CONCLUSIONES

are cultured w ith w ildtype. Cell death in fu s h i tarazu (ftz) m utants effects

f t z expressing cells as well as their neighbours suggesting local cell-cell in teractio n s are necessary for survival (M agrassi, 1988). M any of the n a tu ra lly occurring cell death s in the d ev elo p in g retina req u ire the p ro d u ct of the irregular chiam C-roughest (irreC-rst) gene. M utations in this gene lead to the survival of cells th at fail to occupy a p p ro p ria te p ositio n s in the developing om m atidia (Steller et ah, 1994; W hite et ah,

1995).

In a d d itio n to signals from the extracellular en v iro n m en t, cell d eath in D ro so p h ila is also influenced by intracellular signals th at act au to n o m o u sly w ithin the dying cell. A genetic ap p ro ach w as taken to screen a large fraction of the D r o s o p h ila genom e for genes th at are required for PCD (White et al. 1994; W hite et al., 1994). By exam ining the p a tte rn of apoptosis in em bryos hom ozygous for previously identified chrom osom al deletions, it w as possible to survey approxim ately half of the entire D rosophila genom e for functions involved in PCD. A single region on the th ird chrom osom e w as found to be req u ired for all cell d e a th s th a t n o rm ally occur in the D r o s o p h i l a em b ry o . E m b ry o s hom ozygous for Df(3L0H99), the sm allest cell death defective deletion available in this interval, contained m any extra cells. H99 m u tan ts w ere n o t only deficient in norm al cell d eath b u t w ere also sig n ifican tly protected against the induction of ectopic PCD by X-irradiation and against ectopic PCD th at is otherw ise seen in developm ental m u tan ts. Such a global blockade of PCD indicates that this deletion rem oves a function that is required for the induction of apoptosis in response to m any different signals (W hite et al., 1994). M olecular analysis of the H99 interval has revealed three genes w hose products can induce cell death: rpr (White et a l., 1996), h i d (G ru th er et al., 1995) an d g r i m (C hen et al., 1996). O verexpression of any single one of these genes induces extensive cell death even in the absence of the other two. In all three cases, the deaths seem to be caspase dependent since they are blocked by coexpression of baculovirus p35 (a broad spectrum caspase inhibitor). PCD first becom es a p p aren t d u rin g Drosophila developm ent at about 7hrs after egg laying. H o w ev er it is possible to induce apoptosis at earlier tim es by DNA d a m a g in g ag en ts (x -irradiation) or by o v erex p ressio n of ap o p to tic activators such as rpr or grim . Since all these stim uli activate a caspase-

1. General Introduction

d ep en d en t apoptotic program m e, the caspases required for cell death m ust be present at all times at which death can be induced.

In the past tw o years three Drosophila melanogaster caspases have been cloned drICE (Fraser et a l, 1997), Dcp-1 (Song et a l, 1997) and D redd (C hen et a l , 1998). As w ith all m am m alian caspase tran scrip ts to date, constitutive em bryonic expression has been reported for dr ICE (Fraser et

al., 1997) and for Drosophila caspase, Dcp-1 (Song et a l , 1997). D rIC E and

Dcp-1 are expressed ubiquitously at all stages w here death can occur during d ev elo p m en t and d u rin g the period before any detectable cell d eath is observed in the embryo, consistent w ith the notion th at PCD can occur before developm ental PCD is first observed. In contrast w ith Drosophila

caspases, dr IC E and D c p -1 , pronounced elevation of dredd tr a n s c r ip ts occurs in norm al developm ent in p reap o p to tic cells an d this u n iq u e regulation is tightly linked to apoptotic signaling by Reaper, Grim and Hid. Like drICE, D redd has sequence similarity to caspase-8 and also to caspase- 10. C aspase inhibitors prevent apoptosis b u t not the initial cleavage of D redd (Chen et ah, 1998). This raises the possibility that the initial step in the processing of D redd m ay occur through proteolytic com ponents th at are u p stream of caspase action, i.e. D redd could function as an initiator caspase.

S im ila r to th is , th e m a m m a lia n c a s p a s e , c a s p a s e 8 (proFL IC E /M A C H l) can be activated either via proteolytic processing by an o th er protease or th ro u g h the interaction of its N -term inus w ith the death-dom ain adaptor FA D D /M O R Tl which occurs as p a rt of the CD95- and TNE- killing pathw ays (Muzio, 1996). The cytokine tu m o u r necrosis factor (TNE) and the cytotoxic T cell ligand effector C D 95L /FasL /A PO -lL are p o w erful inducers of PCD w hich u pon b in d in g their specific target receptors induce a suicide response - the classic "cell killing" by TNE or cytotoxic T lym phocytes. There are few m olecules in the p a th w a y connecting the basal cell suicide m achine an d the T N F /C D 95-derived inform ational signals th at initiate it. Both TNE receptor-1 (TNERl) and CD95 share an intracellular "death domain" (DD) that plays a pivotal role in the abilities of these receptors to trigger apoptosis (Tartaglia et a l , 1993). The DD's on TNFRl and CD95 prom ote m ore extensive oligom erization u p o n ligand b in d in g to the receptors. W hereas CD95 d irectly recruits E A D D /M O R T l, TNFRl binds TRADD, w hich then acts as an ad ap to r

1. General Introduction

protein to recruit FA DD/M ORTl. FA D D /M O RTl seems to be the po in t of convergence betw een the CD95 and TNFRl d eath pathw ay. C onsistent w ith this, a dom inant-negative truncation m u tan t of FA D D /M O R T l th at lacks the N -term inal death effector dom ain (DED), blocks both CD95- and T N F R l-induced apoptosis. At its N -term inus, F L IC E /M A C H /casp ase 8 p o ssess regions w hich in teract w ith F A D D /M O R T l, w hile at its C- term inus it possesses a region w ith strong hom ology to caspases including a large prodom ain and all know n residues absolutely required for protease activity (Fig. 1.4.).

D red d could also be activated via proteolytic abscission of its inhibitory N -term inus by an upstream protease, or by interaction via the N -term in u s w ith activators analogous to FA D D /M O R T l, an d hence be a n alo g o u s to caspase 8 (FL IC E /M A C H l). There has b een hom ology re p o rte d b etw een reap er and the death dom ains of CD95 and TN FRl (G oldstein et a l , 1997). In this case reaper could be involved in recruiting D red d resulting in D redd activation and apoptosis. W here m am m alian caspase 8 (FLICE/M ACFIl) activity is regulated by the action of external ligands (CD95 or TNF), Drosophila D redd regulation w ould seem to rely on reaper.

Since the overexpression of m any caspases is sufficient to elicit ap o p to sis, the accum ulation of dredd in cells specified for d eath from signaling by Reaper, Grim and H id could overw helm the capacity of negative regulators such as lAPs (inhibitors of apoptosis), resulting in the initiation of the caspase cascade leading to cell death.

1.2.8. Relevance o f PC D during development.

A cell th at u n d erg o es PCD in anim al d ev elo p m en t is u su ally degraded so rapidly (often disappearing in an hour or less) that even w hen there is large scale PCD, there are surprisingly few dead cells to be seen (Jaco b so n et al., 1997). This m ay help to ex p lain w h y PCD w as u n d erstu d ied for so long. It also suggests that the extent of PCD in anim al d ev elo p m en t is still underestim ated. As it is n o t possible to m easure clearan ce tim es in m ost an im al tissu es, q u a n tify in g PCD rem ain s u n so lv e d .

1. General Introduction

M utant C.elegans which are deficient in PCD can have a norm al life span, even th o u g h they have about 15% m ore cells th an norm al and function less well than w ild-type animals (Ellis et a l , 1991). PCD-deficient flies, by contrast, die early in developm ent (White et ah, 1996). Mice w hich have a targeted m utation in caspase-3 die before birth w ith a vast excess of cells in their CNS as a result of decreased PCD in neuroepithelial cells, although PCD in other organs seems to occur norm ally (Kuida et ah, 1996). It seem s th at w ith increasing complexity of anim als, PCD becom es m ore vital.

ii) Functions of PCD in anim al developm ent

Five functions of PCD in anim al developm ent have been suggested (Jacobson et al, 1997); scu lp tin g stru ctu res, con tro llin g cell n u m b ers, d eletin g re d u n d a n t stru ctu res, elim inating abnorm al, m isplaced, n o n ­ fu n ctio n al or h a rm fu l cells, p ro d u cin g d ifferen tiate d cells w ith o u t organelles. Each of these will be considered in turn.

PCD plays an essential role in sculpting p arts of the body. In the fo rm atio n of digits in som e h ig h er v erteb rates PCD is n ecessary to elim inate the cells betw een the developing digits (Kimura et ah, Shiota et ah, ). PCD is involved in hollow ing out solid structures to create lumina. In early m ouse embryos, for example, the pream niotic cavity is form ed by the d eath of the ectoderm al cells in the core of the developing em bryo (C o u co u v an is, 1995). PCD also occurs w h e re v e r e p ith e lia l sh eets invaginate and pinch off to form tubes or vesicles as in the form ation of the vertebrate neural tube or lens (Ishizaki, et ah, 1998).

In m any organs cells are generated in excess and then elim inated by PCD to ad ju st th eir num bers. In the v erteb rate n erv o u s system for exam ple bo th neurons and oligodendrocytes are overproduced and then reduced by PCD to m atch their num bers to the num ber of target cells they innervate (Burek et ah, 1996; O ppenheim , 1991). Cell death m ay also have a role in size control; the size of an organism largely d ep en d s on how m an y cells there are rath er th an cell size or a m o u n t of extracellular m atrix, an d cell death is one com ponent w hich controls the n u m b er of cells p resen t (Raff, 1996). This h ap p en s d u rin g d ev elopm ent and also o p erates in a d u lt organs. For exam ple if hep ato cy te p ro life ratio n is

1. General Introduction

tra n sie n tly stim u lated in a d u lt rats by p h én o b arb ital trea tm e n t, for exam ple, increased PCD rapidly returns the liver to its norm al size.

In the course of developm ent, various structures are form ed th at are later rem oved by PCD. These include vestigial stru ctu res th at w ere req u ired in an ancestral descendant such as p ro n ep h ric tubules w hich form functioning kidneys in fish and am phibian larvae b u t are not used in m am m als and are elim inated by PCD. Structures that are needed at one stage of developm ent b u t not later, such as the deletion of the tail w hen a tad p o le transform s to a frog (Yaoita et a l , 1997). S tructures th a t are req u ired in one sex b u t not the other are rem oved; the M ullerian duct form s the uterus and oviducts in female m am m als b u t are n o t needed in males and are thought to be lost by PCD. In the developing chick limb, the in te r d ig ita l m esen c h y m e is m a in ta in e d for so m e tim e in an u n d ifferen tiated state and then undergoes m assive PCD. In this m odel TGF-beta bead im plants into the interdigits before the tim e of onset of PCD, diverts the cells from the PCD program to form ectopic cartilage (G anan et al, 1996). Conversely BMPs accelerate death an d cause dig it bifurifcation if placed at the tip of a form ing digit due to the form ation of an ectopic area of death. FGF-2 antagonizes the death inducing effects of the BMP beads suggesting death is controlled by a balance of death and survival signals m ediated by different grow th factors.

PCD also functions as p a rt of a quality-control process in anim al d e v e lo p m e n t, e lim in a tin g cells th a t are a b n o rm a l, m is p la c e d , nonfunctional, or potentially dangerous to the organism . For exam ple in the vertebrate im m une system developing T and B lym phocytes th at fail to pro d u ce potentially useful antigen specific receptors or p roduce self­ reactive receptors that m ake the cells potentially dangerous are elim inated by PCD. If the DNA is sufficiently dam aged in a cell the cell can activate its death pathw ay. This response serves as an anticancer m echanism and also helps prevent the birth of defective offspring.

The d eath program m e m aybe involved in p ro d u cin g specialized differentiated cells w ithout organelles. For exam ple skin kératinocytes die and form a layer of corpses on the surface of the skin, it is th o u g h t th at PCD m ay be involved in these processes as m orphologically the cell death is sim ilar, how ever it has not been determ ined if this is so (Nataraj et al.,

1. General Introduction

organelles w hen they term inally differentiate, at least one m em ber of the caspase family becomes activated (caspase-3). A peptide inhibitor of these p ro teases blocks the nucléation process, suggesting at least p a rt of the m achinery of PCD is involved in lens fibre differentiation (Ishizaki et al.,

1998).

T here are also m an y cell d e ath s th a t occur d u rin g an im al developm ent w here the function is unknow n.

1.2.9. D evelopm ental P C D does not occur u n til after the m id hlastula tr a n s itio n .

In all organism s studied, developm ental cell d eath has not been observed before the m id-blastula transition (MET). In fact blastom eres can survive w ith o u t extracellular signaling molecules: w hen cu ltu red from 1 cell u p to 16 cell m ouse em bryos, they can survive and divide in the absence of exogeneous p ro tein s or signaling m olecules even w h en cultured as isolated single cells (Diggers et a l , 1996).

The m idhlastula transition is characterized by cell-cycle lengthening an d asynchrony, the acquisition of cell m otility and the in itiatio n of zygotic transcription (N ew port et al, 1992). Prior to the MBT, em bryonic d ev elo p m en t is relatively sim ple, consisting of rap id cycles of DN A synthesis and mitosis and is regulated by mRNAs and proteins stored in the egg p rio r to fertilization. At the MBT developm ent becom es m ore com plex as zygotic transcription initiates, the cell-cycle lengthens, and cells differentiate and organize during gastrulation (N ew port et al., 1992). One explanation for a lack of apoptosis until after the MBT is th at cell cycle rem o d elin g is req u ired , as has been p ro p o sed for o th er MBT events (K im elm an et ah, 1987). Prior to the MBT, em bryonic cell cycles lack G phases, d u rin g w hich m any cell cycle checkpoints occur. Therefore, as th ese e a rly cycles occur w ith a lack of c h ec k p o in ts th ey occur in dependently of DNA content and m utations (N ew port et ah, 1989). Thus