EVALUACIÓN VARIABLES DIRECCIÓN PROVINCIAL DE PICHINCHA

Embryos were injected with mRNA encoding soluble Xephrin-A3 into the Xenopus

embryo in one cell at the two cell stage. Again the injected RNA was titrated, such that within a single batch, embryos received 3ng, 2ng, Ing, 500pg and lOOpg of RNA. As with the full length Xephrin-A3 injections, injection of 3ng of soluble Xephrin-A3 can

FUNCTIONAL ANALYSIS OF XEPHRIN-A3

cause non-specific gastrulation phenotypes. These embryos were removed fi*om the analysis.

The phenotypes produced by soluble Xephrin-A3 overexpression were very similar to those observed with full length Xephrin-A3 overexpression (see Fig. 5.10). There were no discernible differences within the phenotypes themselves but the number of embryos exhibiting the phenotypes was less. The trend observed was that embryos injected with high titres of Xephrin-A3 mRNA (3-2ng), showed a greater incidence o f the less severe phenotypes (Fig. 5.10A-D). Here, migration of the third arch neural crest took place but seemed to occupy greater areas of the migration pathway and ectopic cells were seen in the fourth arch region (Fig. 5.10A-D). Embryos injected with lower Xephrin-A3 mRNA (1-0.Ing) concentrations displayed fewer phenotypes and subsequently, larger numbers of embryos showed normal third arch neural crest migration on their injected sides. Injections of lOOpg of soluble Xephrin-A3 were indistinguishable from control RNA injected embryos or their uninjected siblings.

The soluble Xephrin-A3 injected embryos were also analysed with the pan neural crest marker XAP2 and with EphA2. Similar neural crest phenotypes were observed with

XAP2 analysis as had been observed with the full length Xephrin-A3 injections: migration of the first and second arch neural crest appeared unaffected by ectopic expression o f soluble Xephrin-A3 but the migration of the third and fourth arch neural crest streams was delayed and the separation of these streams was lessened (Fig. 5.10E- F). EphA2 expression analysis did not show any disruption of second arch neural crest migration (data n ot sh o w n ). Both results were indistinguishable from phenotypes exhibited

following full length Xephrin-A3 injections.

All the overexpression results with either frill length or soluble Xephrin-A3 were

tabulated (see Table 7). The table shows the number of experiments performed with each Xephrin-A3 construct, the numbers of embryos injected and the spread of phenotypes. Table 8 is an example of a typical injection experiment. It includes the numbers of embryos that die pre- and post- gastrulation. Usually the loss of embryos at gastrulation is related to the concentration of mRNA injected; however, the loss of control p-globin injected embryos injected with the same mRNA concentrations as Xephrin-A3 injected embryos, did not produce the same degree of embryo loss. Further investigation into these observations is described below.

Figure 5.10

Ectopic soluble Xephrin-A3 expression

(A) Side view of the control side and (B) injected side of an embryo injected with 3-2ng of soluble Xephrin-A3. XKroxlO expressing cells occupy a wider area along the migration pathway of third arch neural crest. (C)-(D) Side view of two further examples

XKroxlO analysed embryos injected with 2ng of soluble Xephrin-A3; (C) ectopic cells of the third arch neural crest are present in more caudal positions than normal and (D) a retardation in migration of the third arch neural crest cells. (E)-(F) Control and injected side views respectively of an XAP2 analysed embryo injected with 3ng of soluble Xephrin-A3; (E) The control side indicates normal XAP2 expression in the four neural crest streams. (F) The injected side of the embryo indicates a normal migration of the first and second arch neural crest streams. The third and fourth arch crest streams have not migrated as far as those on the control side of the embryo.

r3-rhombomere 3. r5-rhombomere 5. ncl-first arch neural crest. nc2-second arch neural crest. nc3-third arch neural crest. nc4-fourth arch neural crest.

# - F

^ n c 3 +nc4 •4 nc2

Table 7

Composite table of injections perform ed in 1 cell of the 2 cell Xenopus embryo This table indicates the number o f embryos exhibiting each phenotype in relation to the concentration of mRNA encoding P-globin, full length Xephrin-A3 or soluble Xephrin- A3 injected into the 1 cell of the two cell Xenopus embryo.

The following terms are used;

p-globin - P-globin; Xephrin-A3 - Full length Xephrin-A3; AXephrin-A3 - soluble Xephrin-A3; No. of expts - number of experiments; (N) - number of embryos; No Mig. - No migration of the third arch neural crest cells; Delay - delay in the migration of either the third arch or the third arch plus fourth arch neural crest in respect to the uninjected control side; Aberrant - Migration occurs but incorrectly; Ectopic Spot - single spot of marker expression is separated from the main crest stream being investigated; Merged - Intermingling between the third and fourth arch neural crest cell streams; WT - the percentage of injected embryos exhibiting neural crest migration that is similar to uninjected siblings.

oo O C onstruct RNA injected (ng) No. of Expts (N) No. Mig (N) Delay (N) A berrant (N) Ectopic Spot (N) M erged (N) WT % p-globin 4 1 9 — — — --- — 100 3 3 21 — 2(10) — --- - - 90 2 4 28 — — — — — 100 1 3 14 — — — --- — 100 0.5 3 32 —— — — --- — 100 0.1 2 4 — — --- — 100 Xephrin-A3 4 1 6 5(83) — — --- 1(17) 0 3 4 46 15(33) — 16(35) --- 6(13) 19 2 5 98 13 (13) — 52(21) --- 7(7) 27 1 3 51 — 4(8) 19(33) --- 4 (8 ) 27 0.5 3 58 — 3(5) 26 (22) 3(5) --- 45 0.1 2 27 — 2(7) 11(41) 5(19) --- 33 AXephrin-A3 3 3 23 6(26) --- 5(9) --- 3(22) 30 2 4 78 7(9) --- 13 (13) --- — 76 1 2 25 10 (40) 2(8) — — — 52 0.5 2 26 —— — — --- — 100 0.1 2 15 — — 7(13) — 3(20) 33

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Table 8

Data for a typical injection experiment with reference to gastrula stage embryo death

Included in this table are numbers of embryos that are die at pre-gastrula (from fertilisation inclusive of stage 10) and post gastrula (stage 11 until collection).

* relates to XKrox20 whole mount in situ hybridisations. " relates to XAP2 whole mount in situ hybridisations.

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