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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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All views are ventral with anterior to the left unless otherwise indicated. Gene expression was determined by in situ hybridisation. (A) Tc Dfd expression in a germ band extending embryo as limb buds are just about to form. Expression is present throughout the mandibular (arrowhead) and maxillary segments (arrow). Tc Dfd expression is just beginning to retract from the developing endites (white star). (B) Expression of Tc cnc in a germ band extending stage embryo. There is an anterior cap domain of Tc cnc in the labrum. The posterior collar domain is indicated with an arrowhead, the maxillary segment is marked with an arrow. (C-F) Fluorescent in situ hybridisation of Tc Dfd in developing Tribolium embryos. White star indicates position of developing endites. (C) Early germ band extending stage embryo prior to limb bud formation. Tc Dfd expression is visible in the mandibular and maxillary segments. There is no reduction of Tc Dfd expression in the mandibular segment at this stage. (D) Later germ band extending stage than C with retraction of Tc Dfd expression evident in the developing endites (white star). (E) Later stage than D with progressive loss of Tc Dfd expression. There is no loss of Tc Dfd expression in the maxillary protopodite. (F) Germ band retracting stage embryo. Tc Dfd expression is almost totally gone from the endite at this stage. Expression that is remaining in the mandibular limb bud is present laterally. (G-I) Tc Dfd expression in dissected mandibular and maxillary appendages of germ band retracting embryos. (G) Expression is clearly lost in the inner lobe (arrow) and reduced in the outer lobe. Expression in the maxillary protopodite is ubiquitous. (H) Lateral view. Expression is lost in the inner lobe (arrow), but still present on the lateral part of the mandible. (I) Expression in the maxilla.

Tc Dfd expression is strong in the maxillary protopodite and faint in the palp (arrow).

Fig.4.2. Expression of Tc cnc and Tc Dfd during Tribolium embryogenesis. All views are ventral with anterior to the left. Gene expression was determined by in situ hybridisation. Tc Dfd expression is stained blue, Tc cnc expression is stained red. Overlapping domains of expression are brown.

Mandibular segment indicated with an arrow. (A-E) Series of germ band extending stages of embryogenesis. (A) Very early germ band extending embryo (the heart stage). (B) early germ band extending embryo. Tc cnc is very faintly expressed in the mandibular segment at this stage (arrow). (C-E) Tc cnc is expressed in both the developing labrum and the mandibular segment. Tc Dfd is expressed continually throughout the mandibular segment together with Tc cnc. The limb buds and the endites have not begun to develop. (F) Fully germ band extended stage. Tc Dfd expression retracts from the endites (white star). (G) Fully retracted germ band undergoing dorsal closure. Tc Dfd expression is almost completely gone from the mandibular limb bud, with some residual expression residing at the lateral part of the mandible. Tc Dfd is strongly expressed in the maxillary segment. The two strongly expressed pair of circular domains of Tc Dfd and Tc cnc in the first abdominal segment are in the pleuropods. (H) Close up of the abdominal expression domains of Tc cnc visible in G. The abdominal domains of Tc cnc have a sickle shape which arches around the developing spiracles (arrow).

Fig.4.3. Repression of Tc Dfd expression in the developing mandibular limb bud. All views are ventral with anterior to the left. Gene expression was determined by in situ hybridisation. Tc Dfd expression is stained blue, Tc cnc expression is stained red. Overlapping domains of expression are brown. As soon as the endites start to form, Tc Dfd expression retracts from the mandibular endites (star) whilst Tc cnc expression is maintained throughout the mandibular appendage (C-H). (A,B) prior to limb bud formation, Tc Dfd expression is continuous throughout the mandibular segment. (C) As soon as the mandibular endite forms, Tc Dfd expression retracts from the endite. (G,H) By late embryogenesis, faint Tc Dfd expression is only present in the lateral part of the mandibular limb bud (arrow). Tc Dfd expression is still strongly maintained in the maxillary segment. (A) early germ band extending embryo.

(B) Germ band extending stage embryo prior to limb bud formation. (C) Germ band extending embryo after the limb buds and the endites have started to form. (D) Late germ band extending embryo. (E) Fully extented germ band stage embryo. (F) Germ band retracting embryo. (G) Embryo undergoing dorsal closure with the gnathal appendages moving towards the ventral midline. (H) Lateral view of dissected mandibular and maxillary appendages.

Fig.4.4. Expression of the Hox genes Tc Dfd and mxp in the gnathal appendage segments during embryogenesis. All views are ventral with anterior to the left. Gene expression was determined by in situ hybridisation. Tc Dfd expression is blue, mxp expression is red, overlapping domains of expression are brown. Mandibular (arrowhead) and maxillary (arrow) segments/appendages and labial palp (asterisk) are indicated. Mesodermal mxp expression domain (white arrow) and intercalary domain of mxp (white asterisk) are also indicated. (A) Germ band extending embryo prior to the formation of limb buds. mxp is expressed in the mesoderm of the mandibular segment. (B) Later germ band extending stage embryo as the limb buds are beginning to form. The maxillary and labial segment developing limb buds are marked by mxp expression. mxp overlaps with Tc Dfd in the maxillary segment (brown) and is solely expressed in the labial segment (red). (C) Later germ band extending stage. The mandibular mxp mesodermal expression domain is visible in the mandibular segment (red stain). (D) Germ band retracting embryo. mxp expression is present in the palps of the maxilla and labial appendages. (E) Higher magnification of germ band extending embryo (midway between B and C) highlighting the mesodermal expression of mxp, the loss of Tc Dfd expression in the endite and mxp expression in the developing palp of the maxillary segment. (F) Higher magnification image of the gnathal appendages of a germ band retracting embryo as in D. The loss of Tc Dfd expression in the endite is shown (star). mxp is expressed in the maxilla and labial palps and the distal half of the maxillary and labial protopodites.





Fig.4.5. Expression of Tc Dfd and mxp in the mandible and maxilla during embryogenesis relative to the endites marked by Tc prd expression. All views are ventral with anterior to the left unless otherwise indicated. Gene expression was determined by in situ hybridisation. (A) Tc Dfd (red) and Tc prd (blue) expression in a germ band fully retracted stage embryo. Tc prd is expressed in the developing endites of the mandible, maxilla and labial appendages. The proximal part of the inner lobe (arrow) is lacking Tc prd and Tc Dfd expression. Tc Dfd expression is repressed from the developing mandibular endite. (B) Embryo stained with mxp (red) and Tc prd (blue). mxp is expressed in the maxillary and labial palps and the distal protopodite of both appendages. In the maxilla, protopodite expression relates to the position of the developing galea endite lobe (star) which is marked by the distal domain of Tc prd expression. The mesodermal mandibular domain is visible underneath Tc prd expression (arrowhead). The intercalary domain of mxp expression is also visible (white arrowhead). Mesodermal expression of Tc prd is present in the telopodites of post-antennal appendages but clearly visible in the developing leg appendages (arrow). (C) Tc Dfd expression (red) and Tc prd expression (blue) in the mandibular and maxillary appendages of a germ band retracted stage embryo. Lateral view. Tc Dfd expression is present on the lateral side of the mandible (arrow). (D,E) Tc Dfd expression (red) and Tc prd expression (blue) in a dissected mandible and maxilla of a post germ band retracted stage embryo undergoing dorsal closure.

Distal is top. (D) Lateral view of a dissected mandible. Tc Dfd expression is clearly present on the lateral side of the mandible (arrow). (E) Dissected maxilla, lateral is to the right. Tc Dfd expression is throughout the protopodite and at the base of the palp. The distal part of the palp is lacking or has weak Tc Dfd expression (arrow).

Tc cnc RNAi phenotype

In order to test the role Tc cnc might play in patterning the mandibular segment, Tc cnc was knocked down in developing embryos by injecting Tc cnc dsRNA into female pupae. Injection of Tc cnc dsRNA resulted in homeotic transformation of the mandibular appendages into maxillary identity. Knock down of Tc cnc function also resulted in deletion of the labrum. This result shows that posterior collar domain of RNAi Fig.4.6. Tc cnc results in transformation of the mandibular into maxillary identity and deletion of the labrum. Mandible (arrowhead), maxilla palp (arrow) and ventral branch (star) are indicated. (A-C,F) RNAi Cuticle preparations of a wild type Tribolium first instar larvae. Cuticle preparations of Tc cnc larvae (D,G). (A) Cuticle preparations of gnathal appendages visualized by fluorescence microscopy. The maxillary appendages have a palp with 4 segments (white arrows) attached to a protopodite with the maxillary endites present. (B) Cuticle preparation of a first instar Tribolium larva (C) Cuticle preparation of the larval gnathal appendages of a wild type Tribolium larva visualized by DIC microscopy. (D) Cuticle RNAi preparation of the gnathal appendages of a Tc cnc larva. Knock down of Tc cnc results in transformation of the mandibular appendages into maxillary appendages (arrowheads). The ventral branch is visible on the transformed appendages (white stars). The maxillary appendage is indicated with arrows (palp) and black stars (endite). (E) Cuticle preparation of wild type Tribolium larva visualized by confocal microscopy. The mandible appendage is highlighted in blue, the maxillary appendage is RNAi highlighted in green. (F) Cuticle preparation of a Tc cnc larva visualized by confocal microscopy. The transformed mandibular appendage is highlighted in blue and clearly resembles the maxillary appendage (highlighted in green) (G) SEM of a wild type embryo at fully extended germ band stage. The RNAi labral buds are clearly visible at the anterior of the embryo (arrow). (H) SEM of Tc cnc embryo at germ band extending stage. The labral buds are missing (arrow, compare with G).

Tc cnc differentiates a mandible from a maxillary appendage in the mandibular segment and that the anterior domain of Tc cnc performs a gap gene like role in patterning the labrum. This is shown in fig.4.6D,F, where Tc cncRNAi larvae possess an additional pair of maxillae. The mandibular appendages are clearly transformed into maxillary identity, in possession of the maxillary palp with the same number of segments, with the ventral branch (fused lacinia and galea endites in first instar larvae). There is also deletion of the labrum, which demonstrates that the anterior cap domain of Tc cnc is necessary to pattern this structure (see fig.4.6H, and fig.4.7B,D-F).

There may also be some abdominal defects in some embryos, although it is quite possible that this phenotype was in fact an artefact of the cuticle preparation procedure.

Tc cnc represses Tc Dll and modifies Tc prd expression in the Mandibular segment.

To investigate the transformed mandibular appendage in Tc cnc knockdown embryos, the expression pattern of the homeobox genes Tc Dll and Tc prd was studied as genetic markers of the developing endites and telopodites.

In wild type embryos, Tc prd is expressed in the developing endites of all three pairs of gnathal appendages (see fig.4.5. and fig.4.7A,C). Expression of Tc prd is seen in distinct domains of expression in the lacinia and galea enditic lobes of the maxilla, a single domain in the labial enditic lobe and a larger domain of expression in the mandibular appendage.



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