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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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There appear to be some segmentation defects in the affected maxilla, and some changes to the identity of some of the sensory bristles (fig.5.2). Sensory bristle morphology and segmentation of the maxilla in DfdRNAi larvae was examined to determine the knock down phenotype in the maxilla. It appears as though there are fewer segments present in the affected maxillae although it is difficult to say this with any degree of certainty as the demarcation between the segments that have been suggested are faint and could alternatively represent distortive artefacts such as a fold or crease from the cuticle preparation procedure. Examination of several affected maxilla reveal the crease to be consistently present, and resembling some true segment boundaries (e.g. the postmentum/prementum segment boundary) and is therefore interpreted to represent a true segment boundary (fig.5.2D-G). The boundaries between segments are hard to decipher in the distal-most part of the affected maxilla. It appears there are two palp segments present, the proximal palp Fig.5.1. Tc Dfd patterns the mandible and maxilla in Tribolium. SEMs of Tc Dfd knock down embryos and cuticle preparations of first instar larvae show the transformation of the mandible (arrowhead) to antennal identity and alteration of maxilla morphology, particularly regarding the protopodite. All views are ventral with anterior to the left unless otherwise indicated. Maxilla is indicated with arrows. The antenna is indicated with asterisks. (A) Wild type cuticle preparation of Tribolium larval head showing the mandibular appendages highlighted in blue and the maxillary appendages highlighted in green. (B) Wild type cuticle preparation of Tribolium larval head showing the maxillary and labial appendages. The RNAi mandible (arrowhead) is out of focus. (C) Tc Dfd larva with the transformed mandibular appendage highlighted in green and the affected maxillary appendage highlighted in blue (compare to A). The transformed mandible clearly resembles the antennal appendage, although smaller in size. The maxilla is shorter and has lost the ventral branch endite. (E) Close up of the maxilla of a wild type larva. (F) Close RNAi up of the maxilla of a Tc Dfd larva. (G) SEM of a wild type embryo at the germ band retracting stage.

RNAi The mandible, (inner and outer lobes), maxilla and labial endites are visible. (H) SEM of a Tc Dfd larva at a similar stage to G, lateral view. The mandible has been transformed to an antenna. (I) SEM of a Tc RNAi Dfd larva at a similar stage to G. Ventral-lateral view. The endites of the maxilla have clearly been lost (asterisk). The transformed mandible resembles the antenna, but in a smaller form. (J) SEM of the gnathal appendages of a wild type embryo at a similar stage to G. Lateral view. (K) SEM of the anterior RNAi appendages of a Tc Dfd embryo at a similar stage to G. Ventral-lateral view. The mandible has antennal morphology. The affected maxilla is uniform and relatively undifferentiated along the P/D axis compared to the wild type maxilla (compare with J). Abbreviations are as follows: cardo (ca), stipes (st), palp (p) and ventral branch (vb).

Fig.5.2. Loss of Tc Dfd function affects segmentation and bristle morphology in the larva maxilla. All views of appendages are distal to the top, larval head orientation is anterior to the top. (A) Schematic of wild type larval maxilla and labial appendages derived from B. (B) Wild type cuticle preparation of Tribolium larval head showing typical segmentation and bristle morphology of the maxilla and labial RNAi appendages. (C) Schematic of Tc Dfd larva maxilla and labial appendages. Arrowhead indicates the putative subcoxa/coxa segment boundary, the arrow indicates the labial appendage-like subcoxa bristle present near the base of the affected maxilla. Compare with D. (D-G) Four maxillae from different larval cuticle preparations. The affected maxillae are shorter and missing the ventral branch endites. The putative subcoxa/coxa segmental boundary (white arrowhead) and labial-like proximal bristle (arrow) is visible in each appendage. There is no apparent subcoxa or cardo boundary with the head, it appears as though the affected maxillary appendage is continuous with head cuticle. The palp of the affected maxilla appears to consist of two segments. However, the cuticle preparations are not of sufficient quality to confirm this interpretation. It is clear that the distal most segment of the palp resembles the more elongate maxillary palp and not the shorter, stubby labial palp.

segment having a bristle present on it which is similar to that present on the typical maxilla palp. In light of the above, the affected maxilla is interpreted to have four segments, a two-segmented palp attached to a two-segmented protopodite. This is in contrast to a wild type maxilla that has two protopodite segments and a palp consisting of four segments.

The pattern of bristles on the affected maxilla is also different from the wild type maxilla. There are similarities of the affected maxilla to half the labial appendage (which consists of two fused maxillae). Superficially, the affected maxilla looks as though it has transformed into a labial appendage without fusing with the protopodite of the other affected maxilla. However, the distal-most segment of the palp of the affected maxilla resembles the palp of the normal maxilla and not the labial appendage. There is other genetic evidence in favour of maxilla identity of the affected palp which is discussed below. The pair of large bristles present on the postmentum of the labial appendage are in a similar position to the bristles present in the affected maxilla and is interpreted to represent the distal-most segment of the protopodite, the coxa.

In the wild type maxilla, there are three main bristles present on the stipes. In the transformed maxilla, there are three bristles in a similar position, however the proximal bristle is somewhat enlarged and therefore appears to have been altered by Tc Dfd knock down (arrow in fig.5.2C). There is an apparent segment boundary between the proximal bristle and the other two bristles, which is different from the normal maxilla. The cardo/stipes segment boundary which has a characteristic angular morphology in the wild type maxilla is not recognizable in the affected appendage, which has a more uniform telescopic appearance.

With consideration of gene expression data shown below, the protopodal segment boundary in the affected maxilla (triangle in fig.5.2C-G) is suggested to represent the altered cardo/stipes boundary of the normal maxilla, which is interpreted in this work as the subcoxa/coxa boundary present in post-antennal appendages. The bristle that is present in the subcoxa segment is a bristle that is repressed by Tc Dfd and not normally found in the subcoxa (the cardo) of the wild type maxilla.

Expression of the Hox gene mxp in DfdRNAi embryos

In order to confirm that the maxillary Hox gene is still expressed in the maxillary segment and that the remaining palp was of maxillary identity, mxp was detected in DfdRNAi embryos. mxp is expressed in a domain that includes the telopodite and extends proximally to the galea enditic lobe. mxp is not expressed in the proximal part of the protopodite (fig.5.3A). Expression of mxp did not appear to be significantly affected in Dfd knock down embryos; mxp is still expressed in the palp of the affected maxillary appendage as in wild type embryos. In Tc DfdRNAi embryos, the posterior collar domain of Tc cnc is lost (see chapter four). Therefore, Tc cnc was detected Fig.5.3. Maxillary palp identity is maintained in the absence of Tc Dfd function. Tc Dfd does not activate mxp in the maxillary segment. Gene expression was detected by in situ hybridisation (A) Wild type germ band retracting stage embryo stained for Tc prd (blue) and mxp (red). Tc prd is expressed in the endites of the gnathal appendages. In the maxilla there are two domains, a lacinea endite domain (arrowhead) and a galea endite domain (arrow). mxp expression is present in the palps of the maxillary and labial appendages. Expression is present in the distal part of the protopodite that relates to the galea endite. (B-D) mxp expression remains in the palps of the labial and affected maxillary palps. There is a proximal region of the affected maxilla that is lacking mxp expression (arrowhead).The proximal limit of mxp expression (arrow) and the region free of mxp expression in the affected maxilla is in a similar position to the labial appendage (white arrow in D) which is unaffected by Dfd knock down. This suggests there is no deletion of a proximal region of the P/D axis. The posterior domain of Tc cnc in the RNAi mandibular segment is missing in Tc Dfd embryos. Expression of mxp is present in the posterior of the transformed mandibular appendage (asterisk). (B) Expression of mxp (red) and Tc cnc (blue) in Tc Dfd knock down embryo at late germ band retracting stage. (C) Expression of mxp (red) and Tc cnc (blue) in a later stage (germ band retracted stage) Tc Dfd knock down embryo than B. In both B and C there is a small spot of Tc cnc in the ventral midline, the significance of this is not known and has been observed in RNAi some Tc Dfd embryos. (D) Close up of the expression of mxp (red) and Tc cnc (blue) in the anterior appendages of a Tc Dfd knock down embryo at late germ band retracting stage. The anterior cap domain of Tc cnc is visible out of focus as a dark blue blur at the anterior of the embryo.

simultaneously in doubly stained Tc Dfd knock down embryos to confirm that Tc Dfd was knocked down by the lack of Tc cnc expression in the mandibular segment (fig.5.3B-D).

Whilst the loss of the maxillary endites is quite obvious, it is difficult to tell from cuticle preparations of knock down first instar larvae or scanning electron micrographs of knock down embryos whether the protopodite has been deleted or is merely reduced in size. It is also difficult to determine whether there is a cardo/stipes segmentation boundary present. By studying the expression patterns of several marker genes that are normally expressed in the telopodite or the protopodite, it is possible to determine whether there has in fact been any obvious deletion of the protopodite in a Tc Dfd knock down embryo.

Examination of the expression of mxp in Tc Dfd knock down embryos reveals that there is still a proximal region that lacks mxp expression which is in a similar position to mxp expression in the labial appendage, which shows that there is still a protopodite present (fig.5.3B-D). Expression of mxp in the labial appendage marks the position of the endite possessing prementum segment. Expression of mxp in the maxillary appendage of DfdRNAi embryos is in similar position (white arrow in fig.5.3D).

mxp expression is in the distal half of the protopodite which is consistent with no deletion of the protopodite.

There is faint expression of mxp in the ectoderm of the posterior half of the mandibular appendage (see fig.5.3B-D). The significance of this is not known, and is not expected either as the transformed mandible is of antennal identity which is typically repressed by the presence of a Hox gene.

Tc Dfd upregulates the proximal domain of Tc dac.

Tc Dac is expressed in two domains in the maxilla, a stronger proximal domain that is located in the protopodite with a particularly strong ring of expression around the protopodite located on the distal margin of the lacinea lobe (arrow in fig.5.4A). The distal domain is weaker and is located in the middle of the palp (arrowhead in fig.5.4A). In Tc Dfd knock down embryos, both domains are still present. However, the proximal domain is significantly weaker (fig. 5.4B-D). There is an inversion in the relative strength of expression of these domains, as the distal domain is significantly stronger than the proximal domain which is reminiscent of the expression pattern seen in the legs. The strong expression domain of Tc dac in the mandible is lost. Expression of Tc dac in the transformed mandible (asterisk in fig.5.4C) resembles the Tc dac expression domain of the antenna. The reduction of Tc dac expression in the affected maxilla of DfdRNAi embryos indicates that Tc Dfd up-regulates expression of the proximal domain of Tc dac.

Tc Dfd patterns the endites of the mandible and maxilla

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