A gene expression screen in zebrafish embryogenesis.

A screen for developmentally regulated genes was conducted in the zebrafish, a system offering substantial advantages for the study of the molecular genetics of vertebrate embryogenesis. Clones from a normalized cDNA library from early somitogenesis stages were picked randomly and tested by high-throughput in situ hybridization for restricted expression in at least one of four stages of development. Among 2765 clones that were screened, a total of 347 genes with patterns judged to be restricted were selected. These clones were subjected to partial sequence analysis, allowing recognition of functional motifs in 163 among them. In addition, a portion of the clones were mapped with the aid of the LN54 radiation hybrid panel. The usefulness of the in situ hybridization screening approach is illustrated by describing several new markers for the characteristic structure in the fish embryo named the yolk syncytial layer, and for different regions of the developing brain.

The LN54 radiation hybrid map of zebrafish expressed sequences.

To increase the density of a gene map of the zebrafish, Danio rerio, we have placed 3119 expressed sequence tags (ESTs) and cDNA sequences on the LN54 radiation hybrid (RH) panel. The ESTs and genes mapped here join 748 SSLp markers and 459 previously mapped genes and ESTs, bringing the total number of markers on the LN54 RH panel to 4226. Addition of these new markers brings the total LN54 map size to 14,372 cR, with 118 kb/cR. The distribution of ESTs according to linkage groups shows relatively little variation (minimum, 73; maximum, 201). This observation, combined with a relatively uniform size for zebrafish chromosomes, as previously indicated by karyotyping, indicates that there are no especially gene-rich or gene-poor chromosomes in this species. We developed an algorithm to provide a semiautomatic method for the selection of additional framework markers for the LN54 map. This algorithm increased the total number of framework markers to 1150 and permitted the mapping of a high percentage of sequences that could not be placed on a previous version of the LN54 map. The increased concentration of expressed sequences on the LN54 map of the zebrafish genome will facilitate the molecular characterization of mutations in this species.

A study of Xlim1 function in the Spemann-Mangold organizer.

The Spemann-Mangold organizer is required in amphibian embryos to coordinate cell fate specification, differentiation of dorsal cell types and morphogenetic movements at early stages of development. A great number of genes are specifically expressed within the organizer, most of them encoding secreted proteins and transcription factors. The challenge is now to uncover genetic cascades and networks of interactions between these genes, in order to understand how the organizer functions. The task is immense and requires loss-of-function approaches to test the requirement for a given factor in a specific process. For transcription factors, it is possible to generate inhibitory molecules by fusing the DNA binding region to a repressor or activator domain, which should in principle antagonize the activity of the endogenous protein at the level of the DNA targets. We used this strategy to design activated and inhibitory forms of the LIM homeodomain transcription factor Lim1, which is encoded by an organizer gene involved in head development, as revealed by analyses of knockout mice. We found that Lim1 is a transcriptional activator, and can trigger dorso-anterior development upon ventral expression of hyperactive forms, in which Ldb1 is fused to Lim1. Using inhibitory Lim1 fusion proteins, we found that Lim1, or genes closely related to it, is required for head formation as well as for notochord development. Co-expression experiments revealed that Lim1 is required downstream of the early organizer factor Siamois, first, to establish the genetic program of the organizer and second, to mediate the action of organizer agents that are responsible for blocking ventralizing activities in the gastrula.

Radiation hybrid mapping of the zebrafish genome.

The zebrafish is an excellent genetic system for the study of vertebrate development and disease. In an effort to provide a rapid and robust tool for zebrafish gene mapping, a panel of radiation hybrids (RH) was produced by fusion of irradiated zebrafish AB9 cells with mouse B78 cells. The overall retention of zebrafish sequences in the 93 RH cell lines that constitute the LN54 panel is 22%. Characterization of the LN54 panel with 849 simple sequence length polymorphism markers, 84 cloned genes and 122 expressed sequence tags allowed the production of an RH map whose total size was 11,501 centiRays. From this value, we estimated the average breakpoint frequency of the LN54 RH panel to correspond to 1 centiRay = 148 kilobase. Placement of a group of 235 unbiased markers on the RH map suggests that the map generated for the LN54 panel, at present, covers 88% of the zebrafish genome. Comparison of marker positions in RH and meiotic maps indicated a 96% concordance. Mapping expressed sequence tags and cloned genes by using the LN54 panel should prove to be a valuable method for the identification of candidate genes for specific mutations in zebrafish.

A dominant-negative form of Serrate acts as a general antagonist of Notch activation.

Specification of the dorsal-ventral compartment boundary in the developing Drosophila wing disc requires activation of NOTCH from its dorsal ligand SERRATE and its ventral ligand DELTA. Both NOTCH ligands are required in this process and one cannot be substituted for the other. In the wing disc, expression of a dominant-negative, truncated form of SERRATE called BD(G), is capable of inhibiting NOTCH activation in the ventral but not the dorsal compartments. We demonstrate that BD(G) can act as a general antagonist of both SERRATE and DELTA mediated NOTCH interactions, however, BD(G) retains the SERRATE protein domain targeted by FRINGE, hence its antagonistic effects are restricted in the dorsal wing disc. Our findings suggest a model in which ligand binding to NOTCH is a necessary but insufficient step toward NOTCH activation.

Serrate-mediated activation of Notch is specifically blocked by the product of the gene fringe in the dorsal compartment of the Drosophila wing imaginal disc.

In the developing imaginal wing disc of Drosophila, cells at the dorsoventral boundary require localized Notch activity for specification of the wing margin. The Notch ligands Serrate and Delta are required on opposite sides of the presumptive wing margin and, even though activated forms of Notch generate responses on both sides of the dorsoventral boundary, each ligand generates a compartment-specific response. In this report we demonstrate that Serrate, which is expressed in the dorsal compartment, does not signal in the dorsal regions due to the action of the fringe gene product. Using ectopic expression, we show that regulation of Serrate by fringe occurs at the level of protein and not Serrate transcription. Furthermore, replacement of the N-terminal region of Serrate with the corresponding region of Delta abolishes the ability of fringe to regulate Serrate without altering Serrate-specific signaling.

Beaded of Goldschmidt, an antimorphic allele of Serrate, encodes a protein lacking transmembrane and intracellular domains.

Serrate (Ser) is an essential gene in Drosophila melanogaster best known for the Ser dominant (SerD) allele and its effects on wing development. Animals heterozygous or homozygous for SerD are viable and exhibit loss of wing margin tissue and associated bristles and hairs. The Beaded of Goldschmidt (BdG) allele of Ser, when heterozygous to wild type, will also produce animals exhibiting loss of wing margin material. However, animals homozygous for BdG exhibit a larval lethal phenotype comparable to animals homozygous for loss-of-function Ser alleles. BdG is a partial duplication of the Ser locus with a single 5' Ser-homologous region and two distinct 3' regions. Meiotic recombination between BdG and a wild-type Ser chromosome demonstrated that only one DNA lesion, caused by the insertion of a transposable roo element into the coding regions of the Ser transcript, appears capable of generating BdG phenotypes. Due to the roo insertion, the protein product is predicted to be prematurely truncated and lack an extracellular cysteine-rich region along with the transmembrane and intracellular domains found within the normal SERRATE (SER) protein. The loss of these protein domains apparently contributes to the antimorphic nature of this mutation.

Serrate expression can functionally replace Delta activity during neuroblast segregation in the Drosophila embryo.

Serrate and Delta encode structurally related proteins in D. melanogaster that bind within a common extracellular region on the NOTCH receptor molecule. We used ectopic expression to determine if SERRATE could mediate in vivo functions parallel or antagonistic to those proposed for the putative NOTCH ligand DELTA. Our results demonstrate that Serrate can replace Delta gene function during embryonic neuroblast segregation and that expression of Serrate leads to a NOTCH-dependent suppression of achaete expression in proneural clusters. Our findings strongly suggest that SERRATE functions as an alternative ligand capable of NOTCH activation.