Invertebrate Models - The fly and worm models are enabling highly successful studies of genetic influences because their gene regulatory systems of early development are known in detail (Davidson, 2006; Giudice, 2001; Grant and Wilkinson, 2003). Mutations modify longevity in association with altered mortality rate accelerations (Chapter 5). Some mutations that modify aging involve insulin-like signaling pathways and fat depots (Fig.1.3A). These convergences suggest the importance of energy regulation to aging, as well as to development. The energy-regulating gene circuits have persisted during descent from shared ancestors more than 650 million years ago. Although the causes of death in fly and worm are not well defined, the causes do not include tumors or other abnormal growth during aging.
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| Invertebrate models |
The lab worm C. elegans naturally grows among the roots of plants. Propagation by self-fertilization eliminates more genetic variation than is possible with inbred laboratory mice (Johnson et al, 2005). Free-living larva hatch about 24 h after fertilization, followed by rapid development through larval stages (L1-L4) and maturation by 72 h. If food is limited, or population density is high, the larval development may be arrested for up to 2 m in the dauer larval stages. Dauer larvae cease feeding and utilize fat depots; body movements decrease, but stress resistance increases (Kimura et al, 1997). With improved conditions, dauer larvae complete maturation and proceed to normal life spans. Worm life history has four stages lasting 2–3 w (Huang et al, 2004): I, active egg production by self-fertilization in the first 4 d (Bolanowski et al, 1983; Herndon et al, 2002), followed by several post-reproductive stages: II, postreproductive, with vigorous movements; III, dwindling movement leading to the cessation of feeding; and IV, morbidity with little movement and accelerating mortality. Most eggs are produced during the first 4 days (Croll et al, 1977; Johnson, 1987; Klass, 1977).
While life spans in different genotypes and environments are well documented, less is known about the cellular changes and the pathology of aging. Cell death is not obvious during aging, despite the lack of somatic cell replacement. C. elegans is famous for its almost invariant cell number. Neurons look normal in ultrastructure studies throughout life, including neurons of slowed and decrepit worms (Herndon et al, 2002). However, muscle cells deteriorate in the body wall and in the pharynx, which grinds up bacteria that are the diet (Herndon et al., 2002). Lipids, lipofuscins (aging pigments), and lysosomal hydrolases accumulate in muscle and intestine cells (Bolanowski et al, 1983; Epstein et al, 1972; Garigan et al, 2002; Herndon et al, 2002), implying defects in catabolic pathways. Old worms are less resistant to pathogenic bacteria and show shorter latent period after infection (Kurz et al, 2003; Laws et al, 2004).
Moreover, aging worms become constipated from bacterial packing in the intestine, which may induce oxidative damage. The usual diet of the bacteria Escherichia coli strain OP50 is considered by some to be mildly toxic; life spans are longer on heat-killed bacteria or other media (Section 2.3.2, Section 5.5.2). Long-lived mutants in insulin-like signaling (age-1) have delayed constipation (Section 5.5.2).
Although these worms are isogenic, constitutive variations in the levels of gene expression arise during development that influence later outcomes of aging (Finch and Kirkwood, 2000). Individual worms vary in the duration of these stages and in rates of aging. This extensive variability may be considered to extend variations present at younger ages in egg laying, feeding, and spontaneous movements (Finch, 1990, p. 560; Finch and Kirkwood, 2000). Individual declines of pharyngeal pumping and body movement were strongly correlated with life span in wild-type and longevity mutants (Chapter 5). For example, when fast pumping is maintained one day longer, the odds ratio for death by or later than a specified date is 1.7-fold greater (Huang et al, 2004a). The levels of expression of a stress-protective gene (hsp-16.2) in young worms predicted future life span, over a 2-fold range (Rea et al, 2005). This first example of individual difference in gene expression in the worm model supports the role of epigenetic variations arising during development that may ultimately represent chance variations in the assembly of the multiple proteins present in transcription complexes (Finch and Kirkwood, 2000). In another model, cultured mammalian cells with a reporter gene did not respond synchronously or to the same level to a diffusible inducer (Zlokarnik et al, 1998).
The fly is a more complex animal with a beating heart. At 25 °C, early development takes 24 h to larval hatching. Three mobile feeding larval stages (LILIII) take 7 more d to pupation. During the 4-day pupal stage, without feeding or movement, the adult body is formed from the imaginal disks (metamorphosis).
Adult life spans are about 40 d. Adult flies can over-winter, with extended life span from cool temperature and shorter photoperiods (Flatt et al, 2005; Finch, 1990, p.313; Schmidt et al, 2005). Unlike nematodes, the fly does not have alternate larval stages equivalent to the non-feeding dauer. Juvenile hormone (JH), a series of steroid-like molecules, influences or regulates growth of all developmental stages, particularly the timing of molts and metamorphosis, and also adult diapause (Flatt et al, 2005). JH synthesis is regulated by insulin-like peptides secreted by neurons (Section 5.4). JH also regulates stress resistance and immune responses that are like innate immunity of vertebrates.
Female egg-laying declines exponentially after a fairly stable phase, also observed in the medfly (Ceratitis capitata) (Novoseltsev et al, 2004). Both species have post-reproductive phases that only weakly correlated among individuals with the cessation of egg-laying. As with the worm, somatic cells are not replaced. Major damage is accumulated to the brittle exoskeleton from wearand- tear (Finch et al, 1990). Unlike the worm, the aging fly shows some indication of neuron loss, in the mushroom body (Technau, 1984). The fat body, a key organ of energy reserves and immune function, gradually atrophies (Finch, 1990, p. 63). Apoptosis with DNA fragmentation increases in flight muscles and fat body (Zheng et al, 2005). The heart rate slows during aging and arrests more easily under the stress of electrical pacing, aging sharply (Wessells et al., 2004) (Section 5.6.3, Fig. 5.7). Insulin-signaling mutants with increased life span have delayed cardiac aging (Chapter 5). Little is known about vasculature of aging flies; other aging insects show indications of circulatory blockage (Arnold, 1961, 1964; Finch, 1990, p. 65).
