will become part of the germline, whereas other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is key for germ cell specification and its removal is critical for the development of soma. The molecular mechanism underlying this process in vertebrates is largely unknown. Here we show that germ plasm localization in zebrafish is similar to Xenopus and amniotes but distinct from Drosophila . We identified non muscle myosin II (NMII) and tight junction (TJ) components as interaction candidates of Bucky ball (Buc), which is the germ plasm organizer in zebrafish. Remarkably, we also found that TJ protein ZO1 colocalizes with germ plasm and electron microscopy (EM) of zebrafish embryos uncovered TJ like structures at early cleavage furrows. In addition, injection of the TJ-receptor Claudin-d (Cldn-d) produced extra germ plasm aggregates. Our findings discover for the first time a role of TJs in germ plasm localization.

Protein–protein interactions (PPIs) play a central role in all cellular processes. The discovery of green fluorescent protein (GFP) and split varieties, which are functionally reconstituted by complementation, led to the development of the bimolecular fluorescence complementation (BiFC) assay for the investigation of PPI in vivo. BiFC became a popular tool, as it is a convenient and quick technology to directly visualize PPI in a wide variety of living cells. In combination with the transparency of the early zebrafish embryo, it also permits detection of PPI in the context of an entire living organism, which performs all spatial and temporal regulations missing in in vitro systems like tissue culture. However, the application of BiFC in some research areas including the study of zebrafish is limited due to the lack of efficient and convenient BiFC expression vectors. Here, we describe the engineering of a novel set of Gateway^®-adapted BiFC destination vectors to investigate PPI with all possible permutations for BiFC experiments. Moreover, we demonstrate the versatility of these destination vectors by confirming the interaction between zebrafish Bucky ball and RNA helicase Vasa in living embryos.

Immunohistochemistry has been widely used as a robust technique to determine the cellular and subcellular localization of proteins. This information ultimately helps to understand the function of these proteins and how biological processes are regulated. Antibodies applicable for labeling in zebrafish are limited, making immuno-staining challenging. Recently glyoxal fixation was rediscovered in tissue culture, mouse, rat, and Drosophila, expanding the list of effective antibodies for these species. Here, we compare a protocol for zebrafish staining using glyoxal as a fixative agent with PFA. We demonstrate that glyoxal fixation improves the antigenicity of some epitopes thereby increasing the number of useful antibodies in zebrafish.

Human infantile-onset RNASET2 deficient cystic leukoencephalopathy is a Mendelian mimic of in utero cytomegalovirus brain infection with prenatally developing inflammatory brain lesions. We used a RNASET2 deficient zebrafish model to elucidate the underlying disease mechanisms. Mutant and wildtype zebrafish larvae brain development between 2 and 5 days post fertilisation was examined by confocal live imaging in fluorescent reporter lines of major types of brain cells. In contrast to wild type brains, RNASET2 deficient larvae displayed increased numbers of microglia with altered morphology often containing inclusions of neurons. Furthermore, lysosomes within distinct populations of the myeloid cell lineage including microglia showed increased lysosomal staining. Neurons and oligodendrocyte precursor cells remained unaffected. This study provides first insight into the prenatal onset pathomechanisms of human RNASET2 deficient leukoencephalopathy, linking this inborn lysosomal disease to the innate immune system and other immune-related childhood encephalopathies like Aicardi-Goutieres syndrome (AGS).

Germ cell research in vertebrates has traditionally been challenging, but recent breakthroughs have overcome technical difficulties, demonstrating and expanding the power of the zebrafish experimental system for their analysis in vivo. Exploiting the transparency of the zebrafish embryo, germ cell migration was the first topic that moved the germ cells of this organism into the spotlight of modern research. In recent years, research on teleost germ cells has expanded into additional fields, manifested by a session dedicated to this cell type at the European Zebrafish PI meeting in Trento.

The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. Here, we discover that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, we detect that both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, we show that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm. Multicellular organisms use gametes for their propagation. Gametes are formed from germ cells, which are specified during embryogenesis in some animals by the inheritance of RNP granules known as germ plasm. Transplantation of germ plasm induces extra germ cells, whereas germ plasm ablation leads to the loss of gametes and sterility. Therefore, germ plasm is key for germ cell formation and reproduction. However, the molecular mechanisms of germ cell specification by germ plasm in the vertebrate embryo remain an unsolved question. Proteins, which assemble the germ plasm, are known as germ plasm organizers. Here, we show that the two germ plasm organizers Oskar from the fly and Bucky ball from the fish show similar functions by using a cross species approach. Both are intrinsically disordered proteins, which rapidly changed their sequence during evolution. Moreover, both proteins still interact with conserved components of the germ cell specification pathway. These data might provide a first example of two proteins with the same biological role, but distinct sequence.

Oogenesis is an essential cellular and developmental process to prepare the oocyte for propagation of a species after fertilization. Oocytes of oviparous animals are enormous cells endowed with many, big cellular compartments, which are interconnected through active intracellular transport. The dynamic transport pathways and the big organelles of the oocyte provide the opportunity to study cellular trafficking with outstanding resolution. Hence, oocytes were classically used to investigate cellular compartments. Though many novel regulators of vesicle trafficking have been discovered in yeast, tissue culture cells and invertebrates, recent forward genetic screens in invertebrate and vertebrate oocytes isolated novel control proteins specific to multicellular organisms. Zebrafish is a widely used vertebrate model to study cellular and developmental processes in an entire animal. The transparency of zebrafish embryos allows following cellular events during early development with in vivo imaging. Unfortunately, the active endocytosis of the oocyte also represents a drawback for imaging. The massive amounts of yolk globules prevent the penetration of light-beams and currently make in vivo microscopy a challenge. As a consequence, electron microscopy (EM) still provides the highest resolution to analyze the ultra-structural details of compartments and organelles and the mechanisms controlling many cellular pathways of the oocyte. Among different fixation approaches for EM, High Pressure Freezing (HPF) in combination with freeze substitution significantly improves the samples preservation closest to their natural status. Here, we describe the HPF with freeze substitution embedding method for analyzing cellular processes in zebrafish oocytes using electron microscopy.