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Further analysis of the protopodite of the affected maxillary appendage of a DfdRNAi embryo was conducted by studying the expression of Tc Dll and Tc prd. The expression of Tc Dll in Tc Dfd mutants has previously been described (Brown et al., Fig.5.4. Tc Dfd upregulates the proximal domain of Tc dac in the maxilla. Gene expression was detected by in situ hybridisation. (A) Expression of Tc dac in a wild type germ band retracting stage embryo. Tc dac is expressed in two domains in the maxilla, labial and leg appendages. There is a proximal domain (arrow) and a distal domain (arrowhead). The gnathal appendages have large proximal expression domains. In the legs, the proximal domain is faint and the distal domain in the develping RNAi femur and tibia is markedly stronger. (B-C) Expression of Tc dac in Tc Dfd germ band extending stage embryos. The mandible is transformed into an antenna (star) and has lost the mandible proximal expression domain. The proximal domain in the maxilla is faint. Conversely, the distal domain is larger.
The proximal domain of the labial appendage is unaffected. (C) Close up of the expression of Tc dac in RNAi the gnathal appendages of a Tc Dfd germ band extending stage embryo. (D) Close up of the RNAi expression of Tc dac in the affected maxilla and labial appendage of a Tc Dfd germ band extending stage embryo. Here the reduction in expression of the proximal domain of Tc dac (arrow) relative to the distal domain (arrowhead) can be seen clearly. The labial proximal domain (white arrow) is considerably larger than the distal domain (white arrowhead).
2000). The proximal domain of Tc Dll in the developing lacinea enditic lobe is lost, which indicates that the lacinea endite lobe is lost. In Tribolium DfdRNAi embryos, Tc Dll is expressed in the distal part of the affected maxilla appendage. Tc Dll expression is completely lacking from the proximal region of the maxilla which corresponds to the protopodite.
To analyse the role of Tc Dfd patterning the endites of the maxilla in more detail, an endite marker gene Tc prd was studied in DfdRNAi embryos. Tc prd is expressed in all endites of the gnathal appendages (fig.5.5A,B). Detection of Tc prd transcripts show unambiguously that the mandibular and maxillary endites have been lost in Tc Dfd knock down embryos (asterisk in fig.5.5C,D). The endites of the labial appendage are still present in DfdRNAi embryos, with the expression of Tc prd marking the labial endite (star in fig.5.5C-D). There is no expression of Tc prd in either the Fig.5.5. Tc Dfd patterns the endites of the mandibular and maxillary segments. Gene expression was detected by in situ hybridisation. The mandibular segment appendage is marked with an arrowhead.
The maxillary endites are marked with arrows in wild type embryos (A, B). (A) Expression of Tc prd (blue) and Tc Dfd (red) in a wild type germ band extending embryo. Tc prd is expressed in the developing endites of the gnathal appendages. Tc Dfd is expressed in the mandibular and maxillary segment embryos. (B) Expression of Tc prd (red) and Tc Dll (blue) in a wild type germ band extending embryo. Tc Dll is expressed in the lacinea endite lobe. There is no Tc Dll expression in the mandible (arrowhead). (C) RNAi Expression of Tc prd (red) and Tc Dll (blue) in a Tc Dfd germ band extending embryo. The mandible has been transformed into an antenna which expresses Tc Dll (arrowhead). There is no maxillary endite (asterisk). The labial appendage has an endite (star) marked with Tc prd expression. (D) Enlargement of the altered maxilla in C. There is no Tc prd expression in the affected maxilla. There is a significant region of the base of the affected maxilla excluding Tc Dll expression which indicates that the protopodite is still present.
transformed mandible or the affected maxilla. This result shows that Tc Dfd is necessary to pattern the endites of the mandible and maxilla.
Segmentation is unaffected by loss of Tc Dfd function in the protopodite.
A lack of expression in the proximal part of each limb for both mxp and Tc Dll and the presence of the proximal domain of Tc dac shows that there is still a protopodite in Tc Dfd knock down embryos. It is not obvious that the proximal segments have either been preserved or lost in the affected maxilla in Tc DfdRNAi embryos or larvae (fig.5.2C-F). In order to investigate the role of Tc Dfd in patterning the segments of the protopodite, the expression pattern of Tc ser, a gene involved in the formation of segments in appendages, was investigated in Tc Dfd knock down embryos.
Therefore in order to further study the effects of Tc Dfd knock down on leg appendage segmentation, a marker of leg segmentation, Tc ser, was studied in combination with the PD domain genes Tc Dll and Tc dac to use as markers of segment identity. By determining the expression of Tc ser relative to the domains of Tc Dll and Tc dac, it is possible to determine if maxilla appendage segments have been lost or not, or if they have significantly altered development from wild type maxilla.
Three or four rings of Tc ser expression are present in the maxilla of Tc Dfd knock down embryos (fig.5.6B,D-J). But, due to the morphology of the mutant appendage, it is difficult to identify which ring of Tc ser relates to which segment. The affected maxilla lacks endites and the affected palp is larger and more continuous with the protopodite, which means that there is little morphological frame of reference to identify each Tc ser ring of expression. Consideration of the conclusions from the cuticle preparation maxilla phenotype of DfdRNAi larvae support the first two rings of Tc ser expression represent the subcoxa and coxa domains of Tc ser expression.
To facilitate the identification of the rings of Tc ser expression, Double in situ hybridisations were performed with Tc ser and the leg gap genes Tc Dac and Tc Dll. By comparing the relative positions of both of these genes relative to Tc ser expression in wild type embryos it is possible to identify the rings of Tc ser expression in the mutant maxilla of Tc Dfd knock down embryos.
There are clearly three domains of Tc ser expression in the affected maxilla (fig.5.6B). Comparison of expression to PD domain gene expression confirms that the first three proximal Tc ser domains relate to their wild type counterparts and are therefore not regulated by Tc Dfd. The proximal-most ring of expression is the subcoxal-1 Tc ser domain, and the second Tc ser ring of expression is the coxal-2 ring domain. The third ring of expression relates to the trochanteral-3 ring domain of Tc ser which is expressed at the base of the palp (fig.5.6).
The evidence for this conclusion is based upon the expression of Tc dac and Tc Dll relative to Tc ser ring domains in a manner which is reminiscent of wild type appendages. The proximal domain of Tc dac is expressed between the subcoxal-1 and coxal-2 ring domains in the affected appendage (star in fig.5.6D,G,H). The distal domain of Tc dac is expressed abutting the third ring domain of Tc ser (arrow in fig.5.6DG,H). Tc Dll is expressed in the distal-most region of the affected palp up to and including the trochanteral-3 ring domain of Tc ser (fig.5.6I). A schematic of these
expression domains in both the maxilla of normal and Tc Dfd knock down embryos is shown in fig.5.6J.
In Tc DfdRNAi embryos therefore there is no effect on the expression of the proximal ring domains of Tc ser in the maxilla. The first, second and third rings of Tc ser expression are present and the expression domains of Tc Dac and Tc Dll relative to these domains confirm them to be the subcoxa (1), coxa (2) domains of the protopodite and the third (3) Tc ser ring domain at the base of the palp.
Ring domains of Tc ser expression in the transformed mandible are changed to a pattern typical of the antenna which is consistent with the homeotic transformation of mandible to antenna identity (fig.5.7). Tc dac is expressed in between the first and second rings of Tc ser expression in the transformed mandibular appendage (fig.5.7D).
Tc Dll is expressed in the distal part of the transformed mandible to the first proximal ring domain of Tc ser (fig.5.7E). Expression of the PD domain genes Tc Dll and Tc dac in the transformed mandibular appendage is therefore similar to that of the antenna, confirming homeotic transformation of these appendages (shown schematically in fig.5.7F).
Fig.5.7. The mandible is transformed into antennal identity in Tc Dfd embryos. Transformation of the mandible to antennal identity is confirmed by the expression of the PD domain genes Tc dac and Tc Dll and the segmentation marker Tc ser. Expression of these three genes in the transformed mandible replicate the antennal expression domains of these genes. (A) Tc dac and Tc ser expression in a wildtype mandible. (B-C) Wild type antennae at a slightly later stage than C-E. First antennal domain of Tc ser is in the Antennifer segment. The second antennal domain of Tc ser is in the scapus. (B) Tc dac and Tc ser expression in a wildtype antenna. (C) Tc Dll and Tc ser expression in a wildtype antenna. (D) Tc dac and RNAi Tc ser expression in the transformed mandible of a Tc Dfd embryo. (E) Tc Dll and Tc ser expression in RNAi the transformed mandible of a Tc Dfd embryo. (F) Schematic of the expression patterns of the PD domain genes Tc dac and Tc Dll and the segmentation marker Tc ser in the mandible (Mn), antenna (An) and transformed mandibular appendage (Mn*).
5.3 Discussion General overview Knock down of Tc Dfd by RNAi shows that Tc Dfd is necessary to activate protopodite and endite patterning genes in the maxillary appendages, in addition to repressing antennal development in the mandible as previously described (Brown et al., 2000). Segmentation is unaffected, at least in the proximal part of the affected maxilla as determined by the expression of Tc ser. The affected maxilla therefore retains the protopodite. There are significant morphological changes to the protopodite in Tc Dfd knock down embryos. The protopodite has lost the endites. The affected maxilla retains the protopodite segments, the subcoxa and the coxa, but has been reduced in size (primarily width). It is also apparent that Tc Dfd is necessary to shape the maxilla protopodite. The wild type maxilla protopodite is orientated perpendicular to the palp. In the Tc Dfd knock down embryos, the maxilla appendage has lost its characteristic maxilla shape and is more telescopic in appearance.
Tc Dfd is necessary to pattern the endites as shown by study of the expression of Tc prd in Tc Dfd knock down embryos. Tc Dfd activates Tc prd expression in the mandibular and maxillary endites. Tc prd expression still remains in the labial appendage endites.
Tc Dfd also up-regulates the proximal domain of Tc dac in the maxilla. Tc dac is upregulated by Tc Dfd but not necessarily activated by it. The proximal domain of Tc dac expression is weaker in the maxilla and distal expression domain of Tc dac is stronger in Tc Dfd knock down embryos. As there is still faint expression of the proximal domain of Tc dac in Tc Dfd knock down embryos, it indicates that Tc dac doesn’t require Tc Dfd for initial activation. It is possible that the RNAi knock down phenotype was not fully penetrant in these experiments and as a result there was only partial knock down of Tc Dfd gene function which may reveal itself by partial reduction of Tc dac activation. This criticism could also apply to analysis of the lack of an apparent effect on Tc ser expression. There was however no posterior collar domain of Tc cnc (fig. 3.3B-D) and no obvious partial phenotype like reduced endites, or mandible-antenna hybrid appendages.
The developing limb axis of the maxilla is not controlled by the action of Tc Dfd.
This conclusion can be made as the maxilla of Tc Dfd knock down embryos develops proximal segments which retain subcoxa, coxa and trochanter identity determined by the expression of the PD domain genes relative to the proximal ring domains of Tc ser.
Proximal ser expression is unaffected in Tc Dfd knock down embryos. Using the expression of Tc Dac and Tc Dll as markers for the subcoxa and coxa demonstrates that the subcoxa and coxa domains of Tc ser expression are still present. This situation is unlike that in Drosophila where Dfd has been shown to regulate regulation of Tc ser transcription in the gnathal lobes. Although it is possible that Tc Dfd is required for patterning the segments of the palp and for patterning the palp.