Comparison of the expression patterns of novel genes with that of mirror

In order to study the expression patterns of previously uncharactersied genes, in situ hybridisation in Drosophila embryos to detect the predicted transcripts was performed. In most cases, the full length pACT2 library clone isolated form yeast was first subcloned into pBluescript using the appropriate restriction enzymes. The pBluescript clones which contain both T7 and T3 RNA polymerase promoters, could then be used as templates for the DIG-labeled RNA probe. Due to the fact that the clones often include the 3’UTR and C-terminal portions of the protein, it was hoped that the detected expression patterns would be specific for the selected gene. In some cases, when the library clone was not suitable for use as a template, usually due to problems with restriction sites for linearization, a Drosophila EST clone (ResGen) from the gene was used. All the ESTs used as templates were in the pOT2 vector which uses the SP6 polymerase promoter for anti-sense RNA production.

The in situ hybridisation patterns of most of the genes studied seemed to be ubiquitous. An example of what was termed ubiquitous staining can be seen in figure 3.2 One of the anti-sense probes did not give significant staining over background (in situ hybridisation protocol followed using no RNA or sense probe) and was therefore termed inconclusive. Two of the patterns, although mainly ubiquitous, seemed to show an enrichment for the transcript in some embryonic structures. CG1931 seems to be expressed at a consistent level in the whole embryo up until stage 11-12 before the expression seems to become stronger in the CNS (figure 3.2). CG8448 is also expressed throughout the embryo at earlier stages, but at later stages it also seems to be enriched in narrow segmental bands close to the epidermis (figure 3.2).

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Figure 3.2. Examples of ubiquitous expression patterns

Figure 3.2. In situ hybridisations on Drosophila embryos using RNA probes made from various pACT2 library clones in pBluescript. In all panels anterior is left and dorsal up. A and B: in situ hybridisations using a probe for IV 10a (CGI 135) showing ubiquitous expression at stage 10 (A) and stage 14 (B). C: in situ hybridisation using a probe for IV 29a (CG30337) showing ubiquitous staining and enhanced expression in the CNS at stage 13-14. D: in situ hybridisation using a probe for III 12b (CG8448) showing ubiquitous staining with enhanced expression in a segmental pattern at stage 14-15.

Chapter 3

Three of the previously uncharacterised genes have distinct expression patterns in the embryo. As seen in figure 3.3, CG1962 is expressed exclusively in the pole cells from stage 5 to about stage 10. From stage 10 or 11 the transcript can be seen in a few cells which may be neuroblasts and expression continues and becomes more widespread in cells of the ventral nerve cord and brain until stage 16. Mirror is also expressed in the developing CNS and it is therefore possible that the two proteins are coexpressed. A comparison of the in situ hybridisation patterns of CG 1962 and mirror can be seen in figure 3.4.

Figure 3.5 shows the expression pattern of C G I2919. Expression of GC12919 starts at about stage 4-5 in dorsal regions of the embryo. Expression continues in the dorsal folds and amnioserosa at stages 6-7, but is not present at germband retraction. Later, at stage 12-14, expression reappears in distinct structures in the head region of the embryo and in two dots posteriorly, which could be part of the PNS. The expression pattern of C G I2919 overlaps with the mirror expression patterns in early development as can be seen from the comparison in figure 3.6.

The in situ hybridisation pattern for CG5740 was quite weak, but the transcript seemed to be present in the embryonic gut at the invagination between the proventriculus and the midgut as well as in the hindgut at stages 13-15, figure 3.7. Mirror is also expressed in the area of the proventriculus and could therefore overlap with CG5740 (figure 3.7). Table 4.2 summarises the expression patterns determined for the novel interactors and the possible overlaps with mirror expression.

Chapter 3

Figure 3.3. Expression pattern of CG1962

Figure 3.3. In situ hybridisation on Drosophila embryos using an RNA probe made from the pACT2 library clone IV31b cloned into pBluescript. A-E are lateral views with anterior left and dorsal up. F is a ventral view with anterior left. Embryos of

approximately these stages are show; A) stage 5, B) stage 9, C) stage 10-11, D) late stage 11, E) stage 13-14, F) stage 14-15. The CG1962 transcript can be detected in the pole cells from stage 5 to about stage 10 when expression fades. CG 1962 is expressed in the CNS from about stage 10-11 until stage 16. Initially expression seems limited to a few cells, probably delaminating neuroblasts, but the expression becomes more widespread and by stage 13 the transcript seems to be present in numerous cells in the ventral nerve cord and brain.

Chapter 3

Figure 3,4, Comparison of the expression patterns of CG1962 and mirror

Figure 3,4 Comparison of the in situ hybridisation patterns of CG 1962 and mirror. All panels are lateral views with anterior left and dorsal up. A and C are in situ hybridisations using the pBluescript IV3 lb probe and B and D are in situ hybridisations using the mirror

9B probe. A and C are about stage 11 and B and D are about stage 13-14. Both CG I962 and mirror are expressed in cells of the CNS from stage 10 - 16, so their expression could overlap.

Chapter 3

Figure 3.5. Expression pattern of CG12919

Figure 3.5. In situ hybridisation on Drosophila embryos using a pBluescript clone of library clone pACT2 II48c (CG I2919) as template. In all panels anterior is right, A-D lateral view with dorsal up, E and F dorsal view. Embryos of these stages are shown: A) stage 5, B) stage 6, C) stage 7-8, D) stage 9-10, E) stage 10-11, F) stage 14-15.

Expression of the mRNA can be seen dorsally at stage 5 and in the dorsal folds and amnioserosa at stages 6-10. At about stage 14-15, expression can be seen in two posterior structures as well as in the head.

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Figure 3.6 Comparison of expression patterns of CG12919 and mirror

Figure 3.6 Comparison of the in situ hybridisation patterns of CG 12919 and mirror. In all panels anterior is left and dorsal is up. A-C: in situ hybridisation patterns for CG

12191, D-F: in situ hybridisation patterns for mirror. A and D are stage 5, B and E are stage 7, C and F are stage 9-10. Both CG 12919 and mirror are expressed dorsally early in development and both are expressed in the dorsal folds during germband retraction as well as in the amnioserosa.

chapter 3

Figure 3.7. Expression pattern of CG5740 and

comparison with the mirror expression pattern

i

Figure 3.7. In situ hybridisations of Drosophila embryos using EST G H ll 144from CG5740 and pBluescript clone mirror 9B as templates. All panels are dorsal views of embryos at stage 12-14, anterior is up. A-C: in situ hybridisation patterns of CG5740, D and E: in situ hybridisation pattern of mirror. Expression of CG5740 can be seen at the junction between the proventriculus and the midgut at stages 13-15 and in the hindgut at the same stages (A-C). Mirror is expressed in the proventriculus and may overlap with CG5740 (D and E).

Chapter 3

Table 3.2 Summary of expression patterns of putative interactors Putative

interactor

Expression pattern Overlap with mirror expression pattern

Dichaete Published Neuroectoderm, neuroblasts, brain

Castor Published Neuroblasts (figure 3.1), brain

CG13367 Ubiquitous Yes

CHDl Ubiquitous Yes

CG1962 Expressed in pole cells stage 5-11, CNS from stage 10-11.

Possibly ventral nerve cord and brain (figure 3.4)

CG10395 Ubiquitous Yes

CG30337 Ubiquitous, enriched in CNS late. Yes CG12919 Expressed dorsally stage 4-5, dorsal

folds and amnioserosa stage 6-10, head and posteriorly stage 12-14.

Early dorsal, dorsal folds and amnioserosa (figure 3.6) CG8448 Ubiquitous, possibly enriched in

segmental pattern late.

Yes

CGI 135 Ubiquitous Yes

CG3987 Inconclusive N/D

CG5740 Expressed foregut-midgut boundary and hindgut stage 12-15.

Possibly anterior gut (figure 3.7) Table 3.2. Summary of expression patterns as determined by in situ hybridisation and possible overlaps with m /rm r expression. N/D, not determined.