«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»
2.4E) in late stage Tribolium embryos. The proximal limit of Tc prd expression matches to the developing molar process. This shows that Tc prd expression in the outer lobe relates to the developing incisor process and the inner lobe relates to the developing molar process.
Both gnathal edges marked by Tc prd expression, comprised of the developing incisor and molar processes, are migrating toward each other so that they are in contact at the midline in front of the mouth opening. This is characteristic of the orientation of the gnathal edge in the larva (fig 2.4H,I). In earlier stages of development, Tc prd expression is present throughout the inner and outer lobes (fig.2.5E).
The inner and outer mandible lobes seen in the embryonic Tribolium mandible are also found in other species and appear to be characteristic of the developing mandibular appendage. SEM studies in other insects have demonstrated that the inner and outer lobes are present and even more distinct in the cricket Gryllus assimilis (Liu et al., 2010), the sawfly Athalia rosae (Oka et al., 2010) and the jumping bristletail Pedetontus unimaculatus (Machida, 2000). A study into the expression of PD domain genes in the gnathal appendages of the millipede Glomeris marginata, shows that the mandible has an inner lobe and an outer lobe (Prpic and Tautz, 2003).
Comparison of Tc dac expression to the expression and function of dac in other mandibulates supports the conclusion that the outer lobe relates to the incisor process. dac expression is restricted to the outer lobe and expression appears to be strongest in a ring around the proximal part of the outer lobe adjacent to the inner lobe in the cricket Gryllus in a manner similar to that seen in Tribolium (Ronco et al., 2008). Gm dac expression in the millipede Glomeris is also more strongly expressed in the proximal part of the outer lobe (Prpic and Tautz, 2003).
In a functional study, knock down of dac in the adult Dung beetle resulted in deletion of the region in between the incisor and molar processes. The adult mandibles in this species are characterised by an elongated incisor process. (Simonnet and Moczek, 2011). This result is consistent with the expression domain of Tc dac in the Tribolium mandible in the proximal part of the outer lobe in between the developing molar and incisor processes. Although unpublished functional work performed in Tribolium has not shown a role for Tc dac in patterning the mandible (Simonnet and Moczek, 2011), this does not necessarily detract from conclusions based on its use as a marker for the mandibular gnathal edge.
The mandibular gnathal edge is derived from one endite Evidence provided from several genetic markers supports the conclusion that the mandible possesses one endite. Using Tc prd as a marker for endite development reveals the presence of one domain of Tc prd expression in the mandible compared to two domains in the maxilla. This suggests that there is only one endite present in the mandible. This conclusion contradicts the hypothesis of Machida (2000) which posits that the mandible incisor process and the molar process are homologous to the galea and lacinia maxillary endites, as it implies they are derived from two endites.
A previous study of the Tc wg expression pattern in Tribolium has suggested that the mandible consists of one endite (Jockusch et al., 2004). Tc wg is expressed in a stripe that runs through the middle of the ectoderm of all appendages. Tc wg expression retracts from the endites of the maxilla to form two gaps in Tc wg expression. In the mandible, only one gap in Tc wg expression develops which suggests that there is only one endite present (fig.2.6D).
Comparison of the expression of Tc dac relative to Tc prd expression shows similarities of the mandibular endite to both of the maxilla endites. The co-expression of Tc dac and Tc prd in the mandibular endite resembles the co-expression of these genes in both maxillary endites. Tc dac expression overlaps with the distal half of Tc prd expression in developing endites (arrowhead in fig 2.5H). Tc dac is expressed in the distal half of the maxillary endites whilst Tc prd is expressed throughout both maxillary endites. In the mandible limb bud, Tc dac is expressed in the outer lobe with Tc prd expressed more or less continuously through both inner and outer lobes. The similarity of the relationship of Tc dac expression to Tc prd expression in the maxilla endites therefore supports the conclusion that the mandible has only one endite.
This expression pattern of dac in the distal half of endites appears to be conserved across mandibulates. The expression of dac in the distal half of endites is observed in the cricket Gryllus (Ronco et al., 2008). Expression of Dac in the phyllopodous limbs of Triops is restricted to the distal half of the developing endites (Sewell et al., 2008). Expression of dac is not observed in the distal half of endites in chelicerates (Prpic and Damen, 2004). However, chelicerate endites are not hypothesized to be homologous to mandibulate endites as they derive from unsegmented protopodites as discussed in chapter one (Boxshall, 2004; Sewell et al., 2008).
As there is only one mandibular endite that divides into two lobes that develop into the molar and incisor processes, the mandibular endite has evolved from a typical lobe-like endite to form an endite consisting of two lobes. The mandible endite has therefore expanded proximally to form the molar process (inner lobe) and distally to form the incisor process (outer lobe). The outer lobe is derived from the distal half of the endite (marked by Tc dac and Tc prd expression), and the inner lobe is derived from the proximal half of the endite which is lacking Tc dac expression and is marked by Tc prd expression.
Endite patterning mechanism The expression domain of both Tc dac and Tc prd in the endites suggest a possible functional role for these genes in patterning the gnathal appendage endites of Tribolium, and possibly other arthropods. The functional role of Tc prd and Tc dac to pattern the maxillary endites was tested by knocking down by parental RNAi. However, neither experiment was successful in determining an endite patterning role of these genes (see appendix 5). It would be interesting to investigate the expression of homologs of prd to see if prd expression is conserved in the endites of other arthropods.
The gnathal edge of the mandible is the structure that differentiates the mandible from all other arthropod appendages. This chapter has shown, that the gnathal edge is derived from the inner and outer lobes which themselves derive from one endite. One gene, Tc cnc, that differentiates the mandibular segment from the maxillary segment in Tribolium, including the mandibular endite from maxillary endite identity, will be investigated in chapter four. Another gene that is required to pattern mandibular and maxillary endites, Tc Dfd, will be investigated in chapter five.
3.1 Introduction In this chapter I am interested in the structure of the protopodite of the Tribolium mandible, specifically if there is any evidence of segmentation. The ancestral biramous limb was composed of two rami, an exopodite-derived ramus and a telopodite-derived ramus, which were connected to the protopodite which is located at the base of the appendage. The insect mandible has evolved from a biramous limb by losing both palps. The structure that remains is therefore the remaining protopodite complete with the gnathal edge, the character that distinguishes a mandible from other appendage types. I was also interested in the structure of the protopodites of other post-antennal appendages. By comparing the mandibular protopodite to the protopodites of other appendages it may be possible to show similarities that are suggestive of homology.
The hexapod mandible is an unsegmented appendage in both the larva and the adult. However, it has been observed in the developing mandible of various hexapods that there is a so-called subcoxa/coxa division (Machida, 2000; Liu et al., 2010; Oka et al., 2010). It has also been hypothesized that these subcoxal and coxal segments in the mandible are homologous to segments in the maxillary appendage, the cardo and stipes respectively (see fig.3.1A) (Machida, 2000).
In order to examine the possible existence of a segmental subdivision in the developing embryo, a segmental marker was used to study the developing mandibular appendage. The Notch signalling pathway is involved in the formation of arthropod limb segments and is important in the development of arthropod appendages. There has been no study of the Notch signalling pathway in the gnathal appendages to date.
To determine the precise identity of each appendage segment in the developing embryo, expression of the PD domain genes was studied in conjunction with the Notch signalling pathway.
Fig.3.1 Hypotheses of serial homology of the putative mandibular subcoxa to the cardo of the maxilla and the subcoxa of the leg. Possible homologous appendage structures are indicated with the same colour. Figure is modified from Machida (2000). Subcoxal segments are shown in yellow, distal protopodite segments are shown in blue. (A) Machida has hypothesized that the mandible is divided into a subcoxa (yellow) and coxa (blue) which is serially homologous to the cardo (yellow) and stipes (blue) of the maxilla. (B) If there is a subcoxal segment present in the leg (yellow), there is a possibility to homologize it to other appendage segments, in this example the leg subcoxa is homologized to the putative subcoxa of the mandible.
Appendage segments and the Notch signalling pathway
One clade defining character of the phylum Arthropoda is the presence of limb segments. It has been shown that the Notch signalling pathway is responsible for the creation of leg segment joints in Drosophila. The Notch signalling pathway is often responsible for setting up cell boundaries and defining populations of cells (Bray, 2006).
For the purposes of a genetic marker for segment boundary formation I chose to study the expression of the Tribolium homologue of the gene serrate (Tc ser). Tc ser was significantly easier to detect by in situ hybridization than notch (data not shown).
ser is a transmembrane ligand of the Notch receptor and regulates Notch activation in adjacent cells. ser is expressed in a ring of cells that are on the distal part of each segment. notch is expressed in a ring of cells that form the joint between leg segments and is expressed immediately adjacent to cells expressing ser. ser is therefore expressed slightly proximally to notch and to where the segment boundary will form (Rauskolb, 2001).
The Notch signalling pathway is involved in the formation of leg segments in Drosophila and is regulated by the PD domain genes. For example the first ring of ser expression in Drosophila relates to the coxa and is activated by hth within the presumptive coxa. Dll represses ser in the presumptive telopodite at this stage (Rauskolb, 2001; de Celis Ibeas and Bray, 2003; Hao et al., 2003; Greenberg and Hatini, 2009). Therefore, it is possible to relate PD domain gene expression to precise appendage segments in developing embryos by comparing the expression domains of the PD domain genes with notch rings of expression that relate to specific segment boundaries.
Expression of notch in a segmental fashion in the leg appendages of the spider Cupiennius salei reveals that it is likely that Notch mediated definition of segment boundaries is also present in chelicerates and likely to be conserved across Arthropoda (Prpic and Damen, 2009).
PD domain genes are conserved across Arthropoda in all appendage types
The expression and function of PD domain genes are conserved across arthropod leg development (Angelini and Kaufman, 2005). The expression of PD domain genes in gnathal appendages has been shown to be similar to that observed in the developing legs. The expression patterns of PD domain genes has been studied in the gnathal appendages in several species with mandibulate mouthparts (Abzhanov and Kaufman, 2000b; Beermann et al., 2001; Prpic et al., 2001; Inoue et al., 2002;
Rogers et al., 2002; Prpic et al., 2003; Prpic and Tautz, 2003; Jockusch et al., 2004; Mito et al., 2008; Prpic and Telford, 2008; Ronco et al., 2008; Liubicich et al., 2009). In all studied species the expression domains of PD domain genes are conserved along the proximal-distal axis between gnathal appendages and leg appendages.