Propiedades nutricionales

To attempt to establish when con, you, and syu are first required for Hh signalling I examined myoD and p tcl in these 3 mutants at 5-6 somites. At this stage there is a very slight but consistent reduction in ptcl expression in some embryos (fig. 35 A, C, E, H- J). The numbers were roughly Mendelian for syu and con [syu - 7/36; con 11/52] but were less than Mendelian for you [6/67]. However there is a more noticeable difference in myoD expression at this stage (fig. 35 B, D, F, L - N). In all three mutants myoD

expression in the adaxial cells is reduced and slightly patchy and the numbers were roughly Mendelian for all three of the mutants [syu - 16/80; you 15/64; con 14/56].

Figure 31: Expression of p t c l and M \o D at the end of gastrulation

Transcripts were revealed by in situ hybridisation with antisense RNA probes to ptcl (A, C, E, G, I, J & L) or MyoD + krox-20 (B, D, H, K, M) or just MyoD (F). Expression of krox-20 provides an accurate independent indication of developmental stage and staining levels in individual embryos.

(A - H and K) show a random selection of embryos from an in situ hybridisation to demonstrate that none of the progeny from syu, you or con heterozygous parents have an altered expression of MyoD or p tc l and none of the progeny from igu heterozygous parents have an altered expression of MyoD at this stage: (A & B) are from a syu cross; (C & D) are from a con cross; (E & F) are from a you cross (G & H) are from a ubo

cross and (K) is from an igu cross.

However, the expression of ptcl is reduced in igu homozygotes at this stage ((J) cf. wild type expression in (I)); and yot homozygotes have reduced expression of both p tc l and

MyoD (L & M respectively, wild type embryo on the left in (L) and on the right in (M)), but ptcl is still expressed weakly throughout the adaxial cells, whereas MyoD is only expressed in the tail bud and most posterior adaxial cells.

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Figure 32: p t c l and M vo D expression at 10-15 somites.

Transcripts were revealed by in situ hybridisation with antisense RNA probes to p tcl (A, C, E, G, I, K & M-R) or M yoD (B, D, F, H, J & L). (A, B, M) show expression in wild type embryos at 10-12 somites (B & M) and 15 somites (A).

The expression of MyoD is reduced in the adaxial cells of embryos homozygous for syu, con, igu, you and yot (C, E, G, I, K respectively) at 14-15 somites {syu and con) or 11 -13 somites (you, yot and igu). yot and syu have the most substantial reduction in MyoD

expression, there is less of a reduction in con, igu and especially you embryos. In all cases, even syu and yot, there is persistent expression of MyoD in the tailbud / posterior adaxial cells. The lateral somite expression of MyoD is also slightly reduced in the most rostral somites, most notably at this stage in syu, con and yot.

At 10-12 somites p tcl expression is also reduced in the adaxial cells of embryos homozygous for syu, con, you and yot (D, F, J, L respectively) but in contrast p tc l

expression is expanded in the somites and pre-somitic mesoderm of igu homozygotes (H). The most dramatic loss of ptcl expression is in embryos homozygous for yot which have almost no adaxial expression of ptcl at this stage, though there is still weak neural tube expression (see R).

(O - R) show whole embryo dorsal flat prep views of p tcl expression in con, you, syu, igu, and yot homozygotes respectively, at the same stages as the trunk views shown above. These show that in con, you, syu and yot there is more considerable expression of ptcl in the heads of mutant embryos than in the trunks. This is particularly striking in

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igu I I syu I

Figure 33: ptcl expression in s \ u , con, van, and vot at 32-34 hpf

Transcripts were revealed by in situ hybridisation with antisense RNA probes to p tcl on embryos at 32-34 hpf. (A-E) show flat preps of p tc l expression in the head, whereas (F & G) show p tcl expression in the whole embryo and (H-M) compare expression in the head (H, J, L) with expression in the trunk (I, K, M). (A, F, H and I) show p t c l

expression in wild type embryos for comparison with the expression in homozygous mutant embryos.

(B & G) show that ptcl expression is strongly reduced in syu homozygotes at this stage, most noticeably in the trunk where it is only expressed in the ventral neural tube. The head expression of p tcl is more considerable than the trunk expression, but is still reduced, especially in the very anterior of the brain (behind the eye in (G) where there is normally a peak of ptcl expression that extends up towards the epithesis, also see (B) where the anterior of the diencephalon is hardly expressing any ptcl.

(C, J, And K) show that in con homozygotes p tc l expression is also reduced more considerably in the trunk than in the brain. However, in con homozygotes the anterior expression of ptcl in the brain is still strong and it is the hindbrain expression that is more obviously reduced (C & J). In you homozygotes (D) and yot (B, L) the expression of p tc l is normal in the brain, though in yot homozygotes p tc l expression is still considerably reduced in the trunk in a similar way to con and syu homozygotes (M).