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Hypopharyngeal lobe derived structures are lost in cnc null mutants and are ectopically produced with ectopic expression of cnc (Veraksa et al., 2000). The hypopharyngeal lobes are often thought to be derived from the intercalary segment (Rogers and Kaufman, 2007; Veraksa et al., 2000), however it has recently been shown by our laboratory that the hypopharyngeal lobes are derived from the mandibular segment in agreement with earlier hypotheses (Economou and Telford, 2009).
cnc genetic interactions
Dfd expression is repressed by cnc in the anterior of the mandibular gnathal lobe. The activity of Dfd protein is also repressed by cnc in the mandibular segment. In addition to the repression of Dfd expression and activity in the mandibular segment of the developing fly embryo, cnc represses the expression of the Hox gene proboscipedia (pb) (Rusch and Kaufman, 2000). pb has no known function in the patterning of the fly embryo as pb mutants have no apparent embryonic phenotype (Rusch and Kaufman, 2000). cnc also represses the ventral expression domain of the PD domain gene Dll (McGinnis et al., 1998; Rusch and Kaufman, 2000). cnc itself is activated by gap and pair rule genes and is regulated independently of any Hox genes (Mohler, 1993).
Dfd expression across Mandibulata
Dfd and cnc are required to pattern the mandibular segment in Drosophila. It appears that based on the conserved expression of Dfd and cnc, they will have a similar role in mandibulate insects and myriapods. Dfd is expressed in the mandible and maxilla bearing segments in the majority of mandibulates, although with some variation (Hughes and Kaufman, 2002a). Insect species that have been investigated include Drosophila melanogaster (Diederich et al., 1991) Tribolium castaneum (Brown et al., 2000) Acheta domestica, Thermobia domestica, Oncopeltus fasciatus (Rogers et al., 2002), Bombyx mori (Kokubo et al., 1997) and Apis mellifera (Walldorf et al., 2000).
Crustacean species that have been studied include the woodlouse Porcellio scaber (Abzhanov and Kaufman, 1999a) and the water flea Daphnia pulens (Papillon, unpublished). Two myriapods have been studied, a chilopod Lithobius atkinsoni (Hughes and Kaufman, 2002b) and a diplopod Glomeris marginata (Janssen and Damen, 2006).
There are some differences between the different groups, but in general the expression patterns are conserved with expression limited to the mandibular and maxillary segments. Expression in Lithobius also includes the 2nd maxillary segment which appears to indicate that Dfd is involved with patterning the mandibles and maxillae of this species (Hughes and Kaufman, 2002b). Expression data from crustaceans indicates that the expression domain is more restricted to the mandibular segment.
After early expression in the mandibular and maxillary segments, there is dynamic expression of Dfd in the mandibular segment in mandibulates in a manner that resembles the dynamics of Dfd expression in Drosophila. The region from which Dfd expression retracts is called the distal part or the central region of the limb bud. Tc Dfd expression retracts from the mandibular limb bud in Tribolium (McGinnis et al., 1998; Brown et al., 1999). In Glomeris marginata, there is no expression of Dfd in the distal part of the mandible which relates to the developing mandibular endite (Janssen and Damen, 2006). In Lithobius there is no Dfd expression in the central region of the limb bud (Hughes and Kaufman, 2002b).
However, the dynamics of dfd expression are different in other mandibulates.
For example, there is no reported retraction of Dfd expression from the mandibular appendage in Oncopeltus fasciatus12 or Thermobia domestica (Rogers et al., 2002).
There is the possibility that the late retraction of Dfd expression was not detected because the embryos in the studies in question are too early. In the published in situ hybridisation of Thermobia there appeared to be a reduction of Dfd expression in the central region of the limb (the endite), but this could be an artefact of the plane of focus and the ectodermal expression of Dfd (Rogers et al., 2002).
cnc expression Across Mandibulata
Like Dfd expression, cnc gene expression patterns are highly similar in all studied mandibulates, although the number of taxa sampled is quite small. All mandibulate cnc expression patterns consist of two domains, a cap domain in the labrum and a collar domain in the mandibular segment. There is also cnc expression around the stomodeum that links the cap and collar domains (Mohler et al., 1991;
Mohler, 1993; Mohler et al., 1995; McGinnis et al., 1998; Janssen, 2004; Economou and Telford, 2009).
Oncopeltus has a derived mandibular appendage that develops into a stylet so can be ignored for purposes of comparisons to mandibulates.
Other insects in which cnc has been studied includes the cricket Acheta domestica, the milkweed bug Oncopeltus fasciatus, and the firebrat Thermobia domestica (Rogers et al., 2002). Outside of insects, only one species has been studied to date, the myriapod Glomeris marginata (Janssen et al. 2011). No crustacean homologues of cnc have been investigated.
In order to understand the development of the mandible, I chose to study the red flour Beetle Tribolium castaneum, a mandibulate which possesses a typical insect mandible and is amenable to functional genetic studies. One obvious question is whether the two genes cnc and Dfd which are important in Drosophila to pattern the mandibular segment play a significant role for development of the mandible in Tribolium. Like those of dicondylous insects in general, the Tribolium mandible is derived in a number of respects: it is dicondylic and has lost both the telopodite palp and exopodite ramus. But the Tribolium mandible possesses the one character that defines the mandible apart from any other appendage type, the gnathal edge made up of the incisor and molar processes on the protopodite of the second post-antennulary limb. The possession of this gnathal edge characterizes the Tribolium mandible as resembling the ancestral mandible.
Tc Dfd is necessary to pattern the mandible and maxillary appendages.
Research has demonstrated that the Tribolium orthologue of Dfd, Tc Dfd, is necessary for patterning the mandibular and maxillary segments in Tribolium like in Drosophila. In Tc Dfd mutants there is a homeotic transformation of the mandible to antenna and loss of the maxillary endite (Brown et al., 1999; Brown et al., 2000). In Drosophila however there is no homeotic transformation of the mandible but rather loss of mandibular and maxillary segment derived structures. Tc Dfd expression has been shown to retract from the mandibular limb buds. It is therefore interesting to know whether Tc cnc plays a similar role in differentiating the mandibular segment from the maxillary segment in part by repression of the maxilla patterning function of Tc Dfd.
Another gene, apontic (apt) has been shown to pattern the gnathal appendages in Drosophila (Gellon et al., 1997). This gene acts in parallel with Dfd to pattern ventral gnathal structures. However, there is no orthologue of the Drosophila gene apontic in Tribolium.
mxp is required to pattern the palp of the maxilla and labial appendages If Tc cnc differentiates the mandible by repressing maxilla development then other maxilla patterning genes may possibly interact with cnc. Another Hox gene, maxillopedia (mxp), the Tribolium ortholog of pb, is required to pattern the maxillary palp. Together, Tc Dfd and mxp pattern the maxillary appendage in an additive fashion (Shippy et al., 2000a). Tc Dfd patterns the protopodite, the proximal part of the appendage including the endite, and mxp patterns the telopodite, the palp. Mutants of mxp possess legs instead of palps in both the maxilla and labial segments. These transformed appendages are attached to a protopodite that is unaffected by the loss of mxp.
The orthologue of cnc in Tribolium, Tc cnc, as mentioned above is expressed in the labral and mandibular segment as well as surrounding the stomodeum like in Drosophila (Economou and Telford, 2009). Given that cnc is necessary for patterning the mandibular segment in Drosophila I was interested in the role that Tc cnc might play in patterning the mandibular segment of Tribolium castaneum and whether Tc cnc represses Tc Dfd expression in a manner similar to the mechanism that occurs in Drosophila. In order to test the function of Tc cnc in Tribolium, Tc cnc was knocked down using parental RNAi by injecting dsRNA into female Tribolium pupae (Bucher et al., 2002).
Tc cnc, Tc Dfd, mxp were amplified by PCR and then subsequently cloned.
Antisense labelled RNA probes were synthesized to detect gene expression by in situ hybridisation. Double stranded RNA of Tc cnc was synthesized to inject into female beetles to knock down Tc cnc gene function by RNAi.
Repression of Tc Dfd expression from the mandibular endite and appendage in wild type Tribolium embryos Tc Dfd is expressed throughout the mandibular and maxillary segments in the early developing embryo (see fig.4.1A,C-F, fig.4.2A-E, and fig.4.3A,B). As the limb buds start to form, Tc Dfd expression progressively retracts from the ventral-proximal part of the mandibular limb buds that relates to the position of the developing endites (see fig.4.3C). Some residual expression remains on the lateral part of the limb bud. During dorsal closure, Tc Dfd expression appears to be restricted to the distal portion of the protopodite of the maxillary limb bud (possibly relating to the developing stipes in late stage embryos. Tc Dfd expression is absent (or considerably weaker) in the distal part of the maxillary palps throughout embryogenesis (see fig.4.3G,H and fig.4.5E). Tc Dfd expression retracts from the developing endites on the limb buds as soon as they form (star in fig.4.3C), which develop on the ventral-proximal region. Tc Dfd continues to be repressed (see fig.4.3D-F) until only weak expression of Tc Dfd remains on the lateral side of the mandible (see fig.4.3G,H, fig.4.4D,F and fig.4.5C-D).
Using Tc prd as a marker for endite development shows that the ventral medial region of the mandibular limb bud where Tc Dfd expression is lost encompasses the mandibular endite and the region around it. Tc Dfd expression is retained in the lateral part of the mandibular limb bud, but fades throughout embryogenesis (fig.4.5).
Expression patterns of Tc cnc have been previously described (Economou and Telford, 2009). Tc cnc is expressed in two distinct domains, an anterior cap that includes the developing labrum and stomadeum and a posterior collar domain in the mandibular segment (see fig.4.1B). Tc cnc expression remains constant in these two domains from their first appearance during germ band elongation through late embryogenesis (see fig.4.2 and fig.4.3).
Fig.4.1. Tc Dfd and Tc cnc expression in the mandibular and maxillary segments of Tribolium embryos.