Characterisation o f Rohon Beard cell death Fig Zebrafish explants have normal developm ent of Rohon Beard

neurons as shown by TUNEL labelling, dorsal view (A), HNK-1 labelling, dorsal view (B).

Neutralizing NT-3 with an antibody to NT-3, induces Rohon Beard cell death, as determined by TUNEL labelling. Anti-NT-3 20n g/m l (C, D).

Exogenous NT-3 reduces cell death as determined by TUNEL labelling. NT-3 2ng/m l, (E, F), NT-3 2 0n g/m l (G, H).

Removal of the skin increases cell death in the explants, as determined by TUNEL labelling, (I).

A

B

r

H

4. Characterisation o f Rohon Beard cell death

D iscussion

4.3.1. Z e b r a fis h R o h o n Beard n e u r o n s u n d e r g o a p o p to s is d u r i n g d e v e l o p m e n t .

There is a very predictable p attern of cell death found in the dorsal spinal cord of developing zebrafish. These dying cells are R ohon Beard neurons as show n by double labelling of TUNEL and HNK-1 antibody. The literature indicates that their are varying fates of Rohon Beard neurons in different animals. In Teleosts and A m phibia Rohon Beard cells die during developm ent (Eichler et ah, 1981) how ever in new ts an d lam preys Rohon Beard n eu ro n s survive into ad u lth o o d (Nakao et ah, 1987). This stu d y of zeb rafish Rohon Beard n eu ro n s show s th at all R ohon Beard cells are rem oved from about 20s to 3.5 days in all b u t a few cases. Cell death in the Rohon Beard neurons of zebrafish is slow and gradual, w ith no w aves of death. In contrast, the Rohon Beard cells of X e n o p u s u n d erg o tw o w aves of cell death , one just before the Dorsal Root ganglions (DRGs) innervate, and a second wave before m etam orphic climax (Eickler et ah, 1981).

D eath in zebrafish Rohon Beard cells follows a m ore sim ilar time course to that observed in Rana p ipiens and Ceratophrys ornata in w hich rates of cell death are fairly constant through developm ent. H ow ever one difference is th at Rohon Beard cells of Rana p ip ie n s and C e r a t o p h r y s ornata do undergo an increase in the rate of late cell death (Eickler et ah,

1981). A nother difference in the death of Rohon Beard cells in zebrafish com pared to frogs is that in frogs Rohon Beard cell death takes place in a craniocaudal progression, w ith the last surviving cells all found in the tail. In zebrafish, Rohon Beard cell death took p art in all sections of the em bryo d u rin g the tim e period of death. This m aybe because the developm ent of the zebrafish is m ore rap id th an th a t of the frog. W hereas th e frog p ro d u ce s all its Rohon Beard cells before they die, the zebrafish has p roduction and death of the Rohon Beard cells occurring at the sam e tim e th ro u g h m uch of the period of developm ent w hen Rohon Beard n eu ro n s are present. If an intrinsic clock m echanism determ ines death, in the case of the frog the Rohon Beard cells born near the head w ould be older than the Rohon Beard cells born at the tail and w ould therefore die sooner. This p attern of death w ould not necessary occur in the zebrafish. A lternatively the craniocaudal progression of death found in the frog could be due to the

4. Characterisation of Rohon Beard cell death

w here as in the zebrafish this depletion w ould occur th ro u g h o u t the embryo.

4.3.2. W hat triggers Rohon Beard neuronal death?

The triggers for Rohon Beard cell death have not been determ ined bu t is probably variable betw een the organism s that have been studied. In

X e n o p u s R ohon Beard n eu ro n s u n d e rg o tw o w aves of cell d e a th suggesting th at extrinsic factors determ ine if the cells die, rath er th an an intrinsic clock m echanism (Lam borghini, 1987). The first w ave of d eath occurs just after the innervation of Dorsal Root ganglia (DRGs). The DRGs replace the Rohon Beard cells functionally. It could be hypothesised th at R ohon Beard cells and DRGs com pete for a lim ited su rv iv a l factor resulting in the death of the Rohon Beard cells. H ow ever X e n o p u s Rohon Beard neurons are not im m ediately superseded w hen the neu ro n s of the DRGs send fibres in to the spinal cord and out to the p eriphery, and a proportion of Rohon Beard cells are not effected (Lamborghini, 1987). This suggests that if the DRGs do trigger death of the Rohon Beard neurons, it is not the only controlling factor. In zebrafish DRGs innervate at 2-2.5 days (Kimmel and W esterfield, 1990). At this time an increased rate of Rohon Beard cell death is not detected suggesting th at the innervation of DRGs has no effect on zebrafish Rohon Beard cell death and that their DRGs and Rohon Beard neurons are not com peting for the sam e lim ited survival factor.

The second wave of death observed in Xenopus m ay w ell be caused by the surge of thyroid concentration w hich characterizes m etam orphic climax. Again, the critical factor for the onset of cell death in late larvae of

R an a p ip ie n s and C eratop hrys ornata could be the su rg e in th y ro id concentration (Eichler and Porter, 1981). W hatever the cause of cell death, in X e n o p u s it seems to be a extrinsic factor determ ining w h en the cells die as opposed to an intrinsic clock mechanism. Evidence for this comes from lo o k in g at the loss of R ohon Beard n e u ro n s in em b ry o s w h o se developm ent was arrested by crowding. It was found the rate of cell death w as ap p ro p riate for the developm ental stage of anim al rath er th an their chronological age.

4. Characterisation o f Rohon Beard cell death

how ever a slow depletion of survival factors could account for the demise. A ltern ativ ely , the presence of an "internal clock" m ech an ism could d eterm in e w hen their tim e is up. In this m echanism tim e w o u ld be m easured by the accumulation or loss of a product: at a defined tim e point, a threshold of the p roduct is reached and it acts as a sw itch to trigger a p articular developm ental process. Such a m echanism has been im plicated in, for exam ple, controlling the onset of the m idblasula tran sitio n (MET) an d oligodendrocyte p ro g en ito r differentiation in the ra t optic nerve (Pourquie, 1998).

4 .3 .3 . A caspase inhibitor is able to p reven t Rohon Beard cell death tr a n sie n tly .

The caspase inhibitor, zVADfmk is able to reduce cell d eath in the Rohon Beard cells up until 36 hours of developm ent. A fter this tim e the d ru g offers no protection and eventually all the zebrafish R ohon beard cells die. Caspase inhibitors have previously been show n to p rev en t cell d e ath in m otor n eu ro n s in vivo and in vitro (M illigan, 1995) an d to p rev en t apoptosis of N G F-deprived DRG neu ro n s (M usaka, 1997). The failure of zVADfmk to p erm anently rescue the R ohon Beard n eu ro n s could be due to the caspase inhibitor not being irreversible, as thought. R epetitive additions of zVADfmk do not continue the p rev en tio n of cell death, probably due to perm eability problem s at this stage in zebrafish developm ent. A lternatively there is recent evidence th at in som e cases, cells "rescu ed " from dying eventually w ill die via a necrotic ro u te (Lemaire et al., 1998).

4.3.4. T r k C l receptor is expressed in a subpopulation o f Rohon Beard neurons which are not undergoing cell death.

Triple labelling w ith anti-HNK-1, TrkC l probe and TUNEL show s that T r k C l is expressed in a subpopulation of Rohon Beard n eurons w hich is n o n -o v erlap p in g w ith the subp o p u latio n of cells w hich are TUNEL positive at this time. Expression of T r k C l is never observed in a TUNEL positive cell. Rohon Beard cells first express T r k C l at 16.5 h p f (M artin et al., 1998) a few ho u rs before Rohon Beard TUNEL p o sitiv e cells are observed.

4. Characterisation of Rohon Beard cell death

As well as being expressed in a subset of Rohon Beard cells, Tr kC l is also expressed in the trigem inal ganglia of the zebrafish (M artin et al.,

1998). Rohon Beard cells and Trigeminal ganglia have other sim ilarities. They are the first neurons to differentiate in the embryo and they are both p rim ary sensory neurons that m ediate touch sensitivity (Bertrand et al,

1990). They initiate axogenesis at the same early time and appear to be the first n eu ro n s in the em bryo to do so. Both are labelled by the HNK-1 epitope perm itting the study of their developm ent unobscured by labelling in other cells. Both are embryologically sim ilar how ever all Rohon Beard cells eventually die.

The death of the Rohon Beard neurons could be due to the fact that d u rin g developm ent they change their response to neu ro tro p h in s w hich differs from trigem inal ganglion n euron dependency on n eu rotrophins. As o u tlin e d in the in tro d u c tio n , it has b een sh o w n th a t d u rin g d e v e lo p m e n t p o p u la tio n s an d su b p o p u la tio n s show sp a tia lly an d tem porally different responses to n eu ro tro p h in s (Buchm an and Davies, 1993). For example, during m ouse developm ent trigem inal neurons show a tem p o rally different response to BDNF an d NT-3. First the m ouse trig e m in a ls h av e n e u ro tro p h in in d ep en d en ce, th e n th ey d isp la y a transitory survival response to BDNF and NT-3. This response is lost as the neurons become N G F-dependent (Fig. 4.5.). In this case neurotrophins are needed for cellular differentiation and survival w hile later they are needed for cellular for cellular maintenance.

This data leads m e to hypothesise th at the Rohon Beard n eu ro n s are first tro p h in in d ep en d en t (though pro b ab ly d e p en d en t on o th er factors) w h en HNK-1 is expressed a t IShpf. W hen they sta rt expressing T r k C l at 16.5hpf, they begin to display a transitionary survival response to NT-3. If T r k C l is dow n regulated, w hich occurs in roughly 20% of the population at 26hpf, the neurons die thus there is nearly same percentage of T r k C l negative and TUNEL positive cells at 26hrs. Cells not expressing Trk Cl which are not TUNEL positive could be cells w hich have just sw itched off TrkCl and will soon die. Eventually all the Rohon Beard cells becom e unresponsive to NT-3, and as R ohon Beard cells do not becom e responsive to another neurotrophic factor, they die (Fig. 4.5.). By this n o tio n , the zebrafish trigem inal ganglions are p ro b ab ly tro p h in

4. Characterisation of Rohon Beard cell death

Fig. 4.5. D evelopm ental sequence, from left to right, of the changing survival requirem ents of embryonic m ouse trigem inal ganglion neurons (adapted from Davies, 1997) (A) and proposed for zebrafish Rohon Beard neurons based on data (B).