Briefings in Functional Genomics

Briefings in Functional Genomics Advance Access originally published online on February 22, 2006
Briefings in Functional Genomics 2006 5(1):19-23; doi:10.1093/bfgp/ell008

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Special Issues Papers

Insertional mutagenesis in zebrafish: genes for development, genes for disease

Adam Amsterdam

Adam Amsterdam, Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue E17-340, Cambridge, MA 02139, USA. Tel: +1 617 253 0609; Fax: +1 617 258 0258. E-mail: aha{at}MIT.EDU

	ABSTRACT

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 
In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.

Keywords: zebrafish, retrovirus, vertebrate genetics, cystic kidney, hepatomegaly, cancer

	INTRODUCTION

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 
Genetics, determining the phenotype caused by the mutation of a given gene, has long been one of the most powerful tools for discovering gene function. The past few years have seen the systematic mutation of nearly all the genes in the yeast Saccharomyces cerevisiae (by gene deletion) [1, 2] and the nematode Caenorhabditis elegans (by RNA interference (RNAi)) [3, 4], and one result that emerged is that only a small proportion of genes encoded in either of these organisms is essential either for the life of a yeast cell (19%) or the embryonic development of a worm (7–10%). In order to extend this analysis to vertebrates, we undertook an insertional mutagenesis strategy in zebrafish. Zebrafish is an ideal model organism for vertebrate developmental genetics, combining small size, great fecundity and the external development and optical clarity of its embryos. Embryonic development is complete within 5 days of fertilization, allowing for a rapid assessment of developmental phenotypes. Large-scale screens utilizing ethylnitrosourea (ENU) as a chemical mutagen have yielded mutations in hundreds of genes, affecting every aspect of development [5, 6]. However, the cloning of chemically mutated genes is quite laborious, and thus has only been attempted for mutations with especially interesting phenotypes.

	INSERTIONAL MUTAGENESIS IDENTIFIES 25% OF THE GENES ESSENTIAL FOR EMBRYONIC DEVELOPMENT OF A VERTEBRATE

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 
Insertional mutagenesis provides an alternative whereby the identification of the mutated gene is greatly facilitated by the presence of a molecular tag at the site of the mutagenic lesion. The sequence of the genomic DNA flanking the inserted element can be easily cloned and sequenced, and in most cases rapidly leads to the identification of the mutated gene. The ease of gene cloning has allowed for an unbiased approach to the identification of genes required for development, not only traditional aspects of development such as patterning and cell fate, but also those required for cellular biological processes, as it is not necessary to limit the cloning efforts to a small number of selected mutants. The laboratory of Nancy Hopkins at the Massachusetts Institute of Technology developed an efficient method of insertional mutagenesis using a pseudotyped retrovirus [7–9] and has isolated 500 insertional mutants with recessive embryonic lethal phenotypes. These mutants represent 385 different genes, 333 of which have been identified [10–12]. A full list of these mutants showing the mutated genes and phenotypes can be found at http://web.mit.edu/ccr/pnas_zebrafish_mutant_images. This collection appears to include about 25% of the embryonic lethal loci in the fish, implying that there are about 1400 genes with a unique and essential function in zebrafish embryonic development [11].

This set of embryonic essential fish genes has some interesting characteristics. First, they cover a broad range of biochemical/cellular functions as shown in Table 1. Note that one-fifth of these genes encode proteins that do not yet have a known biochemical function. From an evolutionary perspective, essential genes are far more likely than vertebrate genes at random to have homologues in either yeast or invertebrates. Furthermore, in the cases where a single homologue exists in either S. cerevisiae or C. elegans, those genes are far more likely to be essential for cell viability (yeast) or embryonic development (worm) than yeast or worm genes at random [11]. This indicates that genetically essential genes have a strong tendency to retain this special status through evolution from yeast to vertebrates.

View this table: [in this window] [in a new window]   Table 1: Biochemical/cellular functions of embryonic essential zebrafish genes

 

	DISCOVERING PATHWAYS FOR DEVELOPMENTAL PROCESSES

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 
The patterning and development of many different tissues is best illuminated either by sectioning or by staining with various antibodies, in situ hybridization markers, or other reagents, and a number of ‘shelf screens’ have been conducted on the collection, identifying the subset of mutants affecting the development of a host of structures. As the current insertional mutant collection represents about one-quarter of all of the genes essential for zebrafish embryonic development, it should thus include mutations in about a quarter of all genes that can affect any given developmental process, provided that the overall phenotype is morphologically visible. This should be sufficient to illuminate what biochemical pathways are involved in the formation of a given cell type or structure. Furthermore, when a single pathway is of particular importance for a given developmental process, it will easily be identified as it will contain the preponderance of the mutated genes.

For example, screening through the mutant collection for cartilage differentiation, using Alcian blue staining, eight mutants were identified that form cartilage, but do not fully differentiate (Table 2 and [10, 11]). While one of the mutated genes, sox9a, is a transcription factor known to be an important regulator of cartilage differentiation, half of the genes are all involved in glycosaminoglycan (GAG) synthesis. This indicates that the production of proteoglycans is a key step in cartilage formation. Another screen identified a dozen genes whose mutation leads to the development of cystic kidney; two different mechanisms lead to this phenotype, each with a different pathway indicated by the mutated genes (Table 3 and [13]). One cause of cystic kidney is a defect in anterior–posterior patterning and both mutants with this phenotype had mutations in transcription factors [13–15]. However, the main mechanism leading to cystic kidney appears to be a defect in the differentiation of the kidney epithelium, and nearly all of the genes whose mutation leads to this phenotype have been shown to have a role in the formation or function of the primary cilia (Table 3 and [13, 16–22]). This demonstrated that perturbation of these cilia is the central pathway in embryonic kidney cystogenesis.

View this table: [in this window] [in a new window]   Table 2: Chondrogenesis mutants

 

View this table: [in this window] [in a new window]   Table 3: Cystic kidney mutants

 
One notable feature of the pathways that were illuminated by these screens is that they were clearly co-opted from lower organisms for use in the development and function of vertebrate-specific structures (cartilage, kidney). The C. elegans homologues of the GAG synthesis genes all have a phenotype called ‘squashed vulva’, whereby the gonad does not expand properly [23, 24]; thus, while the gene products presumably perform a similar biochemical function in both worm and fish, they are involved in the formation and structural integrity of very different body parts in the two organisms. Similarly, most of the ‘cilia genes’ whose mutation leads to cystic kidney are present in single-celled motile organisms such as Chlamydomonas and Trypanosoma, where they function in building or maintaining the flagellum [16–21]. In this case, it seems that these genes are reused in vertebrates to make a similar structure, the cilia, which is then adapted to a new function, in part by inclusion of some animal-specific or vertebrate-specific genes, such as pkd2 [22] and scorpion [13]. While in protozoans, the flagella are used for locomotion, in vertebrates, cilia may have taken on a number of different sensory roles. The inputs and outputs of these sensory mechanisms likely differs in different cell types; in the kidney epithelium, it seems that some signal mediated by pkd2 on the cilia is required to prevent overproliferation.

	INSERTIONAL MUTANTS AS DISEASE MODELS

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 
Another notable feature of the two shelf screens mentioned earlier is that several of the mutated genes clearly play a role in human diseases as well (Tables 2 and 3). Two of the cartilage differentiation mutants’ genes are mutated in human diseases that include skeletal defects as part of their manifestation. Similarly, two of the genes mutated in the cystic kidney mutants are mutated in two different human cystic kidney diseases. Thus, for certain phenotypes, even embryonic lethal mutants may serve as models for human disease.

One screen performed with the hope of modelling human disease was to look for embryos with large livers. Seven such mutants were identified, and three of them appear to be models for different human liver diseases [25]. First, mutation of vps18 likely affects sorting of proteins both to endosomes and to the apical membrane in hepatocytes, leading both to the accumulation of large vesicles in these cells and a defect in the canaliculi, the intercellular spaces where secreted bile flows towards the common bile duct. This phenotype is similar to the human disease arthrogryposis-renal dysfunction-cholestasis (ARC), one genetic cause of which can be a mutation in a related vsp gene, vsp33b [26]. Second, mutation of the tumour-suppressor gene nf2 leads to choledocal cysts, possibly due to overproliferation of biliary ductal cells. Finally, mutation of the novel gene foie gras leads to the accumulation of lipid-filled vesicles in the hepatocytes, similar to that seen in fatty liver disease.

In addition to using the embryonic recessive phenotypes as models of human disease, the existing mutant collection can also be used to monitor the long-term effects of heterozygosity of these genes in adults. For example, amongst a large collection of recessive embryonic lethal mutations one might expect to find mutations in which heterozygotes are more prone to develop cancer, because many known tumour suppressor genes are recessive embryonic lethals in mammals [27]. Comfortingly, zebrafish heterozygous for the nf2 mutation referred to earlier are prone to tumourigenesis, demonstrating that this gene acts as a tumour suppressor in fish as well as in mammals [28]. In an effort to identify novel tumour suppressor genes, we screened through all of the mutant lines for heterozygotes with externally visible tumours. This revealed the surprising fact that mutation in any of a dozen ribosomal protein genes leads to a predisposition to the formation of an otherwise rare tumour type [28]. We are continuing to screen for mutations that lead to tumour phenotypes by screening adult heterozygotes for all of the mutations by histology.

	CONCLUSION

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 
Insertional mutagenesis has allowed us to both identify and isolate mutants for about one-quarter of the genes essential for embryonic development in the zebrafish. This has allowed for insight into the nature of essential genes, indicated key pathways for certain developmental processes, and provided a unique resource for further studies in developmental biology as well as disease models.

We have found that essential genes are more likely than genes in general to be evolutionarily conserved, and that the very trait of being an essential gene is evolutionarily conserved as well, indicating that a conserved core group of essential genes has been maintained throughout evolution. We have also demonstrated how genes and pathways that originally evolved in lower organisms are reused in fish for the development of vertebrate-specific structures.

Besides the specific phenotypes reviewed earlier, many additional shelf screens have been performed with this mutant collection, which will hopefully lead to similar insights about other developmental processes. For example, histological examination of the retina identified about 40 genes required for proper eye development [29].

The fact that many of these mutants may represent disease models makes them excellent starting points for additional screens, either genetic screens to identify interacting genes (enhancers or suppressors), or small-molecule screens to identify compounds that can rescue their phenotypes. Small-molecule screens have been performed to find suppressors of embryonic phenotypes in zebrafish [30]; similar screens conducted on some of the mutants described here might discover lead compounds for fighting cystic kidney disease, a number of liver pathologies and some types of cancer.

Key Points Insertional mutagenesis identified about one-quarter of the genes required for embryonic development of the zebrafish. Genes essential for the development of the zebrafish are more likely than genes at random to have homologues in invertebrates and yeast, and these yeast and worm homologues are more likely than genes at random to be essential in those organisms. Key pathways in certain developmental processes can be identified when a preponderance of the mutated genes for a given phenotype are all in one pathway. Zebrafish insertional mutants may be useful as models of human diseases such as cystic kidney, liver disease and cancer.

 

	FOOTNOTES

 
Adam Amsterdam is a Research Scientist in the Center for Cancer Research at the Massachusetts Institute of Technology and is supported by a grant from the National Center for Research Resources at the National Institutes of Health.

	References

TOP ABSTRACT INTRODUCTION INSERTIONAL MUTAGENESIS... DISCOVERING PATHWAYS FOR... INSERTIONAL MUTANTS AS DISEASE... CONCLUSION References

 

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