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«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»

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Tc Dll is expressed in the distal part of all appendages except the mandibular appendage of wildtype Tribolium embryos. In the developing maxilla, there are two domains of Tc Dll expression, a distal domain in the developing palp and a proximal domain in the lacinia endite. Injecting Tc cnc dsRNA results in homeotic transformation of the mandibular appendage into maxillary identity results in complete recapitulation of the maxillary Tc Dll and Tc prd expression patterns in the transformed mandibular appendage of Tc cnc knock down embryos (see fig.4.7B,D-F). The solitary domain of Tc prd expression in the mandible is transformed into two domains of Tc prd expression that relate to the maxillary enditic lobes. As is expected, transformation of the mandible into maxillary identity results in expression of Tc Dll in the palp and proximal enditic lobe (the lacinia enditic lobe).

Fig.4.7. Homeotic transformation of the mandibular appendage to maxillary identity and deletion of the labrum in Tc cnc knock down embryos as revealed by the expression of Tc Dll and Tc prd. Gene expression was determined by in situ hybridization. All views are ventral with anterior to the left. (A-E) The mandibular appendage (arrowhead), lacinia (star), galea (asterisk) and telopodite (arrow) are indicated. (A,B) Germ band retracted stage embryos that are later than the germ band extended embryos shown in C-E. (A) wild type embryo with the expression of Tc Dll (blue) and Tc prd (red). Tc Dll is expressed in the lacinia endite lobe (star) and the maxillary telopodite (arrow). Tc prd is expressed in the RNAi endites of the mandible, maxilla and labial appendages. (B) Tc cnc embryo with expression of Tc Dll (blue) and Tc prd (red). The labral domain of Tc Dll is also missing at the anterior of the embryo. These results indicate that Tc cnc represses Tc Dll and differentiates Tc prd expression in the mandible RNAi appendage. (C) wild type embryo with the expression of Tc Dll (red) and Tc prd (blue). (D,E) Tc cnc RNAi embryo with the expression of Tc Dll (red) and Tc prd (blue). (F) Tc cnc embryo stained for Tc prd and Tc Dll. The telopodite and endite of some appendages are smaller (arrowheads) than their corresponding partner. There is asymmetry between the different transformed mandibular appendages.

The transformed mandibles resemble maxillae at an earlier stage of development and so may have delayed development relative to the maxillary appendages. (G) Extreme phenotype observed in a large proportion of embryos. There is a mass of undifferentiated germ tissue concentrated at the polar ends.

The transformed mandibular appendage develops into a maxilla more slowly than the adjacent true maxillary appendages. This is evident by comparing the relative development of the transformed mandibular appendage to the maxillary appendage (see fig.4.7D-F). The mandibular appendage resembles the maxillary appendage at an earlier stage of embryogenesis. This is evident at several stages of embryogenesis.

There appears to be significant asymmetry of different appendages in Tc cnc RNAi embryos (see fig.4.7D-F). This asymmetry occurs in a random fashion. This does not appear to be an artefact of the RNAi procedure or the in situ hybridisation process as other parental RNAi experiments in Tribolium have not yielded a similar result.

Instead it seems likely that this is another effect of knocking down Tc cnc in Tribolium.

The reasons for this are unclear, but may be related to the housekeeping role that cnc has in protecting the embryo from oxidative stress (Grimberg et al., 2011).

There was an additional strong phenotype observed, where a mass of undifferentiated germ rudiment is present at the anterior of the embryo (see fig.4.7G).

It appears that embryogenesis has been seriously affected in these embryos and halted at this stage. The reason for this is unknown. There is no expression of the genes Tc Dfd, Tc Dll, Tc prd and mxp in the strongly affected embryos. Interestingly, parental injection of Tc cnc dsRNA resulted in the mortality of a significant number of the injected females (see materials and methods).

Tc cnc represses the Hox genes Tc Dfd and mxp in the mandibular limb bud.

The two Hox genes Tc Dfd and mxp pattern the maxilla in an additive manner (Brown et al., 2000; Shippy et al., 2000b). Consistent with these phenotypes, Tc Dfd is expressed in the proximal part of the maxilla, the protopodite, whereas mxp patterns the palp (see fig.4.4 and fig.4.5). As there is a homeotic transformation of the mandible into a maxilla, it is to be expected that the Hox genes responsible for patterning the maxillary appendage will be ectopically expressed in the homeotically transformed appendage.

In wild type embryos, Tc Dfd expression retracts from the mandibular limb bud (see fig.4.8G,H), which is a likely indication that Tc cnc is repressing Tc Dfd expression in the mandibular limb bud, in a similar manner to what occurs in Drosophila. In Tc cncRNAi embryos, expression of the Hox gene Tc Dfd is not repressed from the protopodite of the mandibular appendage which has been transformed into maxillary Fig.4.8. Tc cnc represses the Hox genes Tc Dfd and mxp in the mandibular appendage. Knock down of Tc cnc by RNAi results in transformation of the mandibular appendage to maxillary identity and the expression of Hox genes in a similar manner to that seen in the maxilla. All views are ventral with anterior to the left. Expression of Tc Dfd (blue) and mxp (red) is visualized by in situ hybridisation.





Mandibular segment is indicated with an arrowhead, maxillary segment with an arrow (A,C,E) Wild type RNAi Tribolium embryos. (B,D,F-I) Tc cnc embryos. (A) wildtype germ band extending stage embryo. (B) Tc RNAi cnc embryo at a similar stage to that of A. mxp expression is present in the transformed mandibular appendage (arrowhead) in a telopodite domain consistent with the transformation of the mandible to maxillary identity. (C) Wildtype germ band retracting stage embryo. Tc Dfd expression has retracted RNAi from the majority of the mandibular appendage. (D) Tc cnc embryo at a similar stage to C. Tc Dfd expression does not retract from the transformed mandibular endite (star). mxp is expressed in the transformed mandibular appendage palp in a manner that is identical to that of the maxilla (arrowhead).

(E) Higher magnification of the gnathal appendages of a germ band retracting stage at a similar stage to C, with mesodermal expression of mxp (white arrow). (F, G, H) Higher magnification of the gnathal RNAi appendages of germ band retracting stage Tc cnc embryos. (F) The mesodermal expression domain of mxp (white arrow) is expressed in the transformed mandibular appendage. (G) Tc Dfd expression is throughout the transformed mandibular appendage, in the lacinia endite lobe (star), galea endite lobe (asterisk). mxp expression is present in the palp as well as the galea endite lobe in a manner which is identical to the maxilla. (H) Tc Dfd is expressed throughout the maxilla, the rounded kink at the base of RNAi the maxilla is indicated (star). (I) Higher magnification of the earlier germ band extending stage Tc cnc embryo shown in B.

identity. Removal of Tc cnc function by RNAi abrogates the repressive effect of Tc cnc on Tc Dfd transcription. Tc Dfd expression is retained in the protopodite (see fig.

4.7A,C,E).

In addition to the repression of Tc Dfd, Tc cnc also represses (directly or indirectly) mxp in the mandibular limb bud (see fig.4.8B,D,F-I). In the transformed mandibular appendage, mxp is expressed in the ectopic palp. Interestingly, mxp is expressed in the mesoderm of the mandibular segment of wildtype embryos (see fig.4.8F,I) and this mesodermal expression of mxp is still seen in Tc Dfd knock down embryos (fig. 4.8F,I). This suggests that there is a cnc independent regulation of mxp in the mandibular limb bud, or that Tc cnc is solely expressed in the ectoderm.

Tc Dfd activates the posterior ‘collar’ domain of Tc cnc in the mandibular segment.

In order to investigate whether Tc Dfd has any role in the regulation of Tc cnc, Tc Dfd was knocked down by parental RNAi and Tc cnc expression was detected via in situ hybridisation. In DfdRNAi embryos the posterior collar domain of Tc cnc expression is completely missing from germ band extending embryos through to stages where embryos are undergoing dorsal closure (fig.4.9). The anterior cap domain of expression is unaffected. This result shows that Tc Dfd is necessary for the activation of the posterior domain of Tc cnc in the mandibular segment. The mandibular domain of Tc cnc is therefore dependent on the Hox gene Tc Dfd for activation.

Fig.4.9. Tc Dfd activates the posterior collar domain of Tc cnc in the mandibular segment. Gene expression was determined by in situ hybridisation. (A-C) Tc cnc expression in wild type embryos.

Throughout embryogenesis, Tc cnc expression consists of an anterior cap domain in the labrum (arrowhead) and a collar domain (arrow) in the mandibular segment. (A) Tc cnc (red) and Tc Dfd (blue) expression in a germ band extending embryo. (B) Tc cnc expression (blue) in a germ band extending embryo at a similar but slightly earlier stage to A. (C) Tc cnc expression (blue) in later stage embryo prior RNAi to dorsal closure. (D-F) Tc cnc expression in Tc Dfd embryos. In all stages, from germ band extending (D), germ band retracted (E) and during dorsal closure (F), the posterior domain of Tc cnc is missing in the mandibular segment, whilst the anterior domain of Tc cnc is expressed as normal. This shows that Tc cnc is activated by Tc Dfd in the mandibular segment. There is a faint stripe of Tc cnc in the mandibular segment of D, this may be due to partial knock down effects. (G) Expression of Tc cnc (red) and Tc Dll (blue) in wild type germ band retracting embryo. (H) Expression of Tc cnc (red) and Tc Dll (blue) in a Tc RNAi Dfd germ band extending embryo. The posterior domain of Tc cnc is missing.

4.3 Discussion General overview of results Knock down of Tc cnc transcripts by parental RNAi results in a full homeotic transformation of the mandible into maxillary identity and deletion of the labrum in Tribolium embryos and first instar larvae. Tc cnc represses the Hox genes Tc Dfd and mxp in the developing mandibular limb bud. This result demonstrates that Tc cnc differentiates the mandible from a maxillary appendage in Tribolium in part by repressing Hox genes in a manner similar to that observed in Drosophila.

Tc cncRNAi knock down phenotype

Homeotic transformation of the mandible to maxillary identity is seen in the cuticle preparations of knock down larvae and also by the expression of the genes Tc Dll and Tc prd that are markers for the developing telopodite and endite of the maxilla.

Expression of the gene Tc Dll can be seen in the palp and the endite of the transformed mandibular appendage in the developing embryo. Tc prd is expressed in both of the developing endites of the transformed mandibular appendage. Both Tc Dll and Tc prd are expressed in a maxilla-like manner in the transformed mandible. Tc cnc therefore represses the Tc Dll proximal domain of the endite and distal domain in the palp. Tc cnc also modifies prd expression domain from that of the two domains of maxillary endites to the large single domain of the mandible endite. Whether these effects are achieved directly or indirectly by Tc cnc is not known.

Tc cnc represses the Hox genes mxp and Tc Dfd in the mandibular segment. The Hox genes mxp and Tc Dfd are required to pattern the maxillary appendage. The Hox genes Tc Dfd and mxp are expressed in the transformed mandibular appendage in a manner nearly identical to the maxillary appendage.

There is a mesodermal expression domain of mxp in the mandibular mesoderm, which continues to be expressed in the transformed mandible in a Tc cnc knockdown embryo. This indicates that the mesodermal expression domain of mxp is Tc cncindependent and does not appear to pattern the mandibular appendage.

The ectoderm of the mandibular appendage is fully transformed into an ectopic maxillary appendage. The transformed mandibular appendage mesoderm expresses mxp similarly to that observed in wild type mandibular appendage indicating that there is a cnc independent mxp domain in the mandibular mesoderm that is unaffected in Tc cnc knock down embryos.

These results show that Tc cnc is necessary for the development of the mandible, to differentiate it from a maxilla via the repression of two Hox genes and for the activation of genes that are involved in patterning of the mandible. The manner in which Tc cnc represses the Hox genes Tc Dfd and mxp could be direct or indirect.



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