«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»
For ease of comparison, the nomenclature used to describe the gnathal lobe axis as proximal to distal will be used throughout this thesis in preference to the nomenclature used to study Drosophila.
The structures which are derived from these proximal and distal domains in Drosophila are homologous to structures found in other mandibulate hexapods. The appendage-less larvae of Drosophila still possess sense organs and cirri that are the only remaining structures left from the loss of the gnathal appendages present in the majority of insects (Jurgens et al., 1986; Panganiban, 2000). The palp of mandibulate hexapods is homologous to the distal domain of the maxillary lobe of Drosophila and the endites are homologous to the proximal domain of the maxillary lobe. The sense organs that develop from the gnathal lobes of Drosophila embryos are homologous to those found on insect appendages (Behan and Ryan, 1978; Jurgens et al., 1986). The maxilla sense organ is homologous to the sense organ found on the maxilla palp. The cirri and ventral organ are homologous to structures on the maxilla endites (Jurgens et al., 1986).
To further clarify: The Drosophila maxillary lobe consists of ventral-lateral and dorsal domains which are homologous to the distal and proximal domains of mandibulate Hexapods. The dorsal domain of the Drosophila maxillary lobe is homologous to the Tribolium maxilla palp, whilst the Drosophila ventral-lateral domain is homologous to the Tribolium maxillary endites.
Fig.1.12. Comparison of mandibulate (represented by Tribolium) and Drosophila larval and embryonic morphology. (A-C) adapted from Jurgens et al. (1986). Labrum (Lb), antennal (An) mandibular segment (Mn), maxillary segment (Mx), labial segment (La), first leg (L1) segments are indicated. (A) Drosophila larva head with segment origins indicated by colour. Mouth hooks (MH), cirri (ci) and the ventral organ (VO) are derived from the maxillary segment. Lateralgraten (LG) of the hypopharyngeal skeleton are derived from the mandibular segment. The ventral arms (VA) and T-ribs (T) are derived from the hypopharyngeal lobes (Jurgens et al., 1986; Finkelstein and Perrimon, 1991; Rogers and Kaufman, 1997).
The antennal sense organ (AnSO) is derived from the antennal segment. Most of the maxillary sense organ is derived from the distal portion of the maxillary gnathal lobe. (Jurgens et al., 1986). (B) Tribolium larval head with gnathal appendages highlighted. (C) Schematic of the Drosophila embryo with segmental origins of larval structures indicated. The hypopharyngeal lobes have been shown to derive from the mandibular segment (Economou and Telford, 2009). The maxillary sense organ (MxSO) has diverse segmental origins (shaded in blue). The dorso-medial papilla (DMP) and the dorso-lateral papilla (DLP) are derived from the antennal and mandibular segments respectively. The ventral organ and cirri, are derived from the proximal part of the maxillary lobe (shaded in green) (D) Tribolium embryo schematic showing the developing segmental appendages. (E-F) Expression of Dll (blue) and prd (red) in the gnathocephalon of Drosophila (E) and Tribolium (F). The distal part of the maxillary gnathal lobe is marked by a distal domain of Dll expression. The proximal part of the maxillary gnathal lobe is marked by prd expression. In Drosophila, the ventral organ and cirri, are derived from the proximal part of the maxillary lobe (shaded in green in C) and are homologous to the Tribolium endites.
Expression patterns of two genes, Dll and prd are expressed in proximal domains in the maxillary gnathal lobe similar to that of mandibulate insects. The proximal expression domains of Dll and prd are homologous to the proximal expression domains found in the endites of Tribolium. Dll is also expressed in a distal domain in Drosophila that is likely to be homologous to the distal domain of Dll expression in Tribolium and other insects. These expression patterns are consistent with the homology of the proximal domain of the Drosophila maxillary lobe to the maxillary endites of Tribolium and other mandibulate arthropods (see fig. 1.12E,F).
1.9 Mandibular segment patterning genes in Drosophila.
Deformed Dfd is expressed in the mandible and maxillary segments of all studied mandibulates. The most thorough genetic analysis of Dfd in an insect, crustacean or myriapod has been in Drosophila. Drosophila larvae lack appendages but there are some structures derived from the mandibular and maxillary segments which can be used as markers during the larval stage to determine the segmental patterning function of Dfd (see fig 1.12A,C).
Drosophila that are mutant for Dfd are missing maxillary and mandibular derived structures. In particular, Dfd null mutant flies are missing the cirri, ventral organ, mouth hooks which are of maxillary segment origin and the Lateralgraten and the dorso-lateral papilla of the maxillary sense organ which are of mandibular segment origin (Merrill et al., 1987; Regulski et al., 1987; McGinnis et al., 1998; Brown et al., 1999; Veraksa et al., 2000). Ectopic expression of Dfd results in ectopic maxillary structures such as cirri, ventral organs and mouth hooks on the labial and thoracic segments (O'Hara et al., 1993). Several target genes have been shown to be regulated by Dfd in the developing embryo.
There is no Hox gene that differentiates the mandibular segment from the maxillary segment in Drosophila. Experiments have shown, however, that cnc differentiates the mandibular segment from the maxillary segment. cnc is necessary for the development of mandibular derived structures and achieves this at least in part by repressing the activity of Dfd in the mandibular segment. For its role in modifying Dfd function, it has been labelled a Hox modulator gene (Mohler et al., 1995; McGinnis et al., 1998; Veraksa et al., 2000).
cnc null mutants lose labrum and mandibular segment derived structures and have a duplication of maxilla derived structures. Specifically the duplicated maxillary structures include the mouth hooks of the hypopharyngeal skeleton, cirri and the ventral organ. Mandibular segment derived structures are lost such as the Lateralgraten. Hypopharyngeal lobe derived structures, the ventral arms and T-ribs of the hypopharyngeal skeleton, which are themselves derived from the mandibular segment (Economou and Telford, 2009), are also lost in cnc null mutants (Mohler et al., 1995; McGinnis et al., 1998).
Whilst data from Drosophila are useful for finding candidate genes for mandibular segment patterning, there are limits as to what can be understood from Drosophila. Drosophila does not possess a mandibular appendage in either the larval or adult form. In addition, the Drosophila leg protopodite is lacking endites. Given that the mandibular appendage is a protopodite with a pronounced modified endite, Drosophila is limited as to how much it could inform us about the development of the mandible appendage.
The majority of research into the function of genes patterning arthropod gnathal appendages has focused on insects that possess derived mouthparts. In terms of the evolution of the mandible these studies have discussed mandibular evolution in terms of evolution from a typical mandibular appendage (the ancestral mandible and similarly structured mandibles) into highly derived structures such as the proboscis of adults of the dipteran Drosophila melanogaster (Chadwick et al., 1990; Abzhanov et al., 2001; Joulia et al., 2005; Joulia et al., 2006), the development of the gnathal lobes and pseudocephalon in embryos and larvae of Drosophila (Regulski et al., 1987; Mohler, 1993; Mohler et al., 1995; McGinnis et al., 1998; Veraksa et al., 2000), and the stylet of the hemipteran Oncopeltus fasciatus (Hughes and Kaufman, 2000; Angelini and Kaufman, 2004; Angelini et al., 2005).
1.10 The red flour beetle Tribolium castaneum
There are numerous types of mandible within Mandibulata, but the shared character that links these diverse structural forms to define them as a functional mandible is the presence of the characteristic mandibular endite on the protopodite.
This mandibular endite forms the biting edge. Therefore in order to study the development of the mandible it is necessary to choose a species that has this specialized structure present (Jenner, 2006). For this reason Tribolium is an appropriate model as it possesses a mandible with a typical gnathal edge differentiated into an incisor process and a molar process and is amenable to functional genetics.
Tribolium is an up and coming model organism, easy to culture and maintain in the laboratory and possessing a sequenced genome (Bucher et al., 2002; Bucher and Wimmer, 2005; Richards et al., 2008; Schroder et al., 2008). Whilst functional genetics in Tribolium may not be up to the level of genetic manipulation possible in the fruitfly, it is still at a level that surpasses the majority, if not all other arthropod species.
Considerable work has been done on Tribolium embryogenesis, particularly in comparisons of developmental genes and gene regulatory networks that are of known importance in Drosophila. To date, there has been little study that has investigated the genes that are responsible for patterning the mandibular segment in a member of Mandibulata that unlike Drosophila possesses a typical mandible appendage.
Importantly, then, Tribolium is a mandibulate that possesses a typical insect mandible and is amenable to functional genetic studies. There are inevitably significant differences between the mandible of Tribolium and the probable structure of the ancestral mandible of stem lineage mandibulates. As we have seen, the ancestral mandible has probably evolved from a maxilla-like precursor, and in many ways resembled the ancestral serial homologous biramous limb from which it was derived.
The ancestral mandible was almost undoubtedly biramous and monocondylic (attached to the head with one condyle).
The Tribolium mandible, like all Hexapod mandibles, has lost both palps of the biramous limb structure and consists solely of an unsegmented protopodite. The Tribolium (hexapod) mandible is dicondylic, it has two attachment points to the head.
The defining characteristic of the ancestral mandible that distinguishes it from other appendages is the presence of a heavily sclerotized and developed endite which is in the form of an incisor and molar biting edge, the pars incisiva and pars molaris.
This well-developed endite that forms the biting edge on the protopodite is considered to be a homologous structure amongst Mandibulates (Edgecombe et al., 2003). The development of this structure is therefore of particular interest in understanding the mandible as a synapomorphy of Mandibulata.
Understanding the evolution of the mandible is not only interesting to understand the evolution of a clade-defining novel character, it is also necessary to understand the development of the ancestral mandible to form hypotheses about the evolution of derived gnathal appendages such as those mentioned above. For that purpose it is necessary to turn to an arthropod species that possesses a mandible.
After initial studies of mandible development in Tribolium, comparisons of mandible patterning genes in other taxa in interesting phylogenetic positions relative to Tribolium can be studied to determine whether significant aspects of mandible patterning are shared in diverse taxa. These data could then be used to either support or challenge the notion of mandible appendage and mandibular segment homology across Mandibulata.
Mandible patterning genes in Tribolium
Functional analysis has shown in Tc Dfd mutants there is a homeotic transformation of the mandible to an antenna10 and a loss of the maxillary endite (Brown et al., 1999; Brown et al., 2000). The Tribolium orthologue of cnc, Tc cnc, has an expression pattern that is very similar to the expression pattern of cnc in Drosophila (Economou and Telford, 2009). Given that cnc is necessary to differentiate the mandibular segment from the maxillary segment in Drosophila, I wanted to investigate whether the mandible patterning function of cnc is conserved in other mandiblebearing mandibulates like Tribolium.
Interestingly in Drosophila Dfd mutants there is a duplication of part of the cephalopharyngeal plates (most likely the vertical plate) which is derived from the procephalic lobe. If this derives from the antennal segment, it could indicate further similarity between the Drosophila and Tribolium Dfd mutant phenotype.
1.11 Introduction to results chapters