Contenido de cenizas en el masterbatch

As discussed in chapter one, experimental analysis in Drosophila has established that ptc expression is directly regulated by Hh signalling. When we first cloned p tc l and ptc2 in Zebrafish we wanted to establish whether vertebrate ptc expression is also regulated by Hh signalling. As already described in chapter 4, ptc genes are expressed in embryonic regions that are known to respond to Hh signals and as I showed in chapter 5, overexpression of hh genes is sufficient to induce ectopic transcription of ptc genes. We also wanted to establish whether high level expression of p tc l and ptc2 required Hh signalling. When we started these investigations there was no available mutation in any of the hh genes. Since then a mutation has been isolated in shh, syu, and I will discuss this mutation in the next chapter. However, there is still no known mutation in either twhhor ehh, and as the expression patterns of twhh and ehh overlap that of shh, this means that even in the syu mutation there is still some Hh signalling in most regions of the embryo. Therefore, I examined the expression of p tc l and ptc2 in mutations that are missing all Hh activity in particular regions of the embryo. Embryos homozygous for cyclops (eye) or floating head (flh)lack expression of all of the characterised zebrafish hhgenes, and/or express certain subsets of hhgenes, at different positions along the rostro-caudal axis of the embryo, at particular times of development. I examined the expression of p tc l and ptc2 in these mutants to establish if the high level expression of either of these genes requires Hh activity; and to investigate the spatial relationship between discrete sources of Hh activity and p tc l and ptc2 expression. The initial analysis of p tc l expression in eye and flh homozygotes was conducted by both myself and Jean Paul Concordet (Concordet, J.-P. et al. 1996) but the analysis of ptc2 expression in these mutants is all my own work, and all of the in situsand photographs shown here, except for (fig. 27 D, F, K & M), are my sole work.

c y c lo p s

Mutation of the eyegene disrupts the specification of the prechordal plate mesoderm and concomitantly the induction of the overlying neurectoderm in the brain, and this produces

a cyclopic phenotype, eye mutants also have delayed development of the floor plate in the trunk (Hatta, K. et al. 1991; Hatta, K. et al. 1994; Thisse, C. et al. 1994; Strâhle U. et al 1997). The eye locus has recently been cloned and is a nodal related gene (Sampath, K. et al. 1998; Regbagliati, M. R. et al. 1998).

The disruption of prechordal plate formation in homozygous eye embryos results in a complete loss of shh and of twhh expression in the brain, with the exception of a small stripe of shh expression that extends dorso-ventrally in the diencephalon at about 24 hpf and a very small dot of shh expression in the very anterior of the head (fig. 26 E). At this stage a ventral patch of twhh and shh expression also forms posterior and ventral to this stripe in the presumptive gut (fig. 26 E & F). However, in the hindbrain and the trunk the expression of shh and ehh in the notochord is normal but there is no expression of shh or

twhh in the ventral neural tube, until the floor plate finally forms. At 24 hpf most of the wild type expression of pte2 and p te l is missing from the forebrain and midbrain of homozygous eye embryos (fig. 26 cf. G to C & H to D). The remaining expression of the pte genes correlates closely with the remaining hh expression: p tel and ptel are only expressed around the dorsal ventral stripe of shh expression and the region of the developing gut, where twhh and shh are expressed (fig. 26 G & H). The hindbrain expression of both pte genes is also reduced, though it is still present. However p te l and

ptel are both still expressed at normal levels in the trunk.

F igure 26: p tc expression in cxclops hom ozygotes

Transcripts were revealed by in situ hybridisation with antisense RNA probes to shh (A & E); twhh (B & F); p tel (C & G) and ptel (D & H).

(E - H) show expression in eyelops homozygotes and (A - D) show expression in wild type siblings processed in parallel. All embryos were fixed at 24 hpf. Expression of both

pte genes is lost from all the areas of the anterior brain except for regions around the cells now expressing shh and/or twhh.

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Figure 26

flo a tin g head

flh is a mutation in the zebrafish homologue of X n ot, a homeobox gene which is expressed in the embryonic shield and the notochord, and the main phenotype of flh homozygotes is a disruption of notochord specification (Talbot, W. S. et al. 1995). Cells that would normally form notochord transiently express both notochord and muscle markers and then are mis-specified as muscle (Halpern, M. et al. 1995). This leads to a premature loss of shh expression from the axial mesoderm posterior to the midbrain- hindbrain boundary. In contrast to shh, which is lost gradually from the midline but is initially expressed, ehh is never expressed in f l h (Currie, P. D. and Ingham, P. W. 1996). The expression of hhgenes in the ventral neural tube in the trunk is also affected. In the anterior trunk a continuous floor plate is still induced and expression of shh and twhh is normal. However, in the posterior trunk the development of the floor plate is disturbed and instead of a continuous line of floor plate cells, small discontinuous groups of floor plate like cells develop. These cell express both shh and twhh. In contrast to homozygous eye embryos, expression of shh and twhh is normal in the midbrain and forebrain of homozygous/Z/z embryos.

I analysed pte expression in flh homozygotes to establish whether p te l or pte2 expression in the trunk requires ehh and to compare how the expression of pte2 and p te l relates to discrete sources of twhh and shh activity. As in eye homozygotes, the expression of pte genes correlates with the expression of hh genes 'm flh homozygotes, p te l and pte2 are expressed normally in the brain (fig. 27 B & F), but at 24 hpf, expression of both genes is substantially reduced in the trunk (fig. 27 B, F, H & M). In the caudal region of the trunk, p te l and pte2 are mainly expressed in small groups of neural cells dorsal to those expressing shh and twhh, with the pte2 expression extending slightly more dorsally from the hh expressing cells than the p te l expression, mirroring the more dorsal expression of pte2 in the wild type neural tube. In the rostral trunk where there is a continuous stretch of floor plate, pte2 and p te l are expressed in the ventral neural tube and pte2 is also expressed more dorsally in the neural tube. However, there is no expression of either pte gene in the anterior somites, apart from maybe a few mesodermal cells that are in contact with the shh and twhh expressing cells in the neural tube. Weak expression of pte2 is however sometimes seen in posterior / tail somites of the embryo.

Brief summary and Conclusions

In summary, the analysis presented in this chapter suggests that Hh signals are required for high level expression of both p tc l and ptc2. In addition the expression of p tc l in the trunks o f flh homozygotes suggests that Hh signals can regulate the transcription of p tc l over a longer distance than p tc l, which is also consistent with the wild type expression pattern of the two genes described in chapter four. I will discuss all of these issues in more detail in chapter nine.

Figure 27: ptc expression in flh homozygotes

Transcripts were revealed by in situhybridisation with antisense RNA probes to shh (C, D, J & K); shh + p tc l (N); shh + p tc l (I); p tc l (A, B, G & H) and p tc l (E, F, L & M). Where shh and ptc expression are visualised simultaneously, shhexpression is visualised in red and ptc expression in blue.

(B, D, F, H, I, K, M & N) show expression in flh homozygotes and (A, C, E, G, J and L) show expression in wild type siblings processed in parallel. (A-F) show whole mount lateral views, and the trunk above the yolk extension, at 24 hpf. Expression of both ptc genes is lost from the somites and from most of the caudal trunk apart from around the groups of floor plate like cells that still express shhand twhh. In addition p tc l expression extends more dorsally in the neural tube from these groups of cells than p tc l expression does (cf. I & N).

ANALYSIS OF MIDLINE MUTATIONS ISOLATED IN THE