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Significantly, the labrum is also deleted in Tc cncRNAi embryos in a manner analogous to gap genes. In addition there are also possible abdominal effects, in some cuticle preparations of first instar larvae it was apparent that the abdomen was misshapen. Injection of Tc cnc dsRNA significantly increases mortality of adult beetles.
In Drosophila cnc Protein isoform C is known to be involved in responses to oxidative stress (Grimberg et al., 2011).
Comparison with Drosophila – similarities
There are numerous similarities between the roles of cnc in Tribolium and Drosophila. In both species cnc is required for development of labral and mandibular segment derived structures. With the loss of cnc function there are additional maxillary structures in place of mandibular structures, indicating a homeotic transformation has occurred.
The Hox gene Dfd is repressed by cnc in developing embryos. In Tribolium, knock down of Tc cnc ablates repression of Tc Dfd in the mandibular segment. This result indicates that Tc cnc represses Tc Dfd transcription and is able to regulate Tc Dfd upstream in Tribolium. The situation is similar in Drosophila, where cnc represses Dfd in the anterior of the mandibular lobe, with some Dfd expression remaining in the posterior of the segment.
Dll is also repressed by cnc in both species, although with some differences. The genetic interaction between cnc and Dll is likely to be indirect. Dfd has been shown to activate the proximal domain of Dll in Drosophila (Regulski et al., 1987; McGinnis et al., 1998; Brown et al., 2000). Repression of Dfd by cnc prevents activation of this proximal domain of Dll by Dfd in both species, so cnc indirectly represses the proximal domain of Dll by inhibiting its activation by Dfd.
In Tribolium Tc cnc represses the ectodermal expression of mxp. In the maxilla mxp is expressed in the palp and in the distal part of the protopodite including the endite of the developing galea. In the transformed mandibular appendage, mxp expression is observed both in the palp up to the distal part of the protopodite and the mesoderm.
cnc represses pb in Drosophila, as the homologue of cnc does in Tribolium. pb is expressed in the distal domain of the maxillary lobe, which is homologous to the distal domain of the Tribolium maxillary lobe which becomes the palp. In cnc null mutants, there is ectopic expression in the posterior ectoderm of the mandibular lobe, although not a complete recapitulation of the maxillary expression domain of pb in the mandible. However, pb does not have any discernable function in the Drosophila embryo, it is plausible that the expression of pb in the distal domain of the maxillary lobe regulated by cnc is a remnant of when pb did play a role in patterning structures of the distal (or distal) domain such as the maxillary palp.
In both species, there is a cnc domain free of Dfd expression in the mandibular segment where Dfd expression is repressed. In Tribolium, Tc Dfd is repressed in the part of the mandibular limb bud which relates to the position of the developing endites. In Drosophila the Dfd-free domain includes the anterior of the mandibular gnathal lobe and the hypopharyngeal lobes which are situated anterior to the mandibular lobes. In both species, some Dfd expression remains in the developing mandibular lobe. In Tribolium, Tc Dfd expression remains in the lateral part of the mandibular lobe. In Drosophila, Dfd expression remains in the posterior part of the mandibular lobe.
Whether these domains are homologous between Tribolium and Drosophila is difficult to say, as there are significant morphological differences between the two, in particular the Drosophila mandibular lobe is flatter and pressed against the rest of the embryo (in two dimensions). In contrast to Tribolium the mandibular lobe is developing into an appendage and is raised above the embryo (in three dimensions). It is possible that these two domains are homologous, one domain with solely cnc expression, the other domain with cnc and Dfd co-expression. But given that fly larvae morphology is highly derived, it is unreasonable to interpret homology on the basis of similar gene expression alone.
More likely is that there is homology between these two domains, but that Drosophila embryo has evolved to include new structures such as the hypopharyngeal lobes and lose other structures such as the developing endite lobes.13 Comparison with Drosophila – differences In Drosophila, loss of cnc function does not result in a full homeotic transformation of the mandibular gnathal lobe to maxillary identity. This is in contrast to Tribolium where loss of Tc cnc function results in a complete transformation of mandible to maxillary identity.
In Drosophila, the mandibular gnathal lobe is transformed into the proximal part of the maxillary gnathal lobe, not the entire maxillary gnathal lobe. Structures which derive from the distal part of the maxillary gnathal lobe (like the maxillary sense organ) are not ectopically produced, only proximal derived maxillary structures are produced (like the mouth hooks and cirri). The proximal part of the Drosophila gnathal lobe is supposed to be homologous to the proximal part of the Tribolium maxillary appendage (Jurgens et al., 1986).
Consistent with the above, only the ectopic maxillary ventral domain of Dll is expressed in the mandibular segment of cnc mutant embryos, and not the distal domain of Dll (McGinnis et al., 1998).
In Tribolium, loss of Tc cnc functions results in a complete homeotic transformation of the mandibular appendage to maxillary identity. This is evident in the structure of the transformed mandible which resembles a typical maxillary appendage, complete with a palp and endite.
Tc cnc represses both the proximal and distal maxillary domains of Tc Dll in the mandible. The proximal domain of Tc Dll is activated by Tc Dfd (Brown et al., 2000), the telopodite domain of Tc Dll is likely to be regulated by the Hox gene mxp. cnc is regulating the two domains of Tc Dll by repressing the Hox genes Tc Dfd and mxp.
In Drosophila¸ the reason for the lack of a full homeotic transformation may stem from the loss of any role of pb in patterning the developing embryo. pb is prd can be used as a marker for the homologous region in Drosophila which gives rise to endites in Tribolium: endites are Dfd dependent and from the proximal domain. Hypopharyngeal lobes are not homologous to endites as they are from the anteior of the developing mandibular lobe not the proximal part. Therefore the mandibular endites are probably not homologous to the hypopharyngeal lobes.
expressed in the distal region of the maxillary appendage. cnc represses pb in Drosophila embryos, As pb does not have any function in the developing Drosophila embryo, there is no effect on the distal domain of Dll expression or of ectopic distal maxillary structures present in the transformed mandibular lobe. As a result, cnc represses Dfd and pb, but only the proximal maxillary domain of Tc Dll is ectopically expressed in the mandibular segment. Only the Dfd-dependent domain of Dll expression is affected by cnc mutants.
In Drosophila, the mandibular segment collar domain of cnc is not activated or regulated by Dfd or by any other Hox gene (Mohler, 1993). The situation is different in Tribolium. The posterior collar domain of Tc cnc in the mandibular segment is activated by Tc Dfd. In Tc Dfd knock down embryos the posterior collar domain of Tc cnc is lost.
Tc cnc requires Tc Dfd to pattern all mandibular structures.
In Drosophila, cnc requires Dfd to pattern some mandibular structures but is activated independently of Dfd. Structures derived from the posterior mandibular segment require Dfd. There are other Dfd-independent structures which derive from the hypopharyngeal lobes which do not require Dfd (Mohler et al., 1995; McGinnis et al., 1998; Veraksa et al., 2000). The significance of the dependence of cnc on Dfd for patterning mandibular structures in Drosophila and whether there are any comparable mechanisms in Tribolium is not known.
Another difference between Drosophila and Tribolium is the presence of hypopharyngeal lobes which are derived from the anterior of the mandibular segment in Drosophila. The hypopharyngeal lobes are derived structures that are specific to dipterans and are not present in Tribolium. cnc and a gene called collier (col) are both required to pattern the hypopharyngeal lobes (Mohler et al., 1995; Seecoomar et al., 2000; Economou and Telford, 2009). col has been argued to have a conserved role in patterning the boundary between the intercalary segment and the mandibular segment that is specific to insects (Schaeper et al., 2010). The anterior mandibular domain of cnc is upstream of col in Drosophila (Crozatier et al., 1996; Crozatier et al., 1999; Seecoomar et al., 2000). In Tribolium, it has been recently shown that Tc cnc is not activated by Tc col (Schaeper et al., 2010). Therefore the hypopharyngeal lobe patterning function of cnc and the activation of cnc by col in the anterior mandibular segment of Drosophila is a derived condition that reflects the differences between the anterior mandibular segment of dipterans and jaw bearing mandibulates.
The role of cnc in mandibulates
In mandibulate arthropods that have been studied, the expression patterns of cnc and Dfd are highly conserved. In insects and the myriapod Glomeris marginata, cnc is expressed in two characteristic domains a labral cap domain and a mandibular collar domain. Evidence is lacking from crustacean species. However, considering the phylogenetic position of the crustaceans relative to the insects and myriapods, it is expected that cnc will be expressed in a similar manner to both groups.
Dfd is expressed in the mandibular and maxillary segments of all mandibulate arthropods. The expression of Dfd is highly conserved across insects. Dynamic expression of Dfd expression in the mandibular limb bud, particularly the developing endite, appears to be conserved across mandibulates. The fact that cnc expression and the repression of Dfd expression from the mandibular endite are widely conserved strongly suggests that the mandibular segment patterning mechanism of Tribolium and Drosophila is conserved across mandibulates. It remains to be tested whether cnc will differentiate the mandible from the maxilla in all mandibulates, particularly crustaceans and myriapods, by repressing the Hox genes which are involved in patterning the maxillary segment.
cnc may function as an activator to pattern mandibular structures
Research in Drosophila has shown that despite the importance of cnc for repressing Hox gene transcription and function, cnc has been shown, as a transcription factor, to function as an activator. The role that cnc plays in patterning the mandibular segment in mandibulate insects is likely to be different from Drosophila as Drosophila has lost the appendage of the mandibular segment. In the mandibular segment of mandibulates such as Tribolium that have a mandibular appendage, cnc may be required to pattern and differentiate the mandible from the maxilla by the activation of mandible patterning genes.
Comparison with Drosophila - Molecular Mechanism of cnc regulation of Dfd
Tc cnc differentiates the mandible from the maxillary segment and represses the Hox genes Tc Dfd and mxp in Tribolium. This is very similar to what occurs in Drosophila. Despite the detailed genetic studies which have been performed in Drosophila on the manner in which cnc alters Dfd transcription and function, the precise mechanism by which cnc functions to repress Dfd function and promote development of mandibular segment derived structures is not known. There are however some conclusions that can be drawn, and some implications for the role of cnc in mandibulate arthropods (Mohler, 1993; Mohler et al., 1995; McGinnis et al., 1998; Veraksa et al., 2000).
cnc was originally thought to act in a manner similar to a homeotic gene, with cnc and Dfd acting in a combinatorial manner to pattern the mandibular segment (Mohler et al., 1995; Rogers et al., 2002). As cnc represses the expression and activity of Dfd in the mandibular segment, cnc was reinterpreted to function as a Hox modulator gene (McGinnis et al., 1998). However, detailed experiments to test the role of cnc as a transcription factor have surprisingly shown that it functions as an activator and that the repression of Dfd is likely to be indirect (Veraksa et al., 2000).