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Expression of cnc in diverse mandibulate arthropods such as the myriapod Glomeris and other insect species suggests that the mandible patterning function of the posterior domain of cnc function is conserved in mandibulates.
In Tribolium it has been shown that Tc Dfd patterns the mandible and the protopodite of the maxilla (Brown et al., 2000). mxp (the pb orthologue) patterns the palps of both the maxilla and labial appendages (DeCamillis et al., 2001). Cx (the orthologue of Scr) patterns the protopodite of the labial appendage and the first thoracic segment (Curtis et al., 2001).
The expression patterns of Dfd, pb and Scr in particular segments and parts of appendages are conserved in numerous arthropod species. This suggests that the patterning of the mandible and maxilla in different taxa is conserved. But there are also notable differences.
Some of these differences clearly relate to the derived structure of some gnathal appendages, for example, numerous maxillary appendages have lost the palp and therefore have lost pb expression. Other differences are harder to account for and may represent derived states. For example, there is diversity of Scr expression patterns across mandibulates.
From a consideration of the expression of these genes, the hypothetical expression of these genes in a non-mandibulate and a mandibulate ancestor to all mandibulate arthropods and will be described (shown in fig. 7.6B,C).
Conserved expression of Dfd in maxillary protopodites
Dfd expression is conserved in numerous insect, crustacean and myriapod species. Dfd expression is located in the mandibular and maxillary segments. Typically there is a retraction of Dfd expression during development in the mandibular endite that suggests cnc represses Dfd in the mandibular appendage as described in chapter four (Rogers et al., 2002) (Kokubo et al., 1997; Abzhanov and Kaufman, 1999a;
Walldorf et al., 2000; Hughes and Kaufman, 2002b; Rogers et al., 2002; Mito et al., 2008).
In the majority of these species, Dfd is expressed in the mandibular and maxillary segments. Expression is strongest in the maxillary protopodite and repressed from the mandibular limb bud, although there are exceptions. In the crustacean Porcellio, Dfd expression is limited to the mandibular segment. The hemipteran Oncopeltus, that possesses derived styllate mouthparts and has lost the mandibular endite, has ubiquitous expression of Dfd throughout the appendage (Hughes and Kaufman, 2000; Rogers et al., 2002).
In Lithobius, the homologous segment to the labial segment is the second maxilla. The appendages on this segment are fused to the sternites to form a single coxosternite. Interestingly, Dfd is expressed in the protopodite of the second maxilla appendage as well as the mandible and the first maxilla.
A recent study investigating Dfd expression in an onychophoran Euperipatoides shows that Dfd is expressed in the proximal region of each walking limb bud (Eriksson et al., 2010). This result suggests that Dfd expression in the base of the mandibular and maxillary limbs is ancestral (see fig. 7.5A).
However, functional studies on other mandibulates are required to prove the hypothesis that the protopodite patterning function of Dfd represents the ancestral state. Crustacean taxa are particularly poorly sampled, especially considering the diversity of gnathal appendages present.
Conserved expression of pb in maxillary telopodites
pb expression is conserved in maxillary telopodites which suggests that it is required for patterning these structures (Abzhanov and Kaufman, 1999a; Hughes and Kaufman, 2002b; Rogers et al., 2002; Janssen and Damen, 2006).
Although pb orthologs are commonly expressed in the telopodites of maxillary appendages, there are exceptions, which may relate in part to the morphology of the maxillary appendages found in those species. A number of studied organisms do not have maxillary palps and consequently do not have pb expression in the maxilla. In the case of Glomeris, the maxillary appendages have evolved into a structure called the gnathochilarium which consists of maxillary appendages which have lost their palps and fuse ventrally. A similar situation exists in the Isopod Porcellio, the maxilla has lost its palp, or it has become highly reduced. pb has a small domain of expression in the second antennal segment of Porcellio and no expression in the maxillary appendages (Abzhanov and Kaufman, 1999a).17 Parhyale has a small expression domain located in the second antennal segment, however the data are not published in a peer-reviewed journal, are not annotated and the resolution of the photographs pb has additional conserved expression domains of unknown significance. The in situ hybridisations of mxp transcripts in chapter four show that mxp is expressed in the intercalary/second antennal segment and the mesoderm of the mandibular segment. Mutants for mxp do not have observed effects on these segments. lab knock down embryo phenotypes that affect the intercalary segment are difficult to interpret (Posnien and Bucher, 2010). I anticipate that loss of both the intercalary and mesodermal domains of pb results in phenotypes but the phenotypes will be difficult to detect. Considering that Dfd, mxp and Cx are able to pattern the gnathal segment appendages in a fractional or additive manner it is possible that pb will also be able to pattern mandibular mesodermal derived structures in an additive manner.
The intercalary/second antennal segment expression domain of pb is primitive as this expression domain is conserved in non-mandibulate arthropods. In the spider, pb homologues are expressed in the pedipalp segment which is homologous to the intercalary/second antennal segment of mandibulates, shown in fig.7.5B (Telford and Thomas, 1998b; Abzhanov et al., 1999; Schwager et al., 2007).
In the onychophoran Euperipatoides, an outgroup to the arthropods, the anterior boundary of the pb homologue is expressed in the slime papilla segment (fig
7.5A), which is the homologous segment to the intercalary/second antennal segment (Eriksson et al., 2010). In terms of the colinearity of Hox gene expression this makes sense as the pb gene is the second Hox gene in the Hox cluster (starting from the 3’end) before Dfd and Scr and is expressed more anteriorly to these genes (Hughes and Kaufman, 2002a).
Expression of Scr
Scr has a more diverse expression pattern than that of other anterior Hox genes, but it is likely that the ancestral anterior limit of expression is in the labial/second maxilla segment (homologous to the third leg segment of chelicerates).
Scr in crickets, centipedes and firebrats is expressed mainly in the labial appendage (and the lacinea endite of the maxilla of insects) (Rogers et al., 1997;
Hughes and Kaufman, 2002b). Protein expression of Scr is present in early embryonic stages of the mandibular appendage of Thermobia (Passalacqua et al., 2010). Scr has is quite poor. Serano, J. M. (http://patelweb.berkeley.edu/JuliaSerano.html). It is clear however that there is no expression in the maxillary segments.
broad expression domains in crustacean species, including the maxillary segments and the maxilliped/first thoracic appendage (Abzhanov and Kaufman, 1999a; Abzhanov and Kaufman, 1999b; Abzhanov and Kaufman, 2000a). In the myriapod Glomeris, Scr is expressed in all segments posterior to the mandibular segment (Janssen and Damen, 2006).
Despite this variation of expression, the ancestral anterior limit of expression is likely to be in the fourth post-antennal appendage, homologous to the labial appendage. The anterior boundary of expression of Scr in chelicerates is in the third leg segment (Telford and Thomas, 1998b; Abzhanov et al., 1999; Schwager et al., 2007).
Therefore the expression of Scr in the mandibular and maxillary segments of some mandibulate arthropod species is likely to be derived.
Hox3, the homologue of zen (which has lost its Hox gene function), is expressed in the mesoderm of the developing mandibular appendage of some mandibulates in a Hox-like manner. In Daphnia Hox3 is expressed in the developing mandible (Papillon and Telford, 2007) Hox3 is expressed in the mesoderm of the mandibular and maxillary segments in Thermobia (Hughes et al., 2004) and Glomeris (Janssen and Damen, 2006).
In Lithobius, Hox3 as well as expression in the mandibular segment, is expressed in the intercalary segment during early embryogenesis (Hughes and Kaufman, 2002b).
The function of Hox3 has not been studied in any organism however, considering the mesodermal expression of Hox3 and the conserved ectodermal expression of cnc it seems unlikely that Hox3 will be regulated by cnc, and may have an additive role patterning mandibular mesodermal derived structures.
Role of gnathocephalic Hox genes in chelicerates (and onychophorans) There has been no functional study of anterior Hox gene function in the chelicerates. Expression of the Hox genes in chelicerates is characterized by broad overlapping domains that have their posterior limit at either the posterior of the Fig.7.5 Expression of homologs of Dfd and pb in two outgroups of Mandibulata, the Onychophora and Chelicerata. Onychophoran expression pattern is based on Euperipatoides from Eriksson et al. (2010).
Chelicerate expression is based on Cupiennius and Achaearanea from Schwager et al. (2007) and Abzhanov et al. (1999). The anterior expression of pb is present in the pedipalp (Pp) segment and the slime papilla (SP) segment which are homologous. The anterior expression of Dfd homologues is present in the first leg segment (L1). (A) Expression of Dfd and pb in an onychophoran Euperipatoides. The homologue of Dfd is expressed more strongly at the base of the first walking leg and all segments posterior to it. The pb homologue is expressed in the mesoderm of slime papilla and all segments posterior to it. (B) Expression of the two Dfd homologues, Dfd-1 and Dfd-2, and the homologue of pb in chelicerates.Dfd-1 is expressed in the tips of the walking leg segments. Dfd-2 is expressed in the remaining part of the leg appendages and the ventral ectoderm.
prosoma or the opisthosoma (see fig. 7.5B). In developing spider embryos, the expression of Hox genes is often specific to different parts of limb which is suggestive of additive patterning functions like those revealed in Tribolium. The function of the Hox genes expressed in the prosoma has not been tested to date.
There are two Dfd homologues in spiders. Dfd-1 is expressed in the tips of the L1 to L4 appendages (as shown in chapter six). Dfd-2 is expressed throughout the L1 to L4 ventral ectoderm and the appendages but is mutually exclusive with Dfd-1 and excluded from the distal tips of the leg appendages. Consideration of the conservation of proximal appendage expression domains of onychophoran (see fig. 7.5A) and mandibulate Dfd Hox gene expression patterns suggests that the spider condition is derived. Interestingly, the L1 to L4 segments of spiders have lost endites on these appendages, and are derived in that respect. One chelicerate group, the xiphosurans (horse shoe crabs) possess endites on their leg segments. Considering that the mandible is a modified endite it would therefore be interesting to study expression of Dfd in representatives of this clade. As I have hypothesized above that cnc acquired a function to differentiate the mandibular endite from the maxillary endite, it would be interesting to see if cnc is expressed in the endites of the leg appendages of horse shoe crabs.
Originally, it was planned that the homologues of Dfd and cnc in the spider Achaearanea would be knocked down by RNAi to test their function. However, RNAi proved difficult in the spider. As a positive control for the success of RNAi in the spider, numerous attempts were made at knocking down the orthologue of At Dll. However, the experiment was not successful and so RNAi of Dfd or cnc was not pursued further.
The role of cnc in the ancestor to all mandibulates