Robert K. Ho

A new time-scale for ray-finned fish evolution

The Actinopterygii (ray-finned fishes) is the largest and most diverse vertebrate group, but little is agreed about the timing of its early evolution. Estimates using mitochondrial genomic data suggest that the major actinopterygian clades are much older than divergence dates implied by fossils. Here, the timing of the evolutionary origins of these clades is reinvestigated using morphological, and nuclear and mitochondrial genetic data. Results indicate that existing fossil-based estimates of the age of the crown-group Neopterygii, including the teleosts, Lepisosteus (gar) and Amia (bowfin), are at least 40 Myr too young. We present new palaeontological evidence that the neopterygian crown radiation is a Palaeozoic event, and demonstrate that conflicts between molecular and morphological data for the age of the Neopterygii result, in part, from missing fossil data. Although our molecular data also provide an older age estimate for the teleost crown, this range extension remains unsupported by the fossil evidence. Nuclear data from all relevant clades are used to demonstrate that the actinopterygian whole-genome duplication event is teleost-specific. While the date estimate of this event overlaps the probable range of the teleost stem group, a correlation between the genome duplication and the large-scale pattern of actinopterygian phylogeny remains elusive.

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.

T-box gene tbx5 is essential for formation of the pectoral limb bud

The T-box genes Tbx4 and Tbx5 have been shown to have key functions in the specification of the identity of the vertebrate forelimb (Tbx5) and hindlimb (Tbx4). Here we show that in zebrafish, Tbx5 has an additional early function that precedes the formation of the limb bud itself. Functional knockdown of zebrafish tbx5 through the use of an antisense oligonucleotide resulted in a failure to initiate fin bud formation, leading to the complete loss of pectoral fins. The function of the tbx5 gene in the development of zebrafish forelimbs seems to involve the directed migration of individual lateral-plate mesodermal cells into the future limb-bud-producing region. The primary defect seen in the tbx5-knockdown phenotype is similar to the primary defects described in known T-box-gene mutants such as the spadetail mutant of zebrafish and the Brachyury mutant of the mouse, which both similarly exhibit an altered migration of mesodermal cells. A common function for many of the T-box genes might therefore be in mediating the proper migration and/or changes in adhesive properties of early embryonic cells.

Zebrafish stat3 is expressed in restricted tissues during embryogenesis and stat1 rescues cytokine signaling in a STAT1-deficient human cell line.

Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding‐induced activation of cognate receptors, STAT proteins are phosphorylated, hetero‐ or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal‐responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue‐restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon‐signaling functions in a STAT1‐deficient human cell line, indicating that cytokine‐signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352–370.

m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition

The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.

Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte.

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. We have further characterized one of these mRNAs, zorba, which encodes a homologue of the Drosophila Orb and Xenopus CPEB RNA-binding proteins. Zorba belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization is independent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA.

mRNA localization patterns in zebrafish oocytes

In both invertebrate and vertebrate systems, the localization of maternal mRNAs is a common mechanism used to influence developmental processes, including the establishment of the dorsal/ventral axis, anterior/posterior axis, and the germ line (for review, see Bashirullah et al., 1998. Annu. Rev. Biochem. 67, 335-394). While the existence of localized maternal mRNAs has been reported in the zebrafish, Danio rerio, the precise localization patterns of these molecules during oogenesis has not been determined. In this study, in situ hybridization experiments were performed on zebrafish ovaries and activated eggs to examine different mRNA localization patterns. The results establish that while some maternal mRNAs remain ubiquitously distributed throughout the oocyte, other mRNAs follow specific localization patterns, including localization to the animal pole, localization to the vegetal pole, and cortical localization. The animal/vegetal axis is first apparent in stage II oocytes when the earliest mRNA localization is seen. Unique patterns of localization are seen in mature eggs as well. Some mRNAs maintain their oocyte localization patterns, while others localize upon egg activation (fertilization).

The nieuwkoid gene characterizes and mediates a Nieuwkoop-center-like activity in the zebrafish

BACKGROUND In amphibians, the Nieuwkoop center--a primary inducing region--has a central role in the induction of dorsal mesodermal cells to form the Spemann organizer. In teleosts, such as the zebrafish, Danio rerio, the functional equivalent of the amphibian Spemann organizer is the dorsal shield. Historically, a small region of the teleost yolk syncytial layer (YSL), an extraembryonic tissue that underlies the entire blastoderm, has been implicated in dorsal shield specification. Difficulties in transplanting discrete regions of the YSL and the previous lack of localized expression patterns unique to the YSL have, however, hindered efforts to prove definitively that the YSL possesses Nieuwkoop-center-like activities. RESULTS Here, we describe the isolation and analysis of a new homeobox gene, called nieuwkoid, which is first expressed immediately following the mid-blastula transition on the dorsal side of the zebrafish pregastrula embryo. We found that, by the onset of gastrulation, nieuwkoid expression becomes localized to a restricted region of the YSL, directly underlying the future dorsal shield. Mis-expression of nieuwkoid in early zebrafish embryos was found to be sufficient for the induction of ectopic organizer regions and secondary axes. Mis-expression of nieuwkoid by cell transplantation or by direct injection into the YSL led to the non-autonomous induction of ectopic organizer gene expression. CONCLUSIONS The dynamic and restricted expression of the nieuwkoid gene, combined with its potent dorsalizing activity, suggests that nieuwkoid is an important component in the regionalization of the gastrula organizer, possibly characterizing and mediating an organizer-inducing/Nieuwkoop-center-like activity.

The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

Abstract The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses.

tbx20, a new vertebrate T-box gene expressed in the cranial motor neurons and developing cardiovascular structures in zebrafish

The T-box genes constitute a family of transcriptional regulator genes that have been implicated in a variety of developmental processes ranging from the formation of germ layers to the regionalization of the central nervous system. In this report we describe the cloning and expression pattern of a new T-box gene from zebrafish, which we named tbx20. tbx20 is an ortholog of two other T-box genes isolated from animals of different phyla - H15 of Drosophila melanogaster and tbx-12 of Caenorhabditis elegans, suggesting that the evolutionary origin of this gene predates the divergence between the protostomes and deuterostomes. During development, tbx20 is expressed in embryonic structures of both mesodermal and ectodermal origins, including the heart, cranial motor neurons, and the roof of the dorsal aorta.