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Out of several isoforms, CncB is necessary to pattern mandibular structures and sufficient to pattern anterior mandibular structures derived from the hypopharyngeal lobes. CncB represses both Dfd transcription and Dfd activity. Repression of Dfd activity was detected by studying the effect of CncB on known Dfd response elements (DRE), such as the DREs of the Dfd autoregulatory loop. However, numerous experiments strongly point to the role of CncB as an activator.
cnc represses the activity of the Dfd autoactivation circuit in the anterior mandibular segment, which is necessary for maintenance of Dfd expression.
Repression of Dfd occurs in the anterior of the mandibular gnathal lobe precisely at the same time when the Dfd autoactivation circuit is activated in other tissues (McGinnis et al., 1998).
CncB forms a heterodimer with a small Maf protein and binds a cnc/mafS binding site sequence (CBS) which is conserved in numerous species. The precise manner in which cnc represses Dfd is not known. No CBS sequences have been found near the Dfd locus, known DREs or any other gene to date. It is therefore likely that CncB represses Dfd indirectly.
Veraksa et al. have shown that ectopic activation of cnc results in a reduction or loss of maxilla segment derived structures and a gain of ectopic hypopharyngeal lobe derived structures (Veraksa et al., 2000). They claim that there are no ectopic mandibular structures as they interpret the hypopharyngeal lobes as derived from the intercalary segment. However, the hypopharyngeal lobes have recently been shown to be derived from the mandibular segment (Economou and Telford, 2009). In which case, cnc does result in the production of ectopic mandibular segment derived structures.
The role of cnc in the mandibular segment of mandibulates may include an important role as an activator that patterns specific mandibular segment structures.
One direction of research would be to identify which genes are targets of cnc in Tribolium. One gene that is an obvious candidate based on its mandibular specific endite expression domain is Tc prd. In fact, every gene that is differentially expressed in the mandibular compared to the maxillary segment or any appendage type is a candidate for Tc cnc regulation.
Molecular mechanism of cnc function in Tribolium
Tc cnc represses Tc Dfd transcription in the mandibular lobe of Tribolium just as cnc represses Dfd in the mandibular segment of Drosophila. Based on experiments performed in Drosophila, Tc cnc probably represses Tc Dfd function in a manner similar to that of Drosophila. Tc cnc is likely to have an activating function and therefore to repress Tc Dfd indirectly. The cnc/maf binding sequence (CBS) is highly conserved between diverse taxa although no cnc dependent genes have been discovered (Veraksa et al., 2000). There are no hypopharyngeal lobes in Tribolium, but there are mandibular appendages with endites, which may be patterned by cnc.
It is likely that Tc cnc will repress the autoregulatory loop of Tc Dfd. Expression of Tc Dfd in Drosophila activates the autoregulatory loop of endogenous Dfd (Brown et al., 1999). The dynamics of Tc Dfd expression are similar to those in Drosophila.
Expression of other Hox genes in the mandibular segment
The Hox gene hox3 is expressed in the mandibular segment of some mandibulates outside of higher insects, and may therefore be an important mandible patterning gene. For example Hox3 is expressed in the mesoderm of the developing mandible of the apterygote Thermobia (Hughes et al., 2004), the crustacean Daphnia pulens (Papillon and Telford, 2007) and the myriapod Lithobius (Hughes and Kaufman, 2002b).
However, at present, no Hox3 homolog has ever been subject to functional analysis, so the role that Hox3 might play in embryo development is unknown. It is conceivable that given the diversity of mandibular structures that it may be involved in the development of mandibles of particular organisms.
pb is commonly expressed in the mesoderm of the mandibular segment as well as in the telopodites of developing maxillary appendages. To date no function of pb has been discovered that relates to patterning the mandibular segment. In Tribolium, mxp, the Tribolium ortholog of Pb, is expressed transiently in the mesoderm of the mandibular segment during germ band elongation in Tribolium (Shippy et al., 2000b), but later fades. mxp is also expressed in the mesoderm of the mandibular protopodite.
mxp mutants do not have any observable mandibular segment defects (Rusch and Kaufman, 2000). Scr is also expressed in the mandibular appendage of some species.
Scr is expressed in the mandibular appendage of Thermobia (Passalacqua et al., 2010).
cnc and the evolution of the mandible from a maxilla-like precursor
In some respects, the manner in which cnc functions in Tribolium and Drosophila to differentiate the mandible from a maxilla reflects the evolutionary history of the mandibular appendage. The fossil record indicates that the mandible evolved from a biramous limb and evolved through a maxilla-like appendage. The postantennal limbs of numerous stem lineage arthropods were serially homologous and undifferentiated from each other. Examples of plesiomorphic serially homologous biramous limbs are present in the Cambrian arthropods Ercaia (Chen et al., 2001) and Martinssonia (Muller and Waloszek, 1986).
As stem lineage arthropods diverged during the course of evolution in the Cambrian, post-antennal biramous limbs diverged from the primitive biramous limb structure. The second post-antennal segment, homologous to the mandibular segment, evolved from a biramous limb with a well-defined and sclerotized proximal endite similar to those present on the third and fourth post-antennal segments. At some point the characteristic mandibular gnathal edge evolved on the second postantennal appendage and had differentiated from a maxilla-like precursor into a characteristic mandibular appendage.
If cnc shares a similar mandible patterning function by differentiating the mandible from a maxilla across Mandibulata, it suggests that cnc acquired a new role to differentiate the mandibular appendage from the maxillary appendage in the ancestor to all mandibulates. The similarities of the expression patterns of cnc and Dfd across mandibulates support the notion of a conserved mandible and maxilla patterning mechanism.
It is clear from these results that Tc cnc differentiates the mandible from a maxilla by repressing maxilla patterning function of genes such as Tc Dfd and mxp. The primitive mandible would have resembled the primitive maxilla and possessed two rami, an exopodite-derived ramus and a telopodite-derived ramus (or palp). However, the defining characteristic of the primitive mandibular appendage shared by mandibulate arthropods is the mandibular endite with a molar and incisor process as both the mandible and maxilla possessed palps.
Tc Dfd patterns the protopodite of the maxilla in Tribolium. Tc cnc differentiates the mandibular protopodite from the maxillary protopodite. This differentiating role of cnc to pattern the mandibular protopodite may have been acquired in the ancestor to the Mandibulata.
Considering the similarity of the expression domains in other mandibulates, it is reasonable to expect a similar situation to occur in other species. Given that the mandible has likely evolved from a maxilla-like appendage, the role of cnc could have evolved to include a new function to differentiate a maxilla-like precursor into a mandibular appendage.
The mandibular patterning function of Tc cnc therefore reflects the history of the evolution of the mandible from a maxilla-like precursor. The new role of cnc is likely to include two aspects, that of repressing maxilla patterning Hox genes (such as Dfd and mxp in Tribolium) and that of patterning mandibular specific structures.
To determine whether Tc cnc patterns mandibular segment structures it would be necessary to ectopically express Tc cnc in another segment in Tribolium. This would require the development of genetic techniques such as heat shock transcriptional induction of ectopic gene activation, and mitotic clonal analysis.
The role of cnc in repressing telopodite development in the mandible of Tribolium is not primitive as the primitive mandibular appendage possessed two rami.
If the mandibular differentiating function of cnc is conserved in hexapods, the telopodite repressing function (repression of mxp in Tribolium) has been acquired at some point in the lineage leading to the hexapods. It would be interesting to examine the function of cnc in mandibulates that possess mandibular palps in order to see if cnc differentiates the protopodite of the mandibular appendage. It would also be interesting to study the mandibular appendage of myriapods and crustaceans which have also lost the mandibular palps to determine whether cnc has convergently evolved a similar function of repression of the telopodite patterning genes in the mandibular segment.
cnc in closely related outgroups to Mandibulata
In closely-related outgroups to Mandibulata, such as the chelicerates and onychophorans there is no obvious differentiation between the first leg appendage and the second leg appendage which are homologous to the mandibular and maxillary segments of Mandibulata (Damen et al., 1998; Telford and Thomas, 1998b; Eriksson et al., 2010). If it is shown that the characteristic posterior domain of cnc expression is lacking from the first walking leg segment of these groups, it would provide evidence that the differentiation of the mandibular from the maxillary segment by cnc is specific to the mandibulates and is a synapomorphy of that group. This is examined in chapter six.
Tc Dfd patterns the protopodite of the maxilla and the mandible (which is a protopodite structure). Therefore, in order to understand how Tc cnc differentiates a mandible from a maxilla protopodite, and what genetic interactions Tc cnc is likely to have in the mandible, I studied the genetic interactions of Tc Dfd in the maxillary segment. The results of these experiments are shown in the next chapter.
6 Chapter 5:The protopodite patterning role of Tc Dfd in the maxilla.
5.1 Introduction The previous chapter demonstrated that Tc cnc is necessary to differentiate the mandible from maxillary identity and represses the Hox genes Tc Dfd and mxp, which are required to pattern the maxillary appendages. In this chapter I wanted to explore the role of Tc Dfd in patterning the protopodite of the maxilla, to study the precise morphological phenotype of the affected maxillary appendage and the downstream target genes of Tc Dfd in the maxillary protopodite.
Tc Dfd, although repressed by Tc cnc in the mandibular segment, is required to pattern the mandibular segment in two key respects; repressing antennal development and activating the posterior collar domain of Tc cnc which is required to specify the mandible. Interestingly, in Tc Dfd mutants the maxillary appendages lose the endites but otherwise maintain maxillary identity showing that Tc Dfd specifically patterns the endites of the maxillary appendage (Brown et al., 1999; Brown et al., 2000). This phenotype suggests that Tc Dfd may also have an endite patterning role in the mandibular appendage, as the mandible is in possession of an endite.
However, in Tc Dfd mutants, the mandible is transformed to antennal identity in the absence of Hox gene expression (Brown et al., 1999; Brown et al., 2000). As the mandible is transformed into antennal identity, it is not possible to study the endite or protopodite patterning role of Tc Dfd in the mandible, as Tc Dfd target genes will display antennal specific expression, as well as possible repression of genes that are not involved in patterning the antenna. However, it is reasonable to hypothesize that Tc Dfd performs a similar function in the mandibular segment to the maxillary segment and that Tc cnc modifies expression of these genes to mandible segment specific domains of expression.