ANALISIS DEL REGLAMENTO PARA LOS SISTEMAS

Over-expression experiments were carried out in an effort to study the function of

mD2LIC in X. laevis. t h e first three experiments 100 pg, 200 pg or 400 pg capped

mD2LIC mRNA was injected into Xenopus embryos at either the 2-cell or 4-cell stage

of development and embryos were left to develop at I9°C. mD2LIC proved to cause

defects in gastrulation in a dose-dependent manner (see Table 4.3). Thus, injection of 100 pg, 200 pg and 400 pg lead to 15/21, 19/23 and 33/34 embryos exhibiting

gastrulation defects respectively. Embryos that progressed through the initial stages of gastrulation failed to close the blastopore correctly and had posterior defects, but

Experiment (Exp.) number and details

Exp. 1 100pg total of mD2LIC mRNA

Total number of embryos Total Stage 8-9

number of embryos dead by

Stage Stage Stage

10-11 18-20 34-35 % o f surviving embryos with wt phenotype Predominant phenotype of dead embryos

Uninjected 17 3 82% (14/17) Death prior to

gastrulation Single blastomere of 2-cell stage injected

with p-gal

18 - - 4 - 78% (14/18) Death prior to

gastrulation Single blastomere of 2-cell stage injected

with p-gal + mD2LIC

21 - - 15 - 28.5% (6/21) Gastrulation

failure

Exp. 2 200pg total of mD2UC mRNA

Uninjected 16 0 2 2 2 87.5% (14/16) Gastrulation

failure Single blastomere of 2-cell stage injected

with p-gal

24 3 5 6 6 75% (18/24) Death prior to

gastrulation Single blastomere of 2-cell stage injected

with mD2LIC

23 6 14 18 19 17% (4/23) Gastrulation

failure

Exp. 3 400pg total of mD2LIC mRNA

Both blastomeres of 2-cell stage injected with P-gal

15 1 1 1 1 93% (14/15) Death prior to

gastrulation Both blastomeres of 2-cell stage injected

with 200pg mD2LIC

34 3 22 27 33 3% (1/34) Gatrulation failure

Exp. 4 100pg total of mD2LIC mRNA

Single ventral blastomere of 4-cell stage injected with p-gal

20 - 5 - 75% (5/20) Death prior to or

at gastrulation

Single ventral blastomere of 4-cell stage 21 - - 16 - 24% (5/21) Death prior to or

o

00

injected with p-gal + mD2UC

Table 4.3. Results o f m D2LIC expression inX . laevis embryos

at gastrulation n zr

I

’*«

Figure 4.7 Overexpression of mD2LIC in Xenopus laevis. (a) shows wild-type uninjected

embryos and (b) shows embryos injected with 200pg of mD2LIC mRNA. (c) shows wild-

type unijected embryos after immunostaining of notochordal tissue with MZ15 and (d) shows injected embryos after immunostaining. A variety of phenotypes are produced

following the injection of mD2LIC, but all appear to stem from disrupted gastrulation

movements and failure of the dorsal lip to close correctly. Anterior structures appear less affected than posterior ones and immunostaining reveals that notochord is specified in injected embryos.

Chapter 4: Expression analysis o f D2LIC

anterior structures, including the head and cement gland formed normally (Figure 4.7). Immunostaining with MZ15, a notochord-specific antibody (Smith and Wah, 1985), revealed that notochord was present in such embryos (Figure 4.7)

In a fourth experiment embryos were injected ventrally with 100 pg o f mD2LIC. None

of the embryos (n=21) formed a secondary axis (Table 4.3)

4.3 Discussion

Grissom et al. (2000) have classified D2LIC as a new and highly divergent member of the cLIC family of proteins. This conclusion was based on immunoprécipitation results, which indicate that D2LIC is associated with cytoplasmic Dynein 2 Heavy Chain (DHC2), and the similar expression profiles of D2LIC and DHC2 at the mRNA level, and on the co-localisation of D2LIC and DHC2 at the cellular level to the golgi apparatus.

Data obtained in our study indicate that D2LIC is highly conserved at the protein sequence level between species, but is weakly related to other members of the cLIC family, being both smaller (-350 amino acids compared with -500-700 amino acids) and possessing a 44 amino acid region that is highly related to the yeast small GTPases Temlp and S pglpl, that is not present in other cLICs described to date.

D2LIC is expressed in a similar (but not identical) manner in mouse, chick, Xenopus

and Zebrafish. Expression is in general detected in the late gastrula organiser followed by widespread expression which becomes graded along the A-P axis before becoming restricted to the gut endoderm. It is note worthy that D2LIC is expressed in ciliated cell

types, for example the node of mouse and chick, the neural plate o f Xenopus and the

organiser of Zebrafish. Interestingly, DHC2 loss-of-function mutants in

Chlamydomonas exhibit defects in ciliogenesis due to arrested intra-flagellar transport (Pazour et al., 1999), so one might predict a role for D2LIC in ciliogenesis. However, D2LIC is also expressed in non ciliated cell types suggesting that it might have diverse roles in cell biology. Consistent with this idea is the observation that D2LIC interacts with the Golgi apparatus in a microtubule-independent manner and that some D2LIC localises to vesicular compartments adjacent to or independent of DHC2-associated structures (Grissom et al., 2002).

Mis-expression of mouse D2LIC in X. laevis embryos suggests that this gene may have a role in gastrulation, however the effect of this gene may also be non-specific

In summary, sequence analyses indicate that D2LIC is highly conserved amongst vertebrates; the protein is a new and highly divergent member o f the cLIC family; and its expression pattern indicates that it may play a role in late organiser function and may be involved in cilogenesis.

Chapter 5: Protein studies o f mD2LIC

Chapter 5