Systematic Genetic Screens Research Articles

Nodal signaling and the zebrafish organizer.

Systematic genetic screens in zebrafish have led to the discovery of mutations that affect organizer function and development. The molecular isolation and phenotypic analysis of the affected genes have revealed that TGF-beta signals of the Nodal family play a key role in organizer formation. The activity of the Nodal signals Cyclops and Squint is regulated extracellularly by the EGF-CFC cofactor One-eyed Pinhead and by antagonists belonging to the Lefty family of TGF-beta molecules. In the absence of Nodal signaling, the fate of cells in the organizer is transformed from dorsal mesoderm to neural ectoderm. Differential Nodal signaling also patterns the organizer along the anterior-posterior axis, with high levels required for anterior cell fates and lower levels for posterior fates. In addition, Nodal signaling cooperates with the homeodomain transcription factor Bozozok in organizer formation and neural patterning. The combination of genetic, molecular and embryological approaches in zebrafish has thus provided a framework to understand the mechanisms underlying organizer development.

A modifier screen of ectopic Krüppel activity identifies autosomal Drosophila chromosomal sites and genes required for normal eye development.

Irregular facets (If) is a dominant gain-of-function allele of the Drosophila segmentation gene Krüppel (Kr) that interferes with eye development. In a search for genes that interact with Kr activity, we recently performed a systematic genetic screen to identify dominant enhancers and suppressors of the If eye phenotype that are located on the third chromosome. Here we describe locations and candidate genes of the second chromosome that act as dominant modifiers of ectopic Kr activity during eye development. The collection of more than 40 modifiers of Kr activity located on the second and third chromosomes, from which a total of 16 genes were identified, includes genes encoding transcription factors and components of signal transduction pathways that may regulate or be regulated by Kr activity. We also identified genes coding for more general cellular factors that could interfere with the intracellular transport or the half-life of the Kr protein. The data demonstrate that the If mutation provides a means to screen the Drosophila genome for functional components of developmental pathways that depend on or can be modified by Kr activity. Owing to the bias of the screening system applied, these modifier genes will be expressed and are likely to be required during Drosophila wild-type eye development.

Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection.

The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1-55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in non-idiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eIF-1A, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients.

Genetic analysis of axon guidance and mapping in the zebrafish

Systematic genetic screens have been powerful tools in identifying genes responsible for axon guidance in fruitflies and nematodes. This approach has now been extended to the study of axon guidance and the formation of topographic neuronal connections in the vertebrate brain. A systematic genetic screen was used to identify genes responsible for precise axon pathfinding and targeting in the retinotectal system of the zebrafish (Danio rerio). Over 30 genes were found that affect either: (1) retinal axon pathfinding to the contralateral tectal lobe; or (2) the topographic connection between the eye and the tectum. The zebrafish retinotectal mutants represent a new resource for the study of axon guidance in the vertebrate brain.

Mutations affecting the pattern of the PNS in drosophila reveal novel aspects of neuronal development

Through a systematic genetic screen, we have identified 55 mutations that affect the development of the PNS of Drosophila embryos. These mutations specify 13 novel and 5 previously characterized genes and define new phenotypes for 2 other known genes. Five classes of mutant phenotypes were identified in the screen: gain of neurons, loss of neurons, abnormal position of chordotonal neurons, aberrant neuronal trajectories, and abnormal morphology of neurons. Phenotypic analyses of mutations identified in this study revealed three novel aspects of PNS development. First, we have identified a novel gene that may be required to define glial versus neuronal cell identity. Second, our data indicate that neuronal migration plays an important role in pattern formation in the embryonic PNS. Third, we have identified mutations that cause a lack of sensory organs, but unlike mutations in proneural genes, do not affect the formation of sensory organ precursors. These genes may be required for key aspects of neuronal differentiation. Our studies suggest that approximately 70 essential genes are required for proper PNS development in Drosophila embryos.

Genetic and molecular dissection of axon pathfinding in the Drosophila nervous system

Over the past year, several systematic genetic screens designed to identify mutations that specifically disrupt axon pathfinding processes in the Drosophila nervous system have been described. The analysis of mutations isolated in these screens, in concert with ongoing cellular studies and the ever increasing number of identified proteins expresised in the Drosophila nervous system, is providing further insights into the molecular mechanisms of growth cone guidance.

Analysis of genetic mosaics in developing and adult Drosophila tissues.

We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cell-autonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.