knirps genes as novel players in arthropod head development

Transcription factors encoded by the knirps (kni) gene family constitute a subgroup within the nuclear hormone receptors. Members of this family are uniquely characterised by a shared protein domain encoded by the kni-box (Nauber et al., 1988; Rothe et al., 1989).

Because for none of the kni-encoded hormone receptors that have been analysed so far, ligands were found, this group has also been referred to as “orphan receptors” (Arnosti et al., 1996; Laudet et al., 1992). To date, detailed studies have been done on the Drosophila kni repertoire, consisting of Drosophila knirps (kni, Nauber et al., 1988), knirps-related (knrl, Oro et al., 1988) and eagle (eg, Rothe et al., 1989). Drosophila melanogaster kni is a zygotically expressed gap gene. Its function is necessary for the establishment of posterior segments. Loss of kni function results in the deletion of adjacent abdominal segments, specifically the A2–A6 segments (Nauber et al., 1988). Although it is co-expressed with knrl in a broad anterior domain covering the ventral and lateral parts of the embryonic head during blastoderm stage, loss-of-function does not impede head regionalisation or cause head gap phenotypes. Instead, disruption of both kni and knrl function in the anterior part of the Drosophila embryo leads to severe defects in the differentiation and projection of the labral neuronal elements and the stomatogastric nervous system (Gonzalez-Gaitan et al., 1994). In addition, Drosophila kni and knrl share redundant functions in tracheal placode development (Chen et al., 1998). Specifically, they mediate DPP signalling and coordinate opposing DPP and EGF signals. These functions are required for the establishment, maintenance and morphogenesis of individual tracheal branches (Myat et al., 2005). Drosophila kni and knrl also organise the second wing vein (Lunde et al., 1998). Drosophila eg expression is restricted to the late embryonic gonads (Rothe et al., 1989).

Tribolium possesses two kni family genes, Tc’kni and Tc’eg (e.g., Xu et al., 2010).

Tc’kni is the single ortholog of both Drosophila kni and knrl (Cerny et al., 2008), suggesting that the Drosophila genes derive from a gene locus duplication event specific for the lineage leading to higher dipterans. Like Drosophila kni and knrl, Tc’kni shows early zygotic expression in an anterior gap-like domain during blastoderm stage. In contrast to Drosophila

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kni and knrl, loss of Tc’kni function leads to the deletion of the antennal and mandibular segments. This is a much more severe effect than even the combined loss of Drosophila kni and knrl function in head development. Posterior Tc’kni expression is restricted to the prospective first abdominal segment. However, RNAi-mediated loss of Tc’kni function results in only mild abdominal defects that manifest posterior to its expression domain. Tc’kni is expressed later during Tribolium development in the distal tip of the labrum and, similar to Drosophila kni and knrl, in the putative tracheal placodes and branches (Cerny et al., 2008).

Tc’eg is maternally expressed. It is localised anterior in the embryo. However, RNAi-based loss-of-function of Tc’eg does not result in early anterior patterning defects (Bucher et al., 2005).

Two kni paralogs have been isolated from the milk weed bug Oncopeltus fasciatus.

Early anterior expression has also been found for Oncopeltus kni1, as well as additional expression in central and posterior stripes of the embryo. However, no function of Oncopeltus kni genes has been discovered so far (Jonas Schwirz, personal communication; Ben-David and Chipman, 2010)

Aside from Drosophila, Tribolium and Oncopeltus, kni genes have been isolated from or are present in the genomes of various other arthropod species, more precisely several insects (Ceratitis capitata, diptera, Jonas Schwirz, personal communication; Nasonia vitripennis and Apis mellifera, hymenoptera, 3.4.6, A6.2), crustaceans (Daphnia pulex, cladocera, branchipoda, http://wfleabase.org/; species of the Caligus genus, copepoda, maxillopoda, 3.4.6, A6.2), a myriapod (Strigamia maritima, Carlo Brena, personal communication) and a chelicerate species (Ixodes scapularis, acari, arachnida, 3.4.6, A6.2).

So far, kni genes have not been found in species outside the clade of arthropods. In particular, no sequences similar to kni-box sequences can be found in the sequenced genomes of non-arthropods (3.4.6). Therefore, it is highly probable this group of genes has evolved within arthropods.

Based on known expression data of kni genes in insects, a conserved role in anterior and head patterning in arthropods has been suggested (Cerny et al., 2008). However, the functional range of kni genes varies considerably among these species, as described above. In addition, kni genes appear to conveying pleiotropic functions in arthropod development and to be involved in many diverse developmental processes. To find out whether kni genes share conserved functions in arthropod head development, a close examination of the situation in an outgroup, such as Parhyale hawaiensis, will prove extremely useful.

29 2.7 Aims of this work

To identify conserved elements of insect head development, which might derive from an ancestral molecular and genetic mechanism common to all bilaterian species, the aim of this work was to clone and analyse Parhyale hawaiensis homologs of known conserved genetic regulators of head development. In detail, I wanted to identify Parhyale homologs of the conserved anterior head gene orthodenticle (otd) along with other representatives of paired-class homeodomain encoding factors, as well as homologs of the suggested conserved otd interaction partners optix/six3 and unplugged/gbx and compare my findings to the situation in insects.

In addition, I sought to isolate Parhyale representatives of the knirps gene family, which appear to be an evolutionary novelty of arthropods and convey pleiotropic functions in the insects Drosophila melanogaster and Tribolium castaneum. Here, my aim was to examine whether the divergent roles of insect knirps genes in head development might share or derive from an ancestral, conserved function.

As a prerequisite on which I could base my comparative molecular and genetic studies, I needed to further examine and describe the embryologic and morphological implications of Parhyale hawaiensis head development in detail.

Parhyale hawaiensis represents a promising emerging model system for malacostracans and arthropods in general. Many embryologic, genetic and molecular techniques had been established. However, reliable functional techniques had not been available at the beginning of this work. Therefore, the final aim of my work was to test the efficacy of various functional approaches in Parhyale. In particular, I focused on developing loss-of-function techniques based on RNA interference.

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3 Results