William S. Talbot

The drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily

The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone.

The EGF-CFC Protein One-Eyed Pinhead Is Essential for Nodal Signaling

The zebrafish EGF-CFC gene one-eyed pinhead (oep) is required zygotically for the formation of the ventral neuroectoderm, endoderm, and prechordal plate. Here we report that embryos lacking both maternal and zygotic Oep activity are defective in germ layer formation, organizer development, and the positioning of the anterior-posterior axis. An identical phenotype is displayed by double mutants for the nodal-related genes squint and cyclops. Mutations in oep eliminate the response to Squint and Cyclops overexpression but are suppressed by expression of Activin and activated forms of the type I receptor ActRIB and Smad2. Expression of the murine EGF-CFC gene cripto rescues oep mutants. These results suggest a conserved role for EGF-CFC proteins as essential extracellular cofactors for Nodal signaling during vertebrate development.

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.

Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms

In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.

Positional Cloning Identifies Zebrafish one-eyed pinhead as a Permissive EGF-Related Ligand Required during Gastrulation

The zebrafish one-eyed pinhead (oep) mutation disrupts embryonic development, resulting in cyclopia and defects in endoderm, prechordal plate, and ventral neuroectoderm formation. We report the molecular isolation of oep using a positional cloning approach. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Wild-type oep protein contains a functional signal sequence and is membrane-associated. Following ubiquitous maternal and zygotic expression, highest levels of oep mRNA are found in the gastrula margin and in axial structures and forebrain. Widespread misexpression of both membrane-attached and secreted forms of oep rescues prechordal plate and forebrain development in mutant embryos but does not lead to the ectopic induction of these cell types in wild-type fish. These results establish an essential but permissive role for an EGF-related ligand during vertebrate gastrulation.

Mouse Lefty2 and Zebrafish Antivin Are Feedback Inhibitors of Nodal Signaling during Vertebrate Gastrulation

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

A radiation hybrid map of the zebrafish genome

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Drosophila Ecdysone Receptor Mutations Reveal Functional Differences among Receptor Isoforms

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.

erbb3 and erbb2 Are Essential for Schwann Cell Migration and Myelination in Zebrafish

BACKGROUND Myelin is critical for efficient axonal conduction in the vertebrate nervous system. Neuregulin (Nrg) ligands and their ErbB receptors are required for the development of Schwann cells, the glial cells that form myelin in the peripheral nervous system. Previous studies have not determined whether Nrg-ErbB signaling is essential in vivo for Schwann cell fate specification, proliferation, survival, migration, or the onset of myelination. RESULTS In genetic screens for mutants with disruptions in myelinated nerves, we identified mutations in erbb3 and erbb2, which together encode a heteromeric tyrosine kinase receptor for Neuregulin ligands. Phenotypic analysis shows that both genes are essential for development of Schwann cells. BrdU-incorporation studies and time-lapse analysis reveal that Schwann cell proliferation and migration, but not survival, are disrupted in erbb3 mutants. We show that Schwann cells can migrate in the absence of DNA replication. This uncoupling of proliferation and migration indicates that erbb gene function is required independently for these two processes. Pharmacological inhibition of ErbB signaling at different stages reveals a continuing requirement for ErbB function during migration and also provides evidence that ErbB signaling is required after migration for proliferation and the terminal differentiation of myelinating Schwann cells. CONCLUSIONS These results provide in vivo evidence that Neuregulin-ErbB signaling is essential for directed Schwann cell migration and demonstrate that this pathway is also required for the onset of myelination in postmigratory Schwann cells.

Genetic analysis of zebrafish gli1 and gli2 reveals divergent requirements for gli genes in vertebrate development

Gli proteins regulate the transcription of Hedgehog (Hh) target genes. Genetic studies in mouse have shown that Gli1 is not essential for embryogenesis, whereas Gli2 acts as an activator of Hh target genes. In contrast, misexpression studies in Xenopus and cultured cells have suggested that Gli1 can act as an activator of Hh-regulated genes, whereas Gli2 might function as a repressor of a subset of Hh targets. To clarify the roles of gli genes during vertebrate development, we have analyzed the requirements for gli1 and gli2 during zebrafish embryogenesis. We report that detour (dtr) mutations encode loss-of-function alleles of gli1. In contrast to mouse Gli1 mutants, dtr mutants and embryos injected with gli1 antisense morpholino oligonucleotides display defects in the activation of Hh target genes in the ventral neuroectoderm. Mutations in you-too (yot) encode C-terminally truncated Gli2. We find that these truncated proteins act as dominant repressors of Hh signaling, in part by blocking Gli1 function. In contrast, blocking Gli2 function by eliminating full-length Gli2 results in minor Hh signaling defects and uncovers a repressor function of Gli2 in the telencephalon. In addition, we find that Gli1 and Gli2 have activator functions during somite and neural development. These results reveal divergent requirements for Gli1 and Gli2 in mouse and zebrafish and indicate that zebrafish Gli1 is an activator of Hh-regulated genes, while zebrafish Gli2 has minor roles as a repressor or activator of Hh targets.