Candidate Gene Screen in the Red Flour Beetle Reveals as Ancient Regulator of Anterior Median Head and Central Complex Development

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Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.

Published in the journal: Candidate Gene Screen in the Red Flour Beetle Reveals as Ancient Regulator of Anterior Median Head and Central Complex Development. PLoS Genet 7(12): e32767. doi:10.1371/journal.pgen.1002416
Category: Research Article
doi: https://doi.org/10.1371/journal.pgen.1002416

Summary

Introduction

The insect head is composed of several fused segments, the number of which remains disputed (e.g. [1]–[6]). The posterior labial, maxillary and mandibular segments are patterned by the well-studied trunk segmentation cascade. In contrast, the patterning of the procephalic region (intercalary, antennal, ocular segments and anterior non-segmental region) is less well understood. It has been suggested in the fruit fly Drosophila melanogaster that the head gap-like genes orthodenticle (otd), empty-spiracles (ems), buttonhead (btd) and sloppy-paired (slp) activate segment polarity genes directly or via second order regulators [7]–[9] but an instructive role could not be confirmed for btd and ems [10], [11]. Moreover, the segment polarity interactions of head segments differ from those in the trunk. For example, hedgehog (hh) expression in the intercalary segment is driven by its own unique enhancer element [12], [13] (see [4] and [6] for further details).

However, the development of the larval head of Drosophila is highly derived. During late stages of embryogenesis, the head gets turned outside into the thorax (head involution) and consequently cuticular head structures are highly reduced [14]–[16]. Also the emergence of the everted adult head from imaginal discs is derived within insects [17], [18]. These morphological differences correlate with changes in embryonic pattern formation. Comparisons with other insects have revealed that the upstream levels of head formation differ profoundly (e.g. no bicoid in most insects [19], no torso signaling in head development [20], different input of decapentaplegic (dpp) on head development [21]) while some degree of conservation is found on lower levels like the head gap like genes, second order regulators and segment polarity genes ([4] and references therein). Notably, the head expression of wingless (wg) appears to be reduced in Drosophila correlating with its derived head development. Hence, the evolution of the Drosophila head involved both structural changes and alterations of the gene regulatory network [4], [6], [14], [22].

Intriguingly, orthologs of several genes required for Drosophila head patterning play a role in vertebrate neural plate patterning (e.g. otd/otx, ems/emx, slp/bf1, tailless(tll)/tlx). These data indicated that anterior patterning in insects and vertebrates relies on a strongly overlapping gene set [2], [7], [8], [13], [23]–[36]. Indeed, additional similarities between vertebrate and insect head and brain patterning have subsequently been revealed (e.g. [37]–[47]). Furthermore, an urbilaterian origin of anterior brain patterning has also been supported by similar data in an annelid [48]–[52].

The red flour beetle Tribolium castaneum has recently been established as a model for insect head development because it has an insect typical non-involuted head developing from a ventral-posterior region of the blastoderm, reflecting the ancestral situation (reviewed in [4], [53]). Several orthologs of Drosophila head patterning genes have been studied in Tribolium revealing differences with respect to the head gap-like genes [54] and knirps [55] but also a number of similarities with respect to second order regulators [56], [57]. Furthermore, genes required for vertebrate placode development were found to be active at similar locations in the Tribolium embryonic head [44].

Tc-six3 is a member of the six type homeobox gene family, which has three members in insects [58] while two paralogs of each family are found in mouse (six3/six6, six1/2 and six4/5). It is required for the formation of the labrum, an appendage of the non-segmental part of the head [3]. The Drosophila ortholog, called optix, is required for eye development [59]–[61], and for maxillary and clypeolabral structures of the larval head [62]. However, genetic interactions of this gene in the context of head development have not been analyzed in insects so far. Suggestively, the vertebrate six3 gene is essential for eye development [63]–[70] and anterior neural plate patterning [71]–[75]. Furthermore, vertebrate six3 and its paralog six6 are involved in the development of the neuroendocrine pituitary and hypothalamus [76]–[80]. As the six3 expression domain is anterior to the otd domain in arthropods, annelids and vertebrates, it is likely that this was also a feature of the last common ancestor of bilaterian animals [81].

In order to identify novel insect head patterning genes and with the high degree of conservation in mind, we comprehensively tested Tribolium orthologs of vertebrate neural plate patterning genes for a function in the insect head. Indeed, we find that many of them are required for head development. Closer examination of Tc- six3 reveals that it acts upstream of Tc-wingless (Tc-wg), Tc-otd and Tc-eyeless (Tc-ey) in anterior median head patterning. Further, we find that Tc-six3 is required for median brain development with a specific role in central body formation.

Results

18 out of 24 Vertebrate Neural Plate Patterning Genes Are Expressed in the Tribolium Head

From the literature we identified 24 genes involved in early vertebrate neural plate patterning (see Table S1) [32], [33], [41], [45], [74], [80], [82]–[127]. Three genes do not possess orthologs in either the Tribolium or Drosophila genome (Dmbx1/Atx, Vax1, Hesx1/Rpx; see phylogenetic trees in Figure S1). Tc-FGF8 does not cluster unequivocally with mouse FGF8 but with the Drosophila Pyramus and Thisbe proteins which have previously been identified as FGF8 orthologs [128], [129]. Of the 21 orthologs, Tc-BarH, Tc-Wnt11 and Tc-munster/arx are not expressed in the head anlagen (not shown), while the remaining 18 genes are active in the embryonic head. For these genes, we determined the expression pattern at several stages (determined by Tc-wg counter stain) in order to reveal their dynamics during head development. Genes that had not been described before in Tribolium are shown in Figure 1. In order to produce a comprehensive dataset with comparable staging, we also included previously described genes (Figures S2 and S3) (Tc-otd [130], Tc-ems [54], Tc-tailless (Tc-tll) [131], Tc-six3 [3], Tc-hedgehog (Tc-hh) [132], [133], Tc-cubitus-interruptus (Tc-ci) [132], Tc-wg [134], Tc-fgf8 [128], Tc-sloppy paired (Tc-slp) [135], Tc-eyeless (Tc-ey) [136], Tc-ptx [44], Tc-irx [137]).

**Fig. 1. Novel genes with anterior but not segmental expression.**

Interestingly, a number of these genes are expressed in the head but not segmentally reiterated in the trunk (Tc-otd, Tc-six3, Tc-tll, Tc-lim1, Tc-gsc, Tc-scro, Tc-rx, Tc-fez1) supporting the notion that the anterior patterning system differs from the one of the trunk. However, other genes do have segmentally reiterated expression in addition to anterior expression (Tc-hh, Tc-wg, Tc-ci, Tc-irx/mirr, Tc-fgf8, Tc-slp, Tc-ems, Tc-ey, Tc-dbx, Tc-ptx), linking these two systems.

All Genes But One Are Involved in Head Epidermis Patterning

The embryonic preantennal region gives rise to the lateral and dorsal head capsule (compare white area anterior to the dark grey shaded antennal segment in a flattened germband in Figure 2D with a non-flattened embryo depicted in E) [4], [44]. This region is marked by an invariant bristle pattern in the first instar larval cuticle [54] (see Figure 2F for most prominent bristles and Figure S4 for more details), which allows the localization of cuticle defects, which arose in pre-antennal tissues. Unfortunately, previously published RNAi phenotypes had not been scored for the head bristle pattern except for Tc-otd and Tc-ems [54] and Tc-ey/toy, where a small subset of three setae was scored [136]. Therefore, we performed RNAi for all novel genes as well for those where the head capsule defects had not been described previously. We excluded Tc-hh and Tc-wg because RNAi for these genes induces severe generalized embryonic defects, which impede the analysis of the bristle pattern (data not shown and [132], [138]).

**Fig. 2. Fate map and morphogenesis of the head.**

Tc-ci RNAi interfered with segmentation of the entire embryo as described [132]. Head defects ranged from the total loss of the head (9.1%, n = 11) to the loss and malformation of gnathal segments (90.9%; Figure 3C). Where accessible, the head bristle pattern was analyzed, revealing mainly a disruption of the vertex setae marking the dorsal tissue (Figure 3C′; the numbers for this and other bristle pattern defects are given in Table S2, the names of the setae and bristles are given in Figure S4). Knock down of the pair rule gene Tc-slp resulted in a pair rule phenotype [135] (70%, n = 10; Figure 3D). We found additional head defects in the median part of the vertex, the bell row and the maxilla escort bristles (Figure 3D′). Tc-six3 knock down leads to loss of the labrum and clypeolabral parts of the anterior head capsule [3], [62] (100%, n = 16; Figure 3E). In line with the loss of anterior median cuticle, the anterior vertex seta and the anterior vertex bristle were missing (Figure 3E′), while the median part of the dorsal head cuticle often displayed an irregular pattern of additional bristles and setae. Tc-ey and Tc-toy have been shown to act synergistically in eye formation, while the respective analysis on the head bristle pattern was restricted to 3 bristles [136]. Re-analysis of single and double RNAi revealed more extensive defects than previously described (Figure 3F, 3F′). In comparison with single RNAi experiments, double RNAi revealed a 1,4- to 6-fold increase of penetrance of bristle pattern defects using the same final concentration of dsRNA (Table S2) confirming that the two Tribolium pax6 orthologs also act synergistically in epidermis development. The head of Tc-lim1/5 RNAi larvae was compacted and shortened (16.7%, n = 12; Figure 3G). Head appendages were present but mostly malformed (41.7%). The anterior and median maxilla escort bristles failed to form (Figure 3G′), while in 20.8% no bell row was observed (Figure 3G′). In Tc-scro RNAi larvae the labrum failed to fuse either completely (60%; n = 15) (black arrowheads in Figure 3H) or partially (13.3%; not shown). The bristle pattern remained largely unaltered except for the anterior vertex bristle (Figure 3H′). Interestingly, the labrum quartet bristles were present even on unfused labra. In Tc-rx knock down larvae, the labrum was narrower than in wild type leading to a widened space between the labrum and the antennae (25%, n = 8; arrow in Figure 3I) and the adjacent clypeus bristles of the labrum quartet were lost in more than half of the analyzed RNAi larvae (Figure 3I′). Additionally, the antenna basis bristle and the median maxilla escort seta were sensitive to Tc-rx knock down (Figure 3I′).

**Fig. 3. Genes whose knock down result in severe defects in larval cuticles.**

RNAi against the remaining genes did not elicit large deletions but alterations of the head bristle pattern (Figure 4 and Table S2). Lateral defects were found after RNAi against Tc-dbx, Tc-ey single RNAi and Tc-gsc (Figure 4A–4C). Tc-ptx and Tc-irx led to dorsal defects (Figure 4D, 4E) while the bristle defects of Tc-toy, Tc-fez and Tc-tll were more widespread (Figure 4F–4H). No bristles were missing in Tc-fgf8 RNAi (n = 29), although lethality of most larvae and a bent flagellum phenotype of the antenna in 41.5% (n = 53, not shown) confirmed the RNAi effect. In summary, we showed that all vertebrate neural plate patterning genes investigated here (except for Tc-fgf8) are indeed involved in anterior head epidermis patterning in Tribolium. By and large, the cuticle defects correspond well with the location of the expression domain, but we also find some indication for indirect defects (see red circles in Figure 3 and Figure 4 and discussion for details).

**Fig. 4. Genes whose knock down results in minor defects in larval cuticles.**

Tc-six3 Is a Repressor of Tc-wg and Tc-otd1

Considering its early expression and severe RNAi phenotype, Tc-six3 was likely to play a central role in insect head patterning. Therefore, we centered our subsequent analysis on the epidermal and neural function of this gene. We tested the effect of Tc-six3 RNAi on genes that-based on our expression and RNAi data-were likely to interact. Indeed, the protocerebral neuroectodermal expression domain of Tc-wg (pne) [133] (also called the ocular Tc-wg domain or the head blob in Drosophila [2], [133]) expanded medially and anteriorly in early elongating RNAi embryos (black arrowheads in Figure 5B), resulting in massive median misexpression in fully elongated germbands (Figure 5D). The lateral aspects of Tc-wg expression appeared largely unchanged (open arrows in Figure 5C–5D). Moreover, loss of median embryonic tissue was apparent (white outline in Figure 5C–5D) including the stomodeum (see also Figure 5U) and labrum anlagen (white and black stars in Figure 5C, respectively). As a consequence, the head lobes were not bent outwards and the antennal Tc-wg stripes became perpendicular to the body axis instead of being twisted outwards as in wildtype (compare arrows in Figure 5D with 5C; see Figure S5L–S50 for phenotypes of more advanced stages where the assignment of the antennal stripe becomes obvious). The expression of Tc-otd1 was strongly expanded towards anterior and median tissue (compare Figure 5F, 5H with Figure 5E, 5G) while the lateral aspects appeared normal (open arrowheads in Figure 5G–5H). Despite being partially coexpressed (Tc-tll and Tc-scro) or expressed adjacent to the Tc-six3 domain (Tc-rx), the expression domains of these genes remained unchanged after Tc- six3 RNAi (not shown).

<i>Tc-six3</i> is an upstream regulator of the anterior median region of the embryo. — **Fig. 5. *Tc-six3* is an upstream regulator of the anterior median region of the embryo.**

Early Ocular Tc-ey/Pax6 Is Repressed by Tc-six3 While More Anterior Expression Domains Require Tc-six3 Function

The effect of Tc-six3 RNAi was different with respect to the various domains of Tc-ey (Figure 5I–5N). Tc-ey expression starts in a prominent ocular domain (open arrowheads in Figure S3 H2–3) before an additional anterior median expression arises (black arrowheads in Figure 5I, 5K, 5M and Figure S3 H4–5). We found coexpression of Tc-dachshund with parts of the anterior median domain (white arrowheads in Figure S5C–S5C″), making it possible that it marks mushroom body anlagen as in Drosophila [139], [140]. In Tc-six3 RNAi embryos, the early ocular domain was strongly expanded towards the midline (compare Figure 5J with Figure 5I). Later, a domain remained visible at the midline (black star in Figure 5N). The anterior median domain did not develop in Tc-six3 RNAi embryos (Figure 5J, 5L, 5N). Again, the lateral aspects of the ocular domain as well as the segmental domains appeared unaffected.

Tc-six3 Is Essential for the Expression of Neuroendocrine Marker Genes

six3 has been implicated in neuroendocrine development in vertebrates [80] and protostomes [81]; and in Drosophila, it is coexpressed with the neuroendocrine markers fas2 and chx [81], [141]. The functional relevance of this co-expression remained unknown. We confirmed co-expression in Tribolium (Figure S5D–S5G, S5H–S5K) and found that in Tc-six3 RNAi embryos the anterior median domains of Tc-fas2 and Tc-chx were absent (compare domains marked by black arrowheads in Figure 5O, 5Q to Figure 5P, 5R).

Tc-six3 Is Required for Development of the Central Body and the Median Brain

In insects, epidermal and neural precursor cells are intermingled in the neuroectoderm. The neural stem cells receive spatial patterning cues before they delaminate from the neuroectoderm and contribute to the central nervous system in a cell autonomous way. The remaining epidermal cells eventually secrete the cuticle [142], [143]. This explains why mutations in segment polarity genes elicit both cuticular and CNS defects. With this in mind, it was likely that Tc-six3 knock down would induce brain defects. First, we determined that eight Tc-ase marked neuroblasts are found within the Tc-six3 marked neuroectoderm until 24 hours of development (extended germband stage, white stars and arrows in Figure 6A). Later, in 42–48 hour old embryos, Tc-six3 is expressed in the developing brain lateral and anterior to the stomodeum (Figure 6C, stomodeum marked by black asterisk). Additionally, staining is evident in the overlaying dorsal epidermis (Figure 6B), the stomodeal roof (Figure 6D) and the labrum (Figure 6E).

<i>Tc-six3</i> is required for central body development. — **Fig. 6. *Tc-six3* is required for central body development.**

In order to test the hypothesis of a neural function of Tc-six3, we generated transgenic imaging lines marking neural cells, glia and mushroom bodies (Koniszewski, Kollmann, Averof, in preparation) and we identified the central body at the L1 larval stage (white arrow in Figure 6F). Indeed, Tc-six3 RNAi at low doses led to the loss of the central body in an otherwise normal brain (Figure 6G). Higher doses additionally reduced the distance between the two brain hemispheres (Figure 6H). In weak phenotypes the orientation of the median lobes of the mushroom bodies towards the midline was lost (compare white arrowheads in Figure 6J with Figure 6I) while in strong phenotypes, upon convergence of the brain hemispheres, the mushroom bodies approached each other and were reduced in size (see black arrow in Figure 6K).

In the light of the Tc-six3 expression profile, these brain defects could be due to an early neuroectodermal function of Tc-six3 (see neuroectodermal expression in Figure S2B) or to a later function in developing neural cells (see expression in the brain in Figure 6B–6E). In the first case, epidermal and neural phenotypes would be elicited at the same time and, hence, be tightly linked. In the second case, knockdown at late embryonic stages (when epidermal patterning is already finished) would lead to the induction of neural phenotypes in otherwise unaffected heads. To test this, we injected 1 ug/ul Tc-six3 dsRNA in embryos at 0–2, 4–6, 6–8, 12–14 and 18–20 hours post egg laying and scored the resulting L1 larvae for both cuticle and brain phenotypes (Figure 6L). Indeed, the severity of neural and epidermal phenotypes correlated strongly and we never observed brain phenotypes in embryos without cuticle defects. This indicates that both cuticle and brain phenotype are outcomes of the same early neuroectodermal patterning events.

Discussion

Identification of Novel Insect Head Patterning Genes

With our candidate gene approach we identified five genes that had not been implicated in insect head epidermis patterning before (goosecoid, scarecrow, fez1, dbx, ptx). For four additional genes, we show involvement in embryonic head capsule development while a role in adult Drosophila head patterning had been described previously (ci, Drx, lim1, mirror) [144]–[147]. Based on our fate map, the cuticle defects generally correspond well with the head expression of the respective gene. However, the bell row and the setae of the maxilla escort appear to be sensitive to indirect effects because they were affected in several RNAi experiments with genes, which-based on our fatemap-do not show expression in the respective regions (red circles in Figure 3 Tc-lim1/5, Tc-ptx, Tc-irx and Figure 4 Tc-six3, Tc-rx, Tc-fez). Both regions are located where the head lobes are predicted to fuse either with gnathal segments (maxilla escort) or the trunk (bell row; see black stars in Figure 2C). Hence, primary defects of a gene knockdown in head lobe morphology or size could lead to the observed secondary defects.

A Novel Regulator of Central Complex Formation

We show that Tc-six3 is required for proper formation of the central body, which is a midline spanning neuropile and part of the central complex. To our knowledge, this is the first gene known to be required for embryonic central complex development. Our data are in line with cell lineage tracing experiments in grasshopper embryos, where neuroblasts at a corresponding anterior median position contribute to the central complex [148]. Further, expression of optix/six3 in corresponding neuroblasts was also shown in Drosophila [81]. Together, these data are consistent with the hypothesis that Tc-six3 is required in the neuroectoderm for specifying the identity of central body neuroblasts. However, tools to genetically trace the offspring of these neuroblasts [149] are needed to prove this link.

In hemimetabolous insects, which represent the ancestral mode of embryogenesis, all neuropils of the central complex are formed during embryogenesis. In Drosophila, in contrast, the central complex develops during late larval stages [150]–[153]. Tribolium takes an intermediate position by forming a subset of central complex neuropils during embryogenesis, a situation apparently conserved with another tenebrionid beetle [154]. With the newly available brain imaging lines and its amenability to functional genomics Tribolium is an excellent model to investigate the genetics of embryonic central complex development.

Anterior-Median Development in Insects

We showed that Tc-six3 is expressed in an anterior median domain from earliest stages on and that it acts as an upstream component of anterior median patterning. Drosophila optix/six3 is expressed in an anterior blastodermal ring anterior to otd, which persists at the dorsal side [58], [70], [81] and is required for labral and maxillary structures [62]. Its ring like expression does not support an involvement in median patterning but relevant genetic interactions remain to be studied. The later expression in the labrum and in bilateral dorsal domains, however, is similar in both species.

Interestingly, aspects of dorsal median head patterning are controlled by dpp in Drosophila. Shortly before gastrulation, the action of dpp and its downstream target zen at the dorsal midline separate the neuroectoderm into paired anlagen by medial repression of genes and by promoting median cell death. This results in the establishment of bilateral expression of marker genes of the respective brain parts (e.g. Dchx (pars intercerebralis); Fas2 and Drx (pars lateralis); sine oculis and eyes-absent (visual system)) [46], [141], [155], [156]. Actually, many other anterior patterning genes initiate their expression as unpaired domains across the dorsal midline that are subsequently medially subdivided in Drosophila (e.g. otd [157], tll [158], fezf [159], Dsix4 [58]). In contrast, the Tribolium orthologs of most of these genes are initiated as separate bilateral domains (Tc-rx and Tc-fez (Figure 1E and 1D), Tc-chx and Tc-Fas2 (Figure S5), Tc-tll [131]), Tc-six4 [44], Tc-sine oculis, Tc-eyes-absent [160]). Tc-otd1 starts out with ubiquitous expression related to axis formation [130], [161], [162] but then resolves into paired head lobe domains which are separate as with the aforementioned genes.

Due to differences in topology of the head anlagen (see below), median repression of anterior patterning genes by Tc-dpp is not required in Tribolium. Nevertheless, it is expressed along the rim of the head anlagen at blastoderm stages, some parts of which will become the site of dorsal fusion [163], [164]. However, Tc-Dpp activity (detected by antibodies against pMad) does not occur at the site of expression and is clearly distant from the arising Tc-rx, Tc-chx, Tc-six4, Tc-sine oculis or Tc-fas2 domains [21]. Also the Tc-dpp RNAi phenotypes differ from Drosophila mutants in that the head anlagen are expanded and appear to have lost their dorso-ventral orientation (shown by expansion of Tc-otd1 and the proneural gene Tc-ASH) in an overall ventralized embryo [21]. Hence, the early expression of dpp at the future dorsal midline might be ancestral, but its function with respect to medially repressing gene expression has probably evolved in Drosophila.

Tribolium Probably Reflects the More Ancestral Situation

The difference in generation of paired dorsal domains in these two insect species reflects the different location of the head anlagen. In the long germ insect Drosophila, extraembryonic tissues are reduced to the dorsally located amnioserosa while the head anlagen are situated in the anterior dorsal blastoderm from earliest stages on [165], [166]. Consequently, the head lobes are never separated along the midline. In contrast, in the short germ insect Tribolium, the anterior blastoderm gives rise to extraembryonic amnion and serosa, which eventually ensheath the embryo [166]. In contrast to Drosophila, the Tribolium head anlagen are located in the ventral median blastoderm from where they move towards anteriorly and bend dorsally. The head lobes are separate from the beginning but fuse at late stages at the dorsal midline forming the dorsal head (bend and zipper model, see Figure 2A–2C and [4], [137] for more details). During these morphogenetic movements, the initially separate expression domains of the head lobes eventually come into close proximity at the dorsal midline like in Drosophila (Figure 2D–2F). Both the anterior dorsal location of extraembryonic tissue anlagen and the ventral location of the head anlagen are found in most insects [166] and in the hemimetabolous milkweed bug Oncopeltus fasciatus, gene expression data show a clear separated origin of the head lobes in the blastoderm [167]. Hence, Tribolium is likely to represent the ancestral state in insects.

In striking analogy to Drosophila, the expressions of vertebrate eye field patterning genes start out as one midline spanning domain (e.g. Rx and Pax6 [65], [168], [169]). Later, these domains split medially, which is the prerequisite for the formation of bilateral eye anlagen. shh as well as six3 are involved in medial repression of Pax6 and Rx2 [65], [169] with six3 acting upstream of shh [170]. This appears to be more similar to the derived Drosophila situation than to the ancestral split of head lobe anlagen (see above). However, the molecules involved in median split are different (dpp in Drosophila versus six3 and shh in vertebrates) and we find involvement of Tribolium six3 but not dpp in median patterning. Hence, the molecular data actually suggest a higher degree of conservation between Tribolium and vertebrates and convergent evolution of the similarity between Drosophila and vertebrates.

Conserved Function of six3 in Neuroendocrine and Anterior Median Patterning of Bilaterians

Regarding the likely difference to Drosophila, it is striking that the role of vertebrate six3 in median separation of anterior expression domains is similar to what we find in Tribolium. In vertebrates, six3 represses midbrain derived Wnt signaling [72], [73], which we also find in Tribolium. In vertebrates, six3 and its paralog six6 are involved in pituitary and hypothalamus development [76]–[80]. Based on its expression, six3 has been predicted to contribute to neuroendocrine brain parts in annelids and Drosophila [81]. More generally, the similarity of bilaterian neuroendocrine systems and their common origin from placode like precursors have been noted [44], [47], [171]. Here, we have added functional data showing that Tc-six3 is indeed required for the expression of neuroendocrine markers for the pars intercerebralis (Tc-chx) and pars lateralis (Tc-fas2) [141] placing it high in the hierarchy of neuroendocrine development in bilaterians.

In mouse embryos with reduced levels of six3 and shh expression, median head and brain structures are affected (e.g. median nasal prominence) or absent (e.g. nasal septum, the septum, corpus callosum)(see Figure 5X) [170]. Such holoprosencephaly phenotypes are also seen in some human six3 mutations [172]. Very similarly, we see loss of median brain structures in Tribolium after RNAi for Tc-six3 Overall, these similarities functionally confirm that the ancestral role of six3 orthologs was in the anterior median patterning of the Urbilateria (Figure 5V–5X) [81].

Materials and Methods

Animals

Most experiments were performed using the wild type Tribolium castaneum strain San Bernardino. For brain imaging, a transgenic line for 6XP3-ECFP (marking glia) and elongation factor1-alpha regulatory region-DsRedEx (EF1-B; marking neural cells) and the enhancer trap line Gö-11410 (marking mushroom bodies with EGFP; identified in the GEKU screen [173]) were used (Koniszewski, Kollmann, Averof, in preparation).

Identification of Candidate Genes in Tribolium

Mouse protein sequences of the candidate genes (see Table S1) were obtained from the NCBI database (www.ncbi.nlm.nih.gov/). Tribolium orthologs were identified by BLAST at the Beetle Base server (beetlebase.org/). Test of orthology: Tribolium sequences were blasted against the NCBI protein database and the top 5–15 hits from insects, vertebrate and selected other groups were retrieved, as well as the three most similar Tribolium genes. These were BLASTed against the entire NCBI nucleotide database and the first three hits were retrieved. All these sequences were aligned using the ClustalW algorithm of Mega 4 [174],[175]. Phylogenetic trees were calculated in Mega 4 using the Neighbor-Joining method [176] (bootstrap consensus tree inferred from 10.000 replicates [177]; evolutionary distances computed using the Poisson correction method [178]; all positions containing gaps and missing data were eliminated from the dataset (complete deletion option)). See Figure S1 for phylogenetic trees. Phylogenetic relationships for the following genes were already published: Tc-wnt11 and Tc-wg/wnt1 [179], Tc-otd1/otx and Tc-ems/emx [54], Tc-ey/pax6 and Tc-toy/pax6 [136], Tc-eya [160].

Cloning of Candidate Genes

mRNA of 0–48 h embryos was isolated using the MicroPoly(A)Purist Kit (Ambion) and cDNA was synthesized by using the SMART PCR cDNA Synthesis Kit (ClonTech). Gene fragments obtained by PCR with gene specific primers (see Table S3) were cloned into the pCRII vector using the TA Cloning Dual Promotor Kit (Invitrogen) and their sequence was confirmed.

Whole-Mount In Situ Hybridization

Single (NBT/BCIP) and double in situ stainings (NBT/BCIP & FastRed or INT/BCIP) were performed and documented as described [180], [181].

Knock Down of Gene Function by RNA Interference (RNAi)

RNAi was performed by injection of dsRNA into pupae (pRNAi), adults (aRNAi) or embryos (eRNAi) as described [182]–[184]. Lengths of gene fragments and mode of injection are listed in Table S1. Concentrations used in pupal RNAi and adult RNAi: 2–4 µg/µl; in embryonic RNAi: 1–2 µg/µl. A negative control for pupal RNAi was performed by pricking pupae with the needle or injecting water, injection buffer or dsRNA against tGFP. These controls did not show significant developmental effects in the offspring (not shown). In order to identify potential off-target regions, sequences were BLASTed against the Tribolium genome (BLASTn) at Beetle Base. For five genes, sequence similarity of 21 or more successive identical nucleotides was found to hit other gene predictions. For those, RNAi analysis was repeated by another person using subfragments that did not contain the potentially off-target sequences. The phenotypic effects were very similar with respect to the cuticle phenotype (not shown) and the head bristle pattern (Table S2). See Table S3 for primers for the subfragments. Because Tc-six3 was investigated in more detail, two non-overlapping fragments were cloned, injected and analyzed separately by another person. Staining of Tc-six3 in Tc-six3 RNAi embryos confirmed strongly reduced or imperceptible expression in knock down embryos. The cuticle phenotype (not shown) and the head bristle pattern (Table S2) was very similar, confirming specificity.

Epifluorescent and Confocal Imaging

Image stacks of cleared first instar cuticles were gathered by using a Zeiss LSM 510 or a Zeiss Axioplan 2 microscope and projections were calculated as described previously [44], [181]. Brain imaging was performed using a Zeiss LSM 510.

Supporting Information

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