Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development.

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.

Mitochondrial C1-tetrahydrofolate synthase (MTHFD1L) supports the flow of mitochondrial one-carbon units into the methyl cycle in embryos.

Mitochondrial folate-dependent one-carbon (1-C) metabolism converts 1-C donors such as serine and glycine to formate, which is exported and incorporated into the cytoplasmic tetrahydrofolate (THF) 1-C pool. Developing embryos depend on this mitochondrial pathway to provide 1-C units for cytoplasmic process such as de novo purine biosynthesis and the methyl cycle. This pathway is composed of sequential methylene-THF dehydrogenase, methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase activities. In embryonic mitochondria, the bifunctional MTHFD2 enzyme catalyzes the dehydrogenase and cyclohydrolase reactions, but the enzyme responsible for the mitochondrial synthetase reaction has not been identified in embryos. A monofunctional 10-formyl-THF synthetase (MTHFD1L gene product) functions in adult mitochondria and is a likely candidate for the embryonic activity. Here we show that the MTHFD1L enzyme is present in mitochondria from normal embryonic tissues and embryonic fibroblast cell lines, and embryonic mitochondria possess the ability to synthesize formate from glycine. The MTHFD1L transcript was detected at all stages of mouse embryogenesis examined. In situ hybridizations showed that MTHFD1L was expressed ubiquitously throughout the embryo but with localized regions of higher expression. The spatial pattern of MTHFD1L expression was virtually indistinguishable from that of MTHFD2 and MTHFD1 (cytoplasmic C(1)-THF synthase) in embryonic day 9.5 mouse embryos, suggesting coordinated regulation. Finally, we show using stable isotope labeling that in an embryonic mouse cell line, greater than 75% of 1-C units entering the cytoplasmic methyl cycle are mitochondrially derived. Thus, a complete pathway of enzymes for supplying 1-C units from the mitochondria to the methyl cycle in embryonic tissues is established.

STAR trek: An introduction to STAR family proteins and review of quaking (QKI).

The STAR family has an extremely diverse role during development and in RNA metabolism. We have concentrated on QKI as an example of this pleiotropic activity and also presented some new data on the role of its conserved 3'UTRs gleaned from bioinformatics analysis of theoretical miRNA binding sites. We review the concept ofa direct pathway from signal transduction to activation of RNA, how this pathway could be the cell's quick response to developmental and physiological changes and how it must be tightly regulated.

Tint maps to mouse chromosome 6 and may interact with a notochordal enhancer of Brachyury.

At the proximal part of mouse chromosome 17 there are three well-defined genes affecting the axis of the embryo and consequently tail length: Brachyury, Brachyury the second, and the t-complex tail interaction (T1, T2, and tct). The existence of T1 and tct in fact defines the classical "t-complex" that occupies approximately 40 cM of mouse chromosome 17. Their relationship to each other and various unlinked interacting genes has been enigmatic. The tint gene was the first of the latter to be identified. We report here its genetic mapping using a microsatellite scan together with outcrosses to Mus spretus and M. castaneous followed by a subsequent testcross to T, T1, and T2 mutants. Surprisingly, tint interacts with T2 but not with T1. The implications of our data suggest that T2 may be part of the T1 regulatory region through direct or indirect participation of tint.

Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling.

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.

Mammalian developmental genetics in the twentieth century.

This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas.

Molecular identification of t(w5): Vps52 promotes pluripotential cell differentiation through cell-cell interactions.

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entrée to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.

Targeted disruption of Mib2 causes exencephaly with a variable penetrance.

Mib1 and Mib2 ubiquitin ligases are very similar in their domain construction. They partake in the Notch signaling pathway by ubiquitinating the Notch receptors Delta and Jagged prior to endocytosis. We have created a targeted mutation of Mib2 and show that its phenotype is a variable penetrance, failure to close the cranial neural tube. The penetrance depends on the genetic background but it appears that Mib2 is not completely essential in mouse development.

Function of quaking in myelination: regulation of alternative splicing.

Proteomic diversity is frequently achieved by alternative RNA-splicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The qk locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized qk isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the qk mouse is demonstrated.

A role for KH domain proteins (Sam68-like mammalian proteins and quaking proteins) in the post-transcriptional regulation of HIV replication.

Overexpression of Sam68 functionally substitutes for, as well as synergizes with, human immunodeficiency virus type 1 (HIV-1) Rev in RRE (Rev response element)-mediated gene expression and virus replication. In addition, COOH-terminal deletion and/or point mutants of Sam68 exhibit a transdominant negative phenotype for HIV replication. Sam68 is a member of KH domain family that includes SLM-1, SLM-2 (Sam68 like mammalian); and QKI-5, QKI-6, and QKI-7 (mouse quaking) proteins. The objective of this study was to examine the effects of these KH family proteins on RRE- and CTE (constitutive transport element of type-D retrovirus)-mediated transactivation. We now report that SLM-1 and SLM-2 proteins, which are the closest relatives of Sam68, marginally enhanced RRE-mediated transactivation, while QK isoforms that are distant relatives of Sam68 had no effect. Interestingly, these proteins still enhanced the effect of Rev in RRE-mediated gene expression. The increase in chloramphenicol acetyltransferase activity was also reflected at the levels of cytoplasmic RRE-chloramphenicol acetyltransferase mRNAs, indicating that Sam68 and KH proteins may have been involved in the stability or export of unspliced RNA. The increase in Rev activity was sensitive to leptomycin B, but not to olomoucine, indicating that the effect of SLM-1, SLM-2, QKI-5, QKI-6, and QKI-7 is exerted through a CRM-1-dependent mRNA export pathway. Thus, KH family proteins play an important role in the post-transcriptional regulation of HIV.