«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»
Hox genes pattern the Tribolium maxilla in an additive manner As mentioned above, the Tribolium maxilla is patterned by two Hox genes, Tc Dfd and mxp in an additive fashion. Tc Dfd patterns the protopodite and mxp patterns the telopodite of the maxilla. Consistent with these phenotypes, Tc Dfd is more strongly expressed in the base of the maxilla and weak or non-existent in the palp. mxp is expressed in the maxillary palp and the distal part of the protopodite, the galea endite lobe (Brown et al., 2000; Shippy et al., 2000a; Shippy et al., 2000b; DeCamillis et al., 2001).
Dfd expression is conserved in the mandible and maxillary segments of mandibulate arthropods. In particular the expression of Dfd is conserved in the protopodites of the maxillary appendages of mandibulates. In addition, expression of pb is conserved in the telopodite derived palp of the maxillary segment. The similarity of the expression patterns of these genes suggest that the additive patterning of the maxillary appendage is conserved in diverse mandibulate taxa.
The Tribolium maxilla presents an interesting case regarding the role of Hox genes in patterning segments. The additive function of two Hox genes to pattern one appendage type is in contrast to the more typical manner of Hox gene function, posterior dominance. Posterior dominance refers to the dominance of posterior Hox genes over more anterior Hox genes. When a typically more posteriorly expressed Hox gene is ectopically expressed in more segments, the anterior segments take the identity specified by the posterior Hox gene.
Mandible to antenna transformation in Tc Dfd mutants In Tribolium Tc Dfd mutants, there is a homeotic transformation of the mandible to antennal identity. The reason for the transformation of mandible to antenna is because Hox genes are needed to repress antennal development. In the absence of Hox genes in the ectoderm of developing appendages, the default appendage type specified is antenna (Brown et al., 2002b). Tc Dfd is the only Hox gene which is expressed in the ectoderm of the mandibular segment. In the absence of Hox genes in the mandibular segment in Tc Dfd mutants, there is therefore a homeotic transformation of the mandible to antenna as there is no other Hox gene expressed in the ectoderm of the mandibular appendage. The role of Hox genes in repressing antennal development in post-antennal appendages has been argued to represent the ancestral gene function of Hox genes in arthropods (Brown et al., 2000).
The fact that in Tc Dfd mutant larvae there is a homeotic transformation of mandible to antennal identity means that analysing the downstream effects of Tc Dfd knock down will be complicated by the activation (or de-repression) of the antenna specifying pathway. The antenna patterning pathway involves many of the same genes, such as the PD domain genes and the Notch signalling pathway. Therefore any reduction in expression caused by a loss of Tc Dfd will be masked if these genes are activated by the antenna patterning pathway.
For example, genes that are expressed in the antenna, such as Tc dac, may be activated by Tc Dfd in the mandibular segment. But if those genes are also activated in the antenna specifying pathway then Tc dac will continue to be expressed, but as an ectopic antennal domain of Tc dac. Tc Dfd knock down or mutant phenotypes in the mandible will reveal the genetic interactions which result as a consequence of activating the antenna patterning pathway in the absence of Hox genes.
The Drosophila gnathocephalon
The Drosophila embryonic gnathal lobes of the mandibular, maxillary and labial segments make up the gnathocephalon. The gnathocephalon does not develop into appendages in the larva, instead it forms the pseudocephalon through a complicated process of cell movements (Jurgens et al., 1986; Diederich et al., 1991). Despite the obvious morphological differences between Tribolium and Drosophila larvae, the gnathocephalon of Drosophila is clearly homologous to the gnathocephalon of less derived insects such as Tribolium. The proximal part of the Drosophila gnathal maxillary lobe is homologous to the proximal part of the developing Tribolium maxilla, specifically the endites (Jurgens et al., 1986). There are also significant similarities in the genetic interactions of mandibular and maxillary segment patterning genes in the proximal region of the maxilla between the two species.
Dfd function in the maxillary gnathal lobe of Drosophila
As in Tribolium, Dfd is required to pattern proximal maxillary lobe derived structures in Drosophila larvae (specifically the cirri, ventral organs and mouth hooks).
Dfd has been shown to pattern proximal maxillary lobe derived structures by activating prd, ser and the proximal domain of Dll (Gutjahr et al., 1993; O'Hara et al., 1993;
Vanario-Alonso et al., 1995; Li et al., 1999; Wiellette and McGinnis, 1999).
In Drosophila, Dfd activates the proximal domain of Dll by a maxillary-specific enhancer (called ETD6) and is required for the formation of proximal maxillary lobe derived structures, the cirri (O'Hara et al., 1993). Dfd activates the late expression domain of prd in the proximal region of the gnathal lobes (Gutjahr et al., 1993; Li et al., 1999). prd is necessary for proximal maxillary lobe derived structures (such as the cirri and the ventral organ) (Vanario-Alonso et al., 1995). ser is also a target of Dfd in the mandibular and maxillary lobes. ser is required for normal mouth hook development (Wiellette and McGinnis, 1999).
It is already known that Dfd activates the proximal domain of Tc Dll in Tribolium (Brown et al., 2000). Given that Dfd activates prd and ser in Drosophila, I wanted to find out whether Tc Dfd activated the orthologues of prd and ser in Tribolium.
Regarding Tc ser, I wanted to discover whether Tc Dfd regulated the formation of maxillary segments in the developing maxillary protopodite by regulating or interacting with the Notch signalling pathway as it does in Drosophila.
ser is a component of the Notch signalling pathway required for the formation of arthropod appendage segments. ser expression in the gnathocephalon of Drosophila is different compared to Tribolium as Drosophila larvae do not form segmented appendages. Therefore the appendage segment domains of the notch signalling pathway are missing from the developing gnathal lobes. In Drosophila, ser is expressed strongly in the mandibular segment and the boundaries between each segment of the gnathocephalon (Wiellette and McGinnis, 1999).
There are also differences between late prd expression in Drosophila and Tribolium. prd expression is strongest in the maxillary segment in Drosophila and expression fades in the other gnathal segments during embryogenesis, prd expression is not obviously associated with developing structures in the Drosophila gnathocephalon (Gutjahr et al., 1993). In Tribolium late Tc prd expression is obviously associated with the developing endite lobes and is strongest in the mandibular endite (Aranda et al., 2008).
Experimental introduction I was interested in exploring the role that Tc Dfd has in patterning the protopodite of the maxilla in more detail with a view to the eventual goal of a more complete understanding of the mandibular and maxillary patterning pathways of Tribolium. By understanding the precise functions of Tc cnc and Tc Dfd in patterning the mandible appendage, comparisons across mandibulate taxa will be potentially more informative and conclusions about the homology of mandibular and maxillary patterning mechanisms will be more robust.
Transformation of the mandible into antennal identity with the loss of Tc Dfd function means that it is not possible to use RNAi to study the target genes of Tc Dfd in patterning the mandibular appendage. However, it is not unreasonable to assume that the target genes of Tc Dfd in the maxillary segment will be similar targets of Tc Dfd in the mandibular segment, and therefore informative about the role of Tc Dfd in patterning the mandibular segment. To demonstrate such claims further work will have to be performed using more complex genetic techniques, such as ectopically activating Tc cnc, or using clonal analysis of Tc Dfd mutants for example, to understand the mandible and endite patterning function of Tc Dfd in more detail. Unfortunately, such techniques are currently unavailable for Tribolium.
In order to determine the role of Tc Dfd in patterning the maxillary protopodite, Tc Dfd was knocked down by RNAi and the effect on other genes was determined by in situ hybridisation. Tc prd and the proximal domain of Tc dac are expressed in both the mandible and maxillary protopodites of Tribolium embryos. Both these genes have endite-specific expression domains as described in chapter two. In Drosophila, it has been shown that Dfd activates prd in the maxillary segment. I was therefore interested whether Tc Dfd activates Tc prd and the proximal domain of Tc dac in the maxillary segment.
The mandible and maxilla consist of subcoxal and coxal segments which are marked by Tc ser domains of expression as described in chapter three. In Drosophila, although there are no larval appendages, ser has been shown to be activated by Dfd. I wanted to determine whether in Tribolium Tc Dfd was required to activate protopodite specific domains of Tc ser in the maxillary protopodite.
5.2 Results DfdRNAi embryonic and larval phenotype Knock down of Tc Dfd by parental RNAi results in transformation of mandible to antenna identity and loss of the maxillary endites as previously described (Brown et al., 2000). It is evident from analysis of the cuticle preparations of DfdRNAi larvae (fig.5.1C,D) and SEMs of DfdRNAi embryos (fig.5.1I) that the entire endite is missing. The maxillary palp is also affected in in DfdRNAi embryos. In SEMs of DfdRNAi embryos, the affected maxillary palp is larger than the palp of wild type maxilla (fig.5.1H,K,I). This is particularly true of the base of the palp, which in wild type maxillae is quite narrow at the point where it attaches to the protopodite. Tc Dfd is expressed most strongly in the protopodite of the maxilla but is also expressed in the base of the maxilla palp, which is consistent with the phenotype observed. In DfdRNAi larvae, the palp and maxilla overall are shorter and smaller than the wild type maxilla (fig.5.1C,D,F).
The mandible is transformed into an appendage of antennal identity, which is smaller than the normal antenna. This is seen in both the SEMs and the cuticle preparations (fig.5.1C,D,I).
DfdRNAi phenotype of the affected maxillary appendage