Scott T. Dougan

MAX giant associated protein (MGA) is a dual transcriptional factor containing both T-box and bHLHzip DNA binding domains. In vitro studies have shown that MGA functions as a transcriptional repressor or activator to regulate transcription of promotors containing either E-box or T-box binding sites. BS69 (ZMYND11), a multidomain-containing (i.e., PHD, BROMO, PWWP, and MYND) protein, has been shown to selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3), modulates RNA Polymerase II elongation, and functions as RNA splicing regulator. Mutations in MGA or BS69 have been linked to multiple cancers or neural developmental disorders. Here, by TALEN and CRISPR/Cas9-mediated loss of gene function assays, we show that zebrafish Mga and Bs69 are required to maintain proper Bmp signaling during early embryogenesis. We found that Mga protein localized in the cytoplasm modulates Bmpr1a activity by physical association with Zmynd11/Bs69. The Mynd domain of Bs69 specifically binds the kinase domain of Bmpr1a and interferes with its phosphorylation and activation of Smad1/5/8. Mga acts to antagonize Bs69 and facilitate the Bmp signaling pathway by disrupting the Bs69–Bmpr1a association. Functionally, Bmp signaling under control of Mga and Bs69 is required for properly specifying the ventral tailfin cell fate.

The process of germ layer formation is a universal feature of animal development. The germ layers separate the cells that produce the internal organs and tissues from those that produce the nervous system and outer tissues. Their discovery in the early nineteenth century transformed embryology from a purely descriptive field into a rigorous scientific discipline, in which hypotheses could be tested by observation and experimentation. By systematically addressing the questions of how the germ layers are formed and how they generate overall body plan, scientists have made fundamental contributions to the fields of evolution, cell signaling, morphogenesis, and stem cell biology. At each step, this work was advanced by the development of innovative methods of observing cell behavior in vivo and in culture. Here, we take an historical approach to describe our current understanding of vertebrate germ layer formation as it relates to the long-standing questions of developmental biology. By comparing how germ layers form in distantly related vertebrate species, we find that highly conserved molecular pathways can be adapted to perform the same function in dramatically different embryonic environments.

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.

It has long been known that in a subset of microtubules, α-tubulin is modified post-translationally by acetylation of lysine-40. There is growing evidence that this highly conserved microtubule modification is a key event during cell polarization, especially in the nervous system. The enzyme responsible for this reaction has now been identified as MEC-17, a protein related to the Gcn5 histone receptor acetyltransferase and required for the function of touch receptor neurons in Caenorhabditis elegans. In eukaryotic cells, a subset of microtubules undergo post-translational modifications such as acetylation, which alters microtubule dynamics and trafficking of motors. These authors identify MEC-17 as the enzyme that directly acetylates α-tubulin in vitro and in vivo and in both invertebrates and vertebrates. This is the identification of the long-sought enzyme that acetylates microtubules. 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 microtubules1 that was discovered in the flagella of Chlamydomonas reinhardtii2,3. 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 acetyltransferases4 and required for the function of touch receptor neurons in Caenorhabditis elegans5,6, 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.

Background: The 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. Results: We 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. Conclusion: We 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.

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.