Scott T. Dougan

Zebrafish organizer development and germ-layer formation require nodal-related signals

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells,. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-β (TGF-β) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-β superfamily that is related to mouse nodal. cyc encodes another nodal-related protein,, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation,. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.

MEC-17 is an α-tubulin acetyltransferase

In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the ε-amino group of K40 on α-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the significance of microtubule acetylation have been limited by the undefined status of the α-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases and required for the function of touch receptor neurons in Caenorhabditis elegans, acts as a K40-specific acetyltransferase for α-tubulin. In vitro, MEC-17 exclusively acetylates K40 of α-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R α-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans, MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 α-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of α-tubulin, the only PTM known to occur on the luminal surface of microtubules.

The role of the zebrafish nodal-related genes Squint and Cyclops in patterning of mesendoderm

Nodal signals, a subclass of the TGFβ superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt-/-; cyc+/+ and sqt-/-; cyc+/- mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt-/-; cyc+/- or sqt-/-; cyc+/+ embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.

TTLL3 Is a Tubulin Glycine Ligase that Regulates the Assembly of Cilia

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffers vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish

BackgroundThe vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.ResultsWe show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffers vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated.ConclusionWe conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development

BackgroundThe post-translational addition of the monosaccharide O-linked β-N-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of ogt function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two ogt genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically.ResultsWe manipulated O-GlcNAc levels in zebrafish embryos by overexpressing zebrafish ogt, human oga or by injecting morpholinos against ogt transcripts. Each of these treatments results in embryos with shortened body axes and reduced brains at 24 hpf. The embryos had 23% fewer cells than controls, and displayed increased rates of cell death as early as the mid-gastrula stages. An extensive marker analysis indicates that derivatives of three germ layers are reduced to variable extents, and the embryos are severely disorganized after gastrulation. Overexpression of Ogt and Oga delayed epiboly and caused a severe disorganization of the microtubule and actin based cytoskeleton in the extra-embryonic yolk syncytial layer (YSL). The cytoskeletal defects resemble those previously reported for embryos lacking function of the Pou5f1/Oct4 transcription factor spiel ohne grenzen. Consistent with this, Pou5f1/Oct4 is modified by O-GlcNAc in human embryonic stem cells.ConclusionWe conclude that O-GlcNAc modifications control the activity of proteins that regulate apoptosis and epiboly movements, but do not seem to regulate germ layer specification. O-GlcNAc modifies the transcription factor Spiel ohne grenzen/Pou5f1 and may regulate its activity.

Identification of phagocytic cells, NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish.

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin-EDTA contained 0.80-1.20 x 10(5) and 2.0-3.5 x 10(5) cells, respectively. Media lavage was composed of granulocytes (60-80%), lymphocytes (10-20%), and NCC (4-10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1(+). Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1(-) and NCCRP-1(-). NCC were 3 microm, NCAMP-1(+) and NCCRP-1(+) and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.

The evolutionary origin of nodal-related genes in teleosts.

Because of an extra whole-genome duplication, zebrafish and other teleosts have two copies of genes that are present in a single copy in tetrapod genomes. Some zebrafish genes, however, are present in triplicate. For example, the nodal-related genes encode secreted proteins of the transforming growth factor β superfamily that are required in all vertebrates to induce the mesoderm and endoderm, pattern all three germ layers, and establish the left–right axis. Zebrafish have three nodal-related genes, called ndr1/squint, ndr2/cyclops, and ndr3/southpaw. As part of an analysis of enhancer elements controlling zebrafish nodal-related gene expression, we analyzed the nodal loci in the sequenced genomes of five teleost species and four tetrapod species. Each teleost genome contains three nodal-related genes, indicating that squint, cyclops, and southpaw orthologues were present early in the teleost lineage. The genes flanking the nodal-related genes are also conserved, demonstrating a high degree of conserved synteny. Although we found little homology outside of the coding sequences in this region, pufferfish enhancer sequences work in zebrafish embryos to drive reporter gene expression in the squint expression pattern. This indicates a high degree of functional conservation of enhancer elements within the teleosts. We conclude that the ancestral squint and cyclops genes arose during the teleost-specific whole-genome duplication event and that southpaw emerged from a subsequent duplication event involving ancestral squint.

Embryological Manipulations in Zebrafish

Due to the powerful combination of genetic and embryological techniques, the teleost fish Danio rerio has emerged in the last decade as an important model organism for the study of embryonic development. It is relatively easy to inject material such as mRNA or synthetic oligonucleotides to reduce or increase the expression of a gene product. Changes in gene expression can be analyzed at the level of mRNA, by whole-mount in situ hybridization, or at the level of protein, by immunofluorescence. It is also possible to quantitatively analyze protein levels by Western and immunoprecipitation. Cell behavior can be analyzed in detail by cell transplantation and by fate mapping. Because a large number of mutations have been identified in recent years, these methods can be applied in a variety of contexts to provide a deep understanding of gene function that is often more difficult to achieve in other vertebrate model systems.

correction: arrow encodes an LDL-receptor-related protein essential for Wingless signalling

This corrects the article DOI: 35035110