Experimental Gerontology 38 (2003) 13331341 www.elsevier.

com/locate/expgero

Comparative aspects of zebrash (Danio rerio) as a model for aging research

Glenn S. Gerhard*
Weis Center for Research, Geisinger Clinic, 100 North Academy Avenue, Danville, PA 17822 USA

Abstract The zebrash has emerged over the past decade as a major model system for the study of development due to its invertebrate-like advantages coupled with its vertebrate biology. These features also make it a potentially valuable organism for gerontological research. The main advantages of zebrash include its economical husbandry, small yet accessible size, high reproductive capacity, genetic tractability, and a large and growing biological database. Although zebrash life span is longer than rodents, it shares the feasibility of large-scale mutational analysis with the extremely short-lived invertebrate models. This review compares zebrash with the more widely used model organisms used for aging research, including yeast, worms, ies, mice, and humans. q 2003 Elsevier Inc. All rights reserved.
Keywords: Zebrash; Animal model; Aging

The zebrash has become a premiere model organism for the study of vertebrate development (Anderson and Ingham, 2003). Over the past few years, it has also been used to model a wide variety of disease processes (Rubinstein, 2003). Despite this enthusiasm in closely related elds, gerontology has been slow to embrace zebrash as a model for the study of aging. For example, the number CRISP grant funding database hits for the keyword zebrash for all NIH institutes in 2002 was 32/2, while only ve were found for grants funded by the National Institute on Aging. This review describes the utility of zebrash as a biological model and analyzes its potential usefulness vis-a-vis the current models commonly used for aging research.

1. The origins of zebrash as a biological model The zebrash, a hardy tropical sh commonly kept in home aquariums, has become a major model organism in biomedical research thanks primarily to the late George Streisinger (Grunwald and Eisen, 2002; Streisinger et al., 1981). He was one of a handful of scientists to rst advocate studying neurobiology with a genetic approach using model organisms, which included Sydney Brenners use of Caenorhabditis elegans and Seymour Benzers studies on
* Tel.: 1-570-271-8669; fax: 1-570-271-6701. E-mail address: gsgerhard@geisinger.edu (G.S. Gerhard). 0531-5565/$ - see front matter q 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.10.022

Drosophila. At the University of Oregon, Streisinger developed techniques to produce homozygous diploid zebrash as well as methods to introduce mutations into the germ-line. These tools led to the identication of several mutations affecting embryonic development (Felsenfeld et al., 1990; Grunwald et al., 1988). Despite his progress, Streisinger was constantly embattled to secure federal funding for his zebrash project. His efforts endured only through the prescient and persistent intervention of a handful of scientists, who by chance were involved in the peer review and funding process at NIH (M. C. Capecchi, G. Lark and P. von Hippel, personal communications). (Grunwald and Eisen, 2002). The potential of zebrash as a genetic model did not go unnoticed by Drosophila biologists, who had already performed large-scale mutational analysis of development in the y (Nusslein-Volhard and Wieschaus, 1980). Christian sslein-Volhard, winner of the Nobel prize for her work on Nu Drosophila (Roush, 1995), began a similar mutagenesis screen for embryonic pattern mutations in zebrash in Germany in 1993 (Mullins and Nusslein-Volhard, 1993). Marc Fishman and Wolfgang Driever initiated a parallel screen in Boston (Fishman and Stainier, 1994). Over 4000 embryonic-lethal mutant phenotypes were recovered between the two efforts that are still being studied today. The Trans-NIH Zebrash Initiative was begun soon afterwards to support zebrash research, including the further development of the genetic map in order to facilitate

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the identication of the genes underlying the large number of mutant phenotypes. In 2000, the Sanger Centre initiated a project to sequence the zebrash genome (www.sanger.ac. uk), which will be deposited in a centralized web-based database (www.ZFIN.org) that is maintained, along with a zebrash stock center, at the University of Oregon.

2. Zebrash as a model for development The zebrash is a small (1 2 in.) fresh water tropical sh species native to India (Eisen, 1996). It is a nearly ideal model for systematic mutational analysis (Fishman, 2001). The embryos are transparent (Fig. 1), thus, they may readily screened for morphological abnormalities via gross and

Fig. 1. Development in zebrash. (A) Single cell embryo with evidence of early rst cleavage. (B) 4-cell stage. (C) 8-cell stage. (D) Approximate 64 128 cell stage. (E) 3-h-old blastula stage embryo. (F) 24-h-old embryo curled inside chorion. (G) Free-swimming 48-h-old embryo following hatching from chorion. (H) Adult male zebrash at 8 months of age. (I) Old zebrash at 52 months of age. (J) Following exposure to mitochondrial specic uorescent dye MitoTracker Green (Molecular Probes, Eugene, OR), neuron body, axon, and putative satellite cell are vividly visualized. (K) Punctate mitochondrial staining in an epithelial-type cell, with nuclear sparing seen in some cells.

microscopic visual examination. An individual female can produce hundreds of eggs in each clutch, enabling huge numbers of progeny to be generated, facilitating the detection of rare mutations. The eggs are large enough to be easily manipulated and can be fertilized naturally or in vitro. Rapid ex utero development (Fig. 1) leads to free-swimming larvae with morphological and behavioral features of adults within 4 7 days. Organogenesis is easily visualized. For example, cardiac contractions begin during the second day after fertilization and all major organs recognizable by the fth day. The generation time is 3 4 months and relatively modest amount of resources are required to maintain large numbers of sh (Eisen, 1996). They are also physically large enough to isolate signicant amounts of specic tissues, especially skeletal muscle, yet are sufciently small to allow for substantial economies of scale. Also vital for mutational analysis is the availability of genomic resources. Key for zebrash biologists is the ongoing sequencing of its genome (Vogel, 2000a). The Sanger Institute initiated sequencing of the 1.7 Gb zebrash genome in February 2001 using a two pronged approach, clone-based sequencing from BAC and PAC libraries, and whole genome shotgun sequencing from plasmids (http://www.sanger.ac.uk/Projects/D_rerio/). The clonebased approach is also being used to generate a physical map of the genome and will facilitate positional cloning of mutant genes. Further sequence information is being generated by the Washington University Zebrash Genome Resources Project (http://zsh.wustl.edu/), which is developing cDNA libraries for EST sequencing and mapping genes and ESTs. A number of other complementary genetic resources are available for zebrash. The ability to generate transgenic zebrash allows for overexpression studies as well as for the testing of candidate genes through complementation of mutants (Meng et al., 1999; Yan et al., 1998). While it is not yet possible to generate targeted knock-out mutations for evaluating null phenotypes of candidate genes, transgenic morpholino knock-down techniques allow for the transient suppression of gene expression during early development (Nasevicius and Ekker, 2000). As mentioned above, thousands of genetic mutants from large-scale mutagenesis experiments are also available for study. A key practical consideration is that zebrash sperm can be frozen for future study. A main component of the zebrash armamentarium is the ability to perform high efciency mutagenesis. Chemical mutagenesis protocols are well established (Knapik, 2000), in which single-base substitutions are induced that can result in loss-of-function, decreased-function, gain-of-function, and conditional (e.g. temperature sensitive) mutations. Mutagenesis is primarily accomplished using the DNA alkylating agent ethylnitrosourea (ENU), which has yielded average locus mutation rates of 1 3 mutations per 1000 haploid genomes (Mullins et al., 1994; Solnica-Krezel et al., 1994). Radiation (Walker, 1999) and insertional

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mutagenesis (Amsterdam et al., 1999; Golling et al., 2002) techniques have also been developed. Most induced mutations are recessive, thus, in mutant screens in a diploid organism, recessive mutations must be rendered homozygous to detect their phenotype. Screening mutation-bearing sh for relevant phenotypes is therefore done in individuals carrying homozygous recessive mutations. In the zebrash, this is usually accomplished using a multi-generation back-cross paradigm, the approach used for the rst major screen (Haffter et al., 1996; Stainier et al., 1996). Alternative techniques can be used to produce uniparental progeny, including a variety of gynogenetic (yielding embryos with only maternal genomes), and androgenetic (yielding embryos with only paternal genomes) uniparental techniques (Postlethwait and Talbot, 1997). Uniparental haploid screens utilize zebrash strains devoid of recessive lethal mutations and exploit the ability of haploid embryos to survive for up to 4 days postfertilization to detect subtle developmental phenotypes.

3. The origins of zebrash as a gerontological model Several small tropical sh have been the subject of previous gerontological studies, whose investigators have cited a number of advantages for studying aging including the availability of large cohorts of offspring from single matings, the ectothermic nature of sh (which facilitates environmental modulation by external changes), and their reasonably short life span relative to many mammalian species (Woodhead, 1978). Others have highlighted the low costs for breeding and maintenance, the ability to manipulate life span by both temperature reduction and food restriction, and the large variety of species available as potential models (Patnaik et al., 1994). These advantages hold for many of the more than 24,000 extant sh species, why then the zebrash? One reason is the large and increasing number of investigators who use the model. A critical mass of scientists who generate a wide range of potentially useful reagents and resources for aging studies should not be underestimated. The current surge in zebrash biology was apparent even in the early 1990s prior to the initiation of the rst large-scale screen (Rossant and Hopkins, 1992), after which zebrash leapt into the mainstream of biological research (Chen and Fishman, 2000; Driever et al., 1996; Haffter et al., 1996). Around this time, a life span study was initiated in the authors laboratory with the expectation that the anecdotal reports for zebrash longevity would be 2 4 years, an estimate still referred to and reported (Higgs et al., 2002). The median life span of outbred zebrash (from the age of 17 months) was found to be about 42 months, with the longest living individuals surviving for 66 months (Gerhard et al., 2002). The longevity of a relatively inbred zebrash strain was also assessed and manifested a 10 15% shorter median and maximum life spans than the outbred zebrash, consistent

with the effects of inbreeding depression. This initial study did not assess potential effects of gender and reproductive activity, which could affect life span (Reznick et al., 2001). The life span of outbred zebrash is thus at least 50% longer than that of commonly used mouse strains, and is slightly longer than long-lived mouse mutants (Bartke et al., 2001). A maximum life span of at least 5 years is a major disadvantage relative to the extremely short-lived invertebrate organisms, and to rodents. However, the key utility of the zebrash as a model is that it may be genetically manipulated like invertebrates, yet it offers a life span that is more similar to, and as a vertebrate perhaps more relevant to, mammals. The derivation of large populations can be achieved quickly and cheaply making possible large-scale demographic analyses (Vaupel et al., 1998), an advantage not shared by many vertebrate models. Zebrash also appear to develop various degrees of spinal curvature with age, as reported with aging in other sh species (Comfort, 1961; Liu and Walford, 1969). Muscle degeneration appears to underlie the curvature and may thus serve as a model for sarcopenia (Gerhard et al., 2002). Tens to hundreds of milligrams of skeletal muscle may be isolated from a single sh, providing an ample supply of tissue for study. The pathologies zebrash incur, such as various tumors, are morphologically similar to those found in mammals (Beckwith et al., 2000), yet the costs for such analyses are much less due to the smaller size of zebrash. The next phase of zebrash gerontology should include a more detailed examination of life span and pathology, in parallel with exploitation of some of the available techniques described below. This should avoid the situation that existed for C. elegans, where the detailed phenotype of aging (Herndon et al., 2002) was not characterized for many years after the application of powerful genetic approaches began to yield signicant results.

4. Zebrash as a unicellular model of aging Although a metazoan, zebrash spend about 30 40 min as a readily obtainable, accessible, and manipulable single cell embryo (Fig. 1). Its adult size also enables the accession of specic tissues for in vitro studies. Zebrash may thus serve as a useful model for studies related to two well characterized unicellular models of aging, budding yeast and the cellular senescence of cultured cells. 4.1. Yeast In the budding yeast, Saccharomyces cerevisiae, aging has been studied in two main experimental paradigms, replicative and chronological life span (Jazwinski, 2002; Sinclair, 2002). Replicative life span is measured as the number of daughter cells that bud from a mother cell. On

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an average, a mother cell will give rise to about 20 daughter cells prior to losing the ability to proliferate. The mechanism by which this occurs may be due to the accumulation of extrachromosomal rDNA circles, which may cause senescence by titrating essential transcription and replication factors. The SIR2 gene mediates silencing of rDNA and represses the formation of extrachromosomal rDNA circles. SIR2 is a histone deacetylase that requires NAD as a cofactor, suggesting a link to cellular metabolism (Imai et al., 2000). Overexpression of the SIR2 gene increases rDNA silencing and can extend replicative life span (Tissenbaum and Guarente, 2001). The potential role of rDNA or SIR 2 in zebrash aging has not been reported. The moderately repetitive 5S rDNA sequences have been characterized in zebrash cell lines (Phillips and Reed, 2000) and several zebrash histone deacetylase homologs have been identied (www.TIGR. org). Two single cell phases of zebrash may be useful for such studies. First, similar to other metazoans, specic tissues may be harvested from zebrash, including those that consist primarily of post-mitotic or proliferating tissues (availability of cultured cell lines discussed below). In addition, the fertilized oocyte may serve as a vehicle to track molecular changes occurring in early embryonic cell divisions that could have a subsequent inuence on aging in the adult organism. Chronological aging in yeast is measured in a stationary phase, when glucose becomes limiting following fast logarithmic growth, in which the cells stop dividing but can survive for several months. In this nal phase, they are resistant to heat and oxidative stress and have lower metabolic rates (Sinclair, 2002). Oxidative stress may play a role in determining life span of the stationary phase, since yeast lacking superoxide dismutase have a greatly reduced chronological life span (Longo et al., 1999). A genetic screen for oxidative stress resistant mutants with extended chronological life span identied SCH9, which encodes a protein kinase with homology to C. elegans AKT-1 and AKT-2 part of the IGF-1/DAF-2 insulin-like signaling pathway, and, CYR1, which encodes adenylate cyclase (Fabrizio et al., 2001). The potential role of oxidative stress in zebrash aging has begun with the cloning of the mitochondrial genome and antioxidant defense genes, including catalase (Gerhard et al., 2000). Aging skeletal muscle also appears to manifest abnormal mitochondrial morphology (Gerhard et al., 2002). Potentially important technical advantages of zebrash for studies on oxidative stress, are the ability to accurately measure variables such as caloric intake, metabolic rate, and physical activity, in addition to the use of non-toxic compounds as indicators of reaction oxygen species (Gerhard and Cheng, 2002). The primary drawback for such studies is that the effects of selected mutations or environmental manipulations upon life span cannot be ascertained quickly. Nevertheless, large numbers of

embryos can be generated easily and cheaply and subject to a wide variety of experimental manipulations. 4.2. Cellular senescence Cellular senescence occurs in cells capable of dividing (e.g. broblasts, endothelial cells, etc). Similar to yeast replicative aging, as cell division occurs in vitro, eventually the ability to replicate is lost and a wide variety of related biochemical changes occur (Cristofalo, 1997). It has been proposed that cellular senescence is critical for suppressing tumorigenesis in eukaryotes and might cause or contribute to the aging of tissues that proliferate (Campisi, 2003). Cellular senescence appears to be caused by the progressive shortening of telomeres, which occurs with each cell division (Bodnar et al., 1998). Shortened telomeres may be recognized as damaged DNA by cells, which then triggers an irreversible arrest of cell division and thus senescence. The enzyme telomerase can repair such shortening, but telomerase is not expressed in most human cells. In addition, other stimuli may induce cellular senescence such as oxidative stress and certain oncogenes. Species differences exist in the levels of telomerase expression in post-embryonic tissues (Campisi, 2001). In species with relatively short telomeres and low levels of telomerase expression, such as humans, telomere shortening likely causes cellular senescence. In other species, such as mice, telomeres are longer and telomerase expression is higher. In these species, cellular senescence may occur through non-telomeric pathways. Nothing yet has been published on zebrash cellular senescence or on related aspects of telomeres or telomerase. The zebrash telomere repeat factor sequence has been identied (Genbank AY099522) and telomerase activity has been reported in other sh species. Permanent lymphoid cell lines have been readily obtained from peripheral blood cells of channel catsh (Ictalurus punctatus), which increase telomerase activity upon immortalization (Barker et al., 2000). Cell lines derived from puffersh (Fugu) species have also been found to possess high, perhaps indenite proliferative potential, and high telomerase activity (Bradford et al., 1997). High telomerase activities have been found in multiple tissues in the rainbow trout, attributed to the indeterminate growth in many sh species (Klapper et al., 1998). If indeed linked to proliferative activity, high somatic telomerase activity may then be expected in all species with indeterminate growth. At least several broblast-like zebrash cell lines are publicly available (www.ATCC.org) that were derived from embryos or adult ns, as well as an epithelial line derived from liver. The determination of telomere length and relationship to various aspects of aging, such as the occurrence of neoplasia and cellular senescence in zebrash cells may be of particular interest. Screens for zebrash mutations that affect cell proliferation and differentiation are already underway (Cheng and

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Moore, 1997). Genetic screens in yeast, Drosophila, and C. elegans have identied key genetic pathways regulating cell proliferation and apoptosis. Findings from zebrash could determine whether the function of these genes is conserved, or whether they are only relevant to invertebrates. Also, zebrash provide a model to directly test whether events occurring in embryonic development impact function in adult animals.

5. Worms: debate for sh? The self-fertilizing hermaphrodite nematode C. elegans has become the primary genetic model system for gerontology (Finch and Ruvkun, 2001) due to its short and reproducible longevity (almost 3 weeks at 20 8C), inexpensive maintenance, completely sequenced genome, complete cell fate map, and the identication of a number of genes and pathways that increase its life span (Johnson, 2003; Tissenbaum and Guarente, 2002). Mutations in several components of the daf-2/insulin-like signaling pathway cause an extension of life span (Guarente and Kenyon, 2000; Murakami et al., 2000). Other genes that increase C. elegans life span include those underlying the eat mutants, which have manifest abnormalities in feeding and may operate through caloric restriction, and the clk (biological timing abnormality) mutations, that cause slowing of many physiological processes (Hekimi et al., 2001). Other classes of mutations have also been found (Johnson et al., 2000). Recently reported large-scale systematic RNA interference (RNAi) screens to identify genes that extend life span when suppressed attest to the power of C. elegans as a genetic model (Dillin et al., 2002; Lee et al., 2003). Worms can be grown on bacteria expressing double-stranded RNA (dsRNA), from which decreased expression of the corresponding gene in vivo occurs through consumption of the bacterially expressed double-stranded RNA. In a screen of more than 5600 genes, a number of mitochondrial related transcripts were found to affect C. elegans life span (Lee et al., 2003). These genes appear to act downstream of, or parallel to, the daf-2/insulin-like signaling pathway. Suppression of several mitochondrial genes was also found to extend life span in another RNAi screen (Dillin et al., 2002). Exposure to the RNAi was shown to be required only during development and not throughout the life span, suggesting that a reconguration of mitochondrial function early on in life can extend longevity. The RNAi of mitochondrial genes had other effects as well (Dillin et al., 2002). Body size was reduced, though probably not through a reduction in cell number, and pharyngeal pumping for food consumption was decreased. Lower levels of ATP and oxygen consumption, and marked abnormalities in mitochondrial structure and morphology were noted (Lee et al., 2003). Life span extension was also

accompanied by resistance to heat shock and hydrogen peroxide treatment. Are similar large-scale genetic approaches practical using zebrash? Chemical mutagenesis methods are a major strength of the zebrash model and are being widely exploited for a number of experimental purposes. However, several issues complicate the use of life span as a phenotype used for screening. A maximum life span of more than 5 years exceeds conventional funding intervals. Although this has not prevented aging studies on species with even longer life spans, it is a major practical constraint. The appropriate control group for comparison of mutant longevity also needs to be well dened. Further, baseline data on aging in commonly used zebrash strains is thus needed, including the effects of caloric intake, temperature, housing density, reproductive effort, and physical activity on life span. In addition, to achieve saturation mutagenesis, thousands of sh must be generated. This number is feasible when screening for developmental mutations because phenotypes are assessed only during the rst few days of life and the sh are therefore quite small, minimizing space requirements, and sh without phenotypes can be euthanized freeing space quickly. Using longevity as a phenotype requires maintaining mutation-bearing sh for long periods as adults, dramatically increasing space and husbandry requirements. Enough sh carrying each mutation must be maintained in order to obtain sufcient statistical power to discern potential increases in longevity. Several approaches may be taken to address these disadvantages. Mutation-bearing cohorts that exhibit shortened median longevities can be continually replaced with cohorts carrying new mutations, a sort of rolling mutagenesis strategy to maximize use of space. However, this strategy will miss mutations that extend maximum longevity though decreasing median longevity. Another possibility is to separate the measurement of longevity from the mutagenesis by screening for surrogate phenotypes. Indeed, most of the rst 40 or 50 longevity extending mutations identied in C. elegans weremutants initially identied through screening for developmental abnormalities (Murakami et al., 2000). Characteristics of long-lived worm mutants include resistance to heat and oxidative stress (Johnson et al., 2000), as well as the various changes noted in RNAi treatment with mitochondrial genes described above. Screening for these sorts of phenotypes at a young age could identify a small group for subsequent longevity studies. Zebrash may be a particularly useful model to screen for such surrogate phenotypes. Zebrash are quite small for the rst several months of life, so large numbers of mutant sh may be generated and subject to phenotypic screening. Generation of haploid progeny may also be used for screening to decrease space and time requirements. Non-toxic uorescent compounds may be used to visualize mitochondrial morphology and function (Fig. 1), and various physiological measurements may be performed on individual sh such as oxygen consumption, caloric intake, and physical activity.

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Mutants with potentially relevant phenotypes have already been described, including a temperature preference mutant and two caloric intake mutants (Vogel, 2000b). Initial RNAi approaches in zebrash have not been promising (Zhao et al., 2001), but knock-down morpholino oligonucleotide techniques are well developed (Nasevicius and Ekker, 2000). The reduced expression is only effective for the rst 4 5 days of development, although during this time, essentially all major organs assume adult morphology. Whether a reduction in the expression of a single gene during the rst few days of life could extend the life span of an organism that may live more than 1800 days is not known. However, the nding that the RNAi of mitochondrial genes was required only during development and not throughout life (Dillin et al., 2002) suggests that this may be possible. Indeed, the easy and direct accessibility to early development may make zebrash an extremely useful model to determine how interventions early in development affect subsequent longevity. Expression proling has also been reported in aging C. elegans (Lund et al., 2002). However, the small size and non-compartmentalized vascular system of C. elegans make the isolation of specic cells or tissues for use in microarray studies difcult. Thus, whole organism homogenates have been used to obtain RNA for microarray studies, an option not scientically acceptable for vertebrate systems. For C. elegans, alternative approaches using mutants, transgenics, tissue culture, and computational strategies have been used to interpret whole organism data (Reinke, 2002). An advantage of zebrash for such studies is the ability to obtain signicant amounts of several tissues, especially skeletal muscle, and the availability of large-scale microarrays, both cDNA and oligonucleotide (Lo et al., 2003; Stickney et al., 2002), so that reliance upon cross-species hybridization is not necessary. Until recently, the morphological phenotype of aging worms had not been characterized (Herndon et al., 2002). A major phenotype in old worms appears to be muscle degeneration, akin to sarcopenia in mammals and other species. Muscle degeneration also appears to be a major senescent change in zebrash (Gerhard et al., 2002).

6. The vertebrate Drosophila versus the invertebrate, Drosophila Nusslein-Volhards Nobel Prize-winning work with Drosophila was the precedent for pursuing large-scale mutagenesis of zebrash development. In gerontology, Drosophila has also been well studied for many decades (Helfand and Rogina, 2003). Selective breeding based upon the onset of reproduction has shown that naturally occurring genetic variants can cause large differences in life span (Arking, 2001). Long-lived Drosophila lines are also more resistant to stress, similar to long-lived C elegans mutants.

Selection for specic traits such as onset of reproduction has not yet been reported in zebrash. Large-scale mutational analysis has also been used to identify genes that extend Drosophila life span. As was the case for C. elegans, most of the mutations reported thus far were not identied based upon screening for longevity. In contrast to C. elegans, mutations in most components of the insulin signaling pathway do not extend longevity. Nevertheless, certain insulin receptor mutant alleles do extend life (Tatar et al., 2001) as do mutations in the insulin receptor substrate chico in females (Clancy et al., 2001). The insulin pathway in Drosophila regulates cell, organ, and total body size and affects metabolism (Garofalo, 2002). Zebrash may be a particularly useful species to extend studies on the role of insulin pathways upon aging, given the indeterminate nature of growth in sh. The major proteins involved in insulin signaling appear to be structurally and functionally similar to those found in mammals. Zebrash possess two structurally distinct IGF-I receptor genes that display different expression patterns (Pozios et al., 2001). IGF signaling system has been suggested to play a key role in the indeterminate growth of sh species (Maures et al., 2002). Screening for growth related phenotypes may therefore be a feasible surrogate phenotype for mutations affecting the IGF pathway. Other genes have been identied that extend longevity in Drosophila. Mutation in the Methuselah gene extends life span by about one-third and increases resistance to stress (Lin et al., 1998). The Indy (Im not dead yet) gene also extends longevity and is homologous to a dicarboxylate transporter, suggesting a role in intermediary metabolism and perhaps operating through a mechanism related to caloric restriction (Rogina et al., 2000). Zebrash may be subject to caloric restriction regimens similar to those used in rodent and primate studies (Pugh et al., 1999). Overexpression of the antioxidant defense genes, including Mn superoxide dismutase (Sun et al., 2002), Cu/Zn superoxide dismutase with (Sohal et al., 1995) or without catalase overexpression (Sun and Tower, 1999) has also been reported to increase Drosophila life span, although some controversy exists (Orr and Sohal, 2003). Transgenic overexpression is easily accomplished in zebrash (Udvadia and Linney, 2003) and several antioxidant defense genes have been cloned. Importantly, effects on specic pathologies may be determined using the zebrash model.

7. Zebrash versus lab mice50 times smaller yet live 50% longer The life spans of many commonly used strains of laboratory mice do not extend much beyond 2 3 years. However, several mutations that result in a dwarf phenotype, i.e. the Ames dwarf (Prop1 df/df) and Snell dwarf (Pit1 dw/dw) mice can increase life span by over 50% (Bartke et al., 2001). The growth abnormalities are due to multiple hormonal

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deciencies, including the growth hormone/insulin-like growth factor-1 pathway, suggesting potential common regulatory aspects of life span determination across a wide range of species. Mice selectively bred for post-natal growth rate also had a wide range of life spans (Miller et al., 2000). Zebrash may be a valuable species to investigate the possible relationships between early growth rates and longevity. Wide variation in growth rates is apparent in outbred zebrash populations, suggesting the presence of naturally occurring polymorphisms on which selection could easily be performed. Growth may also be impacted by environmental conditions. Data from a South American annual sh indicate that decreased ambient temperature resulted in both faster growth rates and increased life span (Liu and Walford, 1966). No follow-up studies have been reported on this observation. Mouse and zebrash both offer conventional transgenic overexpression approaches, although the costs and technical difculties associated with generating transgenic zebrash are much lower. The generation of germ-line founder transgenic sh by microinjection of DNA into zebrash embryos is similar to that obtained in mice (Udvadia and Linney, 2003). The injected DNA usually integrates as a concatemer in both species as well. Several alternative transgenic techniques are available for zebrash including retrovirus infection (Linney et al., 1999) and the use of nuclear localization signals (Collas and Alestrom, 1998). The mouse has become the prime vertebrate model system to disrupt gene function through homologous recombination using embryonic stem cells. In zebrash, gene expression may be reduced for the rst several days of embryonic development through microinjection of modied antisense oligonucleotides called morpholinos (Nasevicius and Ekker, 2000). Unfortunately the knock-down of gene expression is only transient. True knock-out techniques have not yet been reported but germ-line chimeras have been generated from embryo cell cultures (Ma et al., 2001) and zebrash have been cloned by substitution of the embryo nucleus with a nucleus from a cultured embryonic broblast (Lee et al., 2002). Accessibility to the sh equivalents of most major mammalian organs is possible in zebrash. Post-mitotic tissues, including skeletal muscle, brain, heart, and eye may thus be easily harvested for study. Replicating tissues may also be obtained, and zebrash develop a wide variety of neoplasms with morphological similarity to mammalian tumors (Beckwith et al., 2000). Pathological analysis is also more cost-effective in zebrash due to the ability to place an entire adult sh on a single microscope slide.

investigators to use mutagenesis screens for phenotypes related to human disease as well as disease therapeutics (Nasevicius and Ekker, 2001; Rubinstein, 2003). Shin and Fishman (2002) have presented zebrash as a modular medical model. They argue that zebrash orthologs have been identied for most human genes, and large regions of conserved chromosomal synteny are present between zebrash and mammals. There are also certain aspects of zebrash mutagenesis screens that facilitate the identication of certain aspects of specic human diseases. For example, complex phenotypes may be sought, paralleling many human disorders, and the mutations induced by chemical mutagenesis often produce hypomorphic mutations rather than the null mutations in mouse knockouts. A number of clinically important age-related human disorders may be modeled in zebrash including heart failure, cardiac ischemia, cancer, diabetes, osteoporosis, Parkinsons disease, Alzheimers disease, hearing loss, and macular degeneration. Most of the work done so far has been to identify mutations that affect anatomically important cell types related to these various disorders. Little work has been done on screening for agerelated phenotypes at older ages. Another potential use that zebrash, may have in the study of age-related human disease, is the ability to perform high throughput screening for compounds affecting a specic biochemical or morphological phenotype (Moon et al., 2002). Zebrash embryos are permeable to a wide variety of small molecules that may be dissolved directly in the water. The high reproductive capacity, economy, and small size all facilitate the use of zebrash for such screening.

9. Summary The zebrash is a prime organism for the study of embryonic development that is now being used to model a number of physiological and pathological processes. It is anticipated that the experimental attributes that have led to its widespread use in many diverse disciplines will also make it a fruitful gerontological model in the near future.

Acknowledgements This work was supported by grants AG14731 and R03AG021292 (GSG).

References 8. Zebrash, a new angle on human disease Despite nearly a half a billion years of evolutionary divergence, zebrash possess many anatomical and functional features that are similar to humans. This has led
Amsterdam, A., Burgess, S., Golling, G., Chen, W., Sun, Z., Townsend, K., Farrington, S., Haldi, M., Hopkins, N., 1999. A large-scale insertional mutagenesis screen in zebrash. Genes. Dev. 13, 27132724. Anderson, K.V., Ingham, P.W., 2003. The transformation of the model organism: a decade of developmental genetics. Nat. Genet. 33, 285 293.

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G.S. Gerhard / Experimental Gerontology 38 (2003) 13331341 Gerhard, G., Kauffman, E., Wang, X., Stewart, R., Moore, J., Kasales, C., Demidenko, E., Cheng, K., 2002. Life spans and senescent phenotypes in two strains of Zebrash (Danio rerio). Exp. Gerontol. 37, 10551068. Golling, G., Amsterdam, A., Sun, Z., Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K., Farrington, S., Lin, S.Y., Nissen, R.M., Hopkins, N., 2002. Insertional mutagenesis in zebrash rapidly identies genes essential for early vertebrate development. Nat. Genet. 13, 13. Grunwald, D.J., Eisen, J.S., 2002. Headwaters of the zebrashemergence of a new model vertebrate. Nat. Rev. Genet. 3, 717 724. Grunwald, D.J., Kimmel, C.B., Westereld, M., Walker, C., Streisinger, G., 1988. A neural degeneration mutation that spares primary neurons in the zebrash. Dev. Biol. 126, 115128. Guarente, L., Kenyon, C., 2000. Genetic pathways that regulate ageing in model organisms. Nature 408, 255262. Haffter, P., Granato, M., Brand, M., Mullins, M.C., Hammerschmidt, M., Kane, D.A., Odenthal, J., van Eeden, F.J., Jiang, Y.J., Heisenberg, C.P., Kelsh, R.N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C., Nusslein-Volhard, C., 1996. The identication of genes with unique and essential functions in the development of the zebrash, Danio rerio. Development 123, 136. Hekimi, S., Benard, C., Branicky, R., Burgess, J., Hihi, A.K., Rea, S., 2001. Why only time will tell. Mech. Ageing Dev. 122, 571 594. Helfand, S.L., Rogina, B., 2003. Molecular genetics of aging in the y: is this the end of the beginning? Bioessays 25, 134 141. Herndon, L.A., Schmeissner, P.J., Dudaronek, J.M., Brown, P.A., Listner, K.M., Sakano, Y., Paupard, M.C., Hall, D.H., Driscoll, M., 2002. Stochastic and genetic factors inuence tissue-specic decline in ageing C. elegans. Nature 419, 808814. Higgs, D.M., Souza, M.J., Wilkins, H.R., Presson, J.C., Popper, A.N., 2002. Age- and size-related changes in the inner ear and hearing ability of the adult zebrash (Danio rerio). J. Assoc. Res. Otolaryngol. 3, 174 184. Imai, S., Armstrong, C.M., Kaeberlein, M., Guarente, L., 2000. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795800. Jazwinski, S.M., 2002. Growing old: metabolic control and yeast aging. Annu. Rev. Microbiol. 56, 769792. Johnson, T.E., 2003. Advantages and disadvantages of Caenorhabditis elegans for aging research. Experimental Gerontology, this issue. Johnson, T.E., Cypser, J., de Castro, E., de Castro, S., Henderson, S., Murakami, S., Rikke, B., Tedesco, P., Link, C., 2000. Gerontogenes mediate health and longevity in nematodes through increasing resistance to environmental toxins and stressors. Exp. Gerontol. 35, 687 694. Klapper, W., Heidorn, K., Kuhne, K., Parwaresch, R., Krupp, G., 1998. Telomerase activity in immortal sh. FEBS Lett. 434, 409 412. Knapik, E.W., 2000. ENU mutagenesis in zebrashfrom genes to complex diseases. Mamm. Genome. 11, 511 519. Lee, K.Y., Huang, H., Ju, B., Yang, Z., Lin, S., 2002. Cloned zebrash by nuclear transfer from long-term-cultured cells. Nat. Biotechnol. 20, 795 799. Lee, S.S., Lee, R.Y., Fraser, A.G., Kamath, R.S., Ahringer, J., Ruvkun, G., 2003. A systematic RNAi screen identies a critical role for mitochondria in C. elegans longevity. Nat. Genet. 33, 40 48. Lin, Y.J., Seroude, L., Benzer, S., 1998. Extended life-span and stress resistance in the Drosophila mutant methuselah. Science 282, 943 946. Linney, E., Hardison, N.L., Lonze, B.E., Lyons, S., DiNapoli, L., 1999. Transgene expression in zebrash: a comparison of retroviral-vector and DNA-injection approaches. Dev. Biol. 213, 207216. Liu, R.K., Walford, R.L., 1966. Increased growth and life-span with lowered ambient temperature in the annual sh, Cynolebias bellottii. Nature 212, 12771278. Liu, R.K., Walford, R.L., 1969. Laboratory studies on life-span, growth, aging, and pathology of the annual sh, Cynolebias bellottii Stendachner. Zoolog.: N. Y. Zoolog. Soc. 54, 1 19.

Arking, R., 2001. Gene expression and regulation in the extended longevity phenotypes of Drosophila. Ann. N. Y. Acad. Sci. 928, 157 167. Barker, K.S., Quiniou, S.M., Wilson, M.R., Bengten, E., Stuge, T.B., Warr, G.W., Clem, L.W., Miller, N.W., 2000. Telomerase expression and telomere length in immortal leukocyte lines from channel catsh. Dev. Comp. Immunol. 24, 583595. Bartke, A., Coschigano, K., Kopchick, J., Chandrashekar, V., Mattison, J., Kinney, B., Hauck, S., 2001. Genes that prolong life: relationships of growth hormone and growth to aging and life span. J. Gerontol. A Biol. Sci. Med. Sci. 1, B340B349. Beckwith, L.G., Moore, J.L., Tsao-Wu, G.S., Harshbarger, J.C., Cheng, K.C., 2000. Ethylnitrosourea induces neoplasia in zebrash (Danio rerio). Lab. Invest. 80, 379 385. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., Wright, W.E., 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349352. Bradford, C.S., Miller, A.E., Toumadje, A., Nishiyama, K., Shirahata, S., Barnes, D.W., 1997. Characterization of cell cultures derived from Fugu, the Japanese puffersh. Mol. Mar. Biol. Biotechnol. 6, 279288. Campisi, J., 2001. From cells to organisms: can we learn about aging from cells in culture? Exp. Gerontol. 36, 607 618. Campisi, J., 2003. Cellular senescence and apoptosis: how cellular responses might inuence aging phenotypes. Exp. Gerontol. 38, 5 11. Chen, J.N., Fishman, M.C., 2000. Genetics of heart development. Trends Genet. 16, 383388. Cheng, K.C., Moore, J.L., 1997. Genetic dissection of vertebrate processes in the zebrash: a comparison of uniparental and two-generation screens. Biochem. Cell Biol. 75, 525 533. Clancy, D.J., Gems, D., Harshman, L.G., Oldham, S., Stocker, H., Hafen, E., Leevers, S.J., Partridge, L., 2001. Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292, 104 106. Collas, P., Alestrom, P., 1998. Nuclear localization signals enhance germline transmission of a transgene in zebrash. Transgenic Res. 7, 303 309. Comfort, A., 1961. The longevity and mortality of a sh (Lebistes reticularis Peters) in captivity. Gerontologia 5. Cristofalo, V.J., 1997. Cell culture as a model for gerontological research. Aging (Milano) 9, 441442. Dillin, A., Hsu, A.L., Arantes-Oliveira, N., Lehrer-Graiwer, J., Hsin, H., Fraser, A.G., Kamath, R.S., Ahringer, J., Kenyon, C., 2002. Rates of behavior and aging specied by mitochondrial function during development. Science 298, 23982401. Driever, W., Solnica-Krezel, L., Schier, A.F., Neuhauss, S.C., Malicki, J., Stemple, D.L., Stainier, D.Y., Zwartkruis, F., Abdelilah, S., Rangini, Z., Belak, J., Boggs, C., 1996. A genetic screen for mutations affecting embryogenesis in zebrash. Development 123, 3746. Eisen, J.S., 1996. Zebrash make a big splash. Cell 87, 969977. Fabrizio, P., Pozza, F., Pletcher, S.D., Gendron, C.M., Longo, V.D., 2001. Regulation of longevity and stress resistance by Sch9 in yeast. Science 292, 288290. Felsenfeld, A.L., Walker, C., Westereld, M., Kimmel, C., Streisinger, G., 1990. Mutations affecting skeletal muscle myobril structure in the zebrash. Development 108, 443459. Finch, C.E., Ruvkun, G., 2001. The genetics of aging. Annu. Rev. Genomics Hum. Genet. 2, 435462. Fishman, M.C., 2001. Genomics. Zebrashthe canonical vertebrate. Science 294, 12901291. Fishman, M.C., Stainier, D.Y., 1994. Cardiovascular development. Prospects for a genetic approach. Circ. Res. 74, 757 763. Garofalo, R.S., 2002. Genetic analysis of insulin signaling in Drosophila. Trends Endocrinol. Metab. 13, 156 162. Gerhard, G.S., Cheng, K.C., 2002. A call to ns! Zebrash as a gerontological model. Aging Cell 1, 104111. Gerhard, G.S., Kauffman, E.J., Grundy, M.A., 2000. Molecular cloning and sequence analysis of the Danio rerio catalase gene. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 127, 447457.

G.S. Gerhard / Experimental Gerontology 38 (2003) 13331341 Lo, J., Lee, S., Xu, M., Liu, F., Ruan, H., Eun, A., He, Y., Ma, W., Wang, W., Wen, Z., Peng, J., 2003. 15,000 unique zebrash EST clusters and their future use in microarray for proling gene expression patterns during embryogenesis. Genome Res. 13, 455466. Longo, V.D., Liou, L.L., Valentine, J.S., Gralla, E.B., 1999. Mitochondrial superoxide decreases yeast survival in stationary phase. Arch. Biochem. Biophys. 365, 131142. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S.K., Johnson, T.E., 2002. Transcriptional prole of aging in C. elegans. Curr. Biol. 12, 15661573. Ma, C., Fan, L., Ganassin, R., Bols, N., Collodi, P., 2001. Production of zebrash germ-line chimeras from embryo cell cultures. Proc. Natl Acad. Sci. USA 98, 24612466. Maures, T., Chan, S.J., Xu, B., Sun, H., Ding, J., Duan, C., 2002. Structural, biochemical, and expression analysis of two distinct insulin-like growth factor I receptors and their ligands in zebrash. Endocrinology 143, 18581871. Meng, A., Jessen, J.R., Lin, S., 1999. Transgenesis. Meth. Cell Biol. 60, 133148. Miller, R.A., Chrisp, C., Atchley, W., 2000. Differential longevity in mouse stocks selected for early life growth trajectory. J. Gerontol. A Biol. Sci. Med. Sci. 55, B455B461. Moon, H.S., Jacobson, E.M., Khersonsky, S.M., Luzung, M.R., Walsh, D.P., Xiong, W., Lee, J.W., Parikh, P.B., Lam, J.C., Kang, T.W., Rosania, G.R., Schier, A.F., Chang, Y.T., 2002. A novel microtubule destabilizing entity from orthogonal synthesis of triazine library and zebrash embryo screening. J. Am. Chem. Soc. 124, 11608 11609. Mullins, M.C., Nusslein-Volhard, C., 1993. Mutational approaches to studying embryonic pattern formation in the zebrash. Curr. Opin. Genet. Dev. 3, 648 654. Mullins, M.C., Hammerschmidt, M., Haffter, P., Nusslein-Volhard, C., 1994. Large-scale mutagenesis in the zebrash: in search of genes controlling development in a vertebrate. Curr. Biol. 4, 189202. Murakami, S., Tedesco, P.M., Cypser, J.R., Johnson, T.E., 2000. Molecular genetic mechanisms of life span manipulation in Caenorhabditis elegans. Ann. N. Y. Acad. Sci. 908, 40 49. Nasevicius, A., Ekker, S.C., 2000. Effective targeted gene knockdown in zebrash. Nat. Genet. 26, 216 220. Nasevicius, A., Ekker, S.C., 2001. The zebrash as a novel system for functional genomics and therapeutic development applications. Curr. Opin. Mol. Ther. 3, 224228. Nusslein-Volhard, C., Wieschaus, E., 1980. Mutations affecting segment number and polarity in Drosophila. Nature 287, 795801. Orr, W.C., Sohal, R.S., 2003. Does overexpression of Cu,Zn-SOD extend life span in Drosophila melanogaster? Exp. Gerontol. 38, 227230. Patnaik, B.K., Mahapatro, N., Jena, B.S., 1994. Ageing in shes. Gerontology 40, 113 132. Phillips, R.B., Reed, K.M., 2000. Localization of repetitive DNAs to zebrash (Danio rerio) chromosomes by uorescence in situ hybridization (FISH). Chromosome Res. 8, 27 35. Postlethwait, J.H., Talbot, W.S., 1997. Zebrash genomics: from mutants to genes. Trends Genet. 13, 183 190. Pozios, K.C., Ding, J., Degger, B., Upton, Z., Duan, C., 2001. IGFs stimulate zebrash cell proliferation by activating MAP kinase and PI3kinase-signaling pathways. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1230R1239. Pugh, T.D., Klopp, R.G., Weindruch, R., 1999. Controlling caloric consumption: protocols for rodents and rhesus monkeys. Neurobiol. Aging 20, 157165. Reinke, V., 2002. Functional exploration of the C. elegans genome using DNA microarrays. Nat. Genet. 32, 541546. Reznick, D., Buckwalter, G., Groff, J., Elder, D., 2001. The evolution of senescence in natural populations of guppies (Poecilia reticulata): a comparative approach. Exp. Gerontol. 36, 791812.

1341

Rogina, B., Reenan, R.A., Nilsen, S.P., Helfand, S.L., 2000. Extended lifespan conferred by cotransporter gene mutations in Drosophila. Science 290, 21372140. Rossant, J., Hopkins, N., 1992. Of n and fur: mutational analysis of vertebrate embryonic development. Genes Dev. 6, 113. Roush, W., 1995. Nobel prizes: y development work bears prize-winning fruit. Science 270, 380 381. Rubinstein, A.L., 2003. Zebrash: from disease modeling to drug discovery. Curr. Opin. Drug. Discov. Dev. 6, 218223. Shin, J.T., Fishman, M.C., 2002. From zebrash to human: modular medical models. Annu. Rev. Genomics Hum. Genet. 3, 311340. Sinclair, D.A., 2002. Paradigms and pitfalls of yeast longevity research. Mech. Ageing Dev. 123, 857867. Sohal, R.S., Agarwal, A., Agarwal, S., Orr, W.C., 1995. Simultaneous overexpression of copper- and zinc-containing superoxide dismutase and catalase retards age-related oxidative damage and increases metabolic potential in Drosophila melanogaster. J. Biol. Chem. 270, 1567115674. Solnica-Krezel, L., Schier, A.F., Driever, W., 1994. Efcient recovery of ENU-induced mutations from the zebrash germline. Genetics 136, 14011420. Stainier, D.Y., Fouquet, B., Chen, J.N., Warren, K.S., Weinstein, B.M., Meiler, S.E., Mohideen, M.A., Neuhauss, S.C., Solnica-Krezel, L., Schier, A.F., Zwartkruis, F., Stemple, D.L., Malicki, J., Driever, W., Fishman, M.C., 1996. Mutations affecting the formation and function of the cardiovascular system in the zebrash embryo. Development 123, 285 292. Stickney, H.L., Schmutz, J., Woods, I.G., Holtzer, C.C., Dickson, M.C., Kelly, P.D., Myers, R.M., Talbot, W.S., 2002. Rapid mapping of zebrash mutations with SNPs and oligonucleotide microarrays. Genome Res. 12, 19291934. Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F., 1981. Production of clones of homozygous diploid zebra sh (Brachydanio rerio). Nature 291, 293296. Sun, J., Tower, J., 1999. FLP recombinase-mediated induction of Cu/Znsuperoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster ies. Mol. Cell. Biol. 19, 216 228. Sun, J., Folk, D., Bradley, T.J., Tower, J., 2002. Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 161, 661 672. Tatar, M., Kopelman, A., Epstein, D., Tu, M.P., Yin, C.M., Garofalo, R.S., 2001. A mutant Drosophila insulin receptor homolog that extends lifespan and impairs neuroendocrine function. Science 292, 107 110. Tissenbaum, H.A., Guarente, L., 2001. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227230. Tissenbaum, H.A., Guarente, L., 2002. Model organisms as a guide to mammalian aging. Dev. Cell 2, 9 19. Udvadia, A.J., Linney, E., 2003. Windows into development: historic, current, and future perspectives on transgenic zebrash. Dev. Biol. 256, 117. Vaupel, J.W., Carey, J.R., Christensen, K., Johnson, T.E., Yashin, A.I., Holm, N.V., Iachine, I.A., Kannisto, V., Khazaeli, A.A., Liedo, P., Longo, V.D., Zeng, Y., Manton, K.G., Curtsinger, J.W., 1998. Biodemographic trajectories of longevity. Science 280, 855860. Vogel, G., 2000a. Genomics. Sanger will sequence zebrash genome. Science 290, 1671. Vogel, G., 2000b. Zebrash earns its stripes in genetic screens [news]. Science 288, 11601161. Walker, C., 1999. Haploid screens and gamma-ray mutagenesis. Meth. Cell Biol. 60, 43 70. Woodhead, A.D., 1978. Fish in studies of aging. Exp. Gerontol. 13, 125 140. Yan, Y.L., Talbot, W.S., Egan, E.S., Postlethwait, J.H., 1998. Mutant rescue by BAC clone injection in zebrash. Genomics 50, 287 289. Zhao, Z., Cao, Y., Li, M., Meng, A., 2001. Double-stranded RNA injection produces nonspecic defects in zebrash. Dev. Biol. 229, 215 223.