Insect Tc-Six4 Marks a Unit with Similarity to Vertebrate Placodes

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Developmental Biology 350 (2011) 208–216

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Developmental Biology

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Evolution of Developmental Control Mechanisms Insect Tc-six4 marks a unit with similarity to vertebrate placodes

Nico Posnien a,b, Nikolaus Koniszewski a, Gregor Bucher a,⁎

a Center of Molecular Brain Physiology, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany b Institute for Populationgenetics, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria

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Article history: Cranial placodes are specialized ectodermal regions in the developing vertebrate head that give rise to both Received for publication 11 August 2010 neural and non-neural cell types of the neuroendocrine system and the sense organs of the visual, olfactory Revised 18 October 2010 and acoustic systems. The cranial placodes develop from a panplacodal region which is specifically marked by Accepted 19 October 2010 genes of the eyes absent/eya and two “six homeobox” family members (sine oculis/six1 and six4). It had been Available online 27 October 2010 believed that cranial placodes are evolutionary novelties of vertebrates. However, data from non-vertebrate chordates suggest that placode-like structures evolved in the chordate ancestor already. Here, we identify a Keywords: Cranial placodes morphological structure in the embryonic head of the beetle Tribolium castaneum with placode-like features. Six4 It is marked by the orthologs of the panplacodal markers Tc-six4, Tc-eya and Tc-sine oculis/six1 (Tc-six1) and Six1 expresses several genes known to be involved in adenohypophyseal placode development in vertebrates. Sine oculis Moreover, it contributes to both epidermal and neural tissues. We identify Tc-six4 as a specific marker for this Eya structure that we term the insect head placode. Finally, we reveal the regulatory gene network of the neuroendocrine panplacodal genes Tc-six4, Tc-eya and Tc-six1 and identify them as head epidermis patterning genes. Our Insect head finding of a placode-like structure in an insect suggests that a placode precursor was already present in the Tribolium last common ancestor of bilaterian animals. © 2010 Elsevier Inc. All rights reserved.

Introduction The cranial placodes arise at the interface between the anterior neural plate (the neurogenic ectoderm) and the epidermal ectoderm Vertebrate cranial placodes are specialized ectodermal regions in while the neural crest cells are specified at the same interface in more the vertebrate head that contribute to a variety of cell types of the posterior regions (Baker and Bronner-Fraser, 1997; Brugmann and neuroendocrine system and sense organs of the visual, olfactory and Moody, 2005; Litsiou et al., 2005; Schlosser, 2006; Schlosser, 2008; acoustic systems (reviewed in Baker and Bronner-Fraser, 2001; Streit, 2007). The PPE is marked by genes of the eyes absent/eya and Brugmann and Moody, 2005; Schlosser, 2006; Streit, 2007). Cranial sine oculis homeobox/six1/4 family (Ghanbari et al., 2001; Pandur and placodes are first evident as regions with thickened columnar Moody, 2000; Schlosser and Ahrens, 2004). Panplacodal Eya1 and Six1 epithelium within the non-neural ectoderm at the border of the activity has been shown to promote both the proliferation of neural neural plate (Baker and Bronner-Fraser, 2001; Le Douarin et al., 1986). progenitors by activating SoxB1 and neural differentiation in a dose This initially uniform preplacodal ectoderm (PPE) region becomes dependent manner (Schlosser et al., 2008). The specification of subdivided and contributes to different structures of the head different placodes occurs by stepwise refinement of transcription (e.g. reviewed in Brugmann and Moody, 2005; Streit, 2004). The factor expression from panplacodal to multiplacodal areas to anterior cranial placodes are the adenohypophyseal and olfactory expression of placode-specific sets of transcription factors (Schlosser, placodes, which give rise to the adenohypophysis and the olfactory 2006; Streit, 2004). system, respectively. Other placodes are the lens and profundal/ In their “New Head” theory Northcutt and Gans propose that many trigeminal placodes, the otic and the epibranchial placodes. derived features of the vertebrate body plan are based on the Hypobranchial and lateral line placodes have only been identified in evolution of cranial placodes and the neural crest in the vertebrate some amphibians and fish (e.g. reviewed in Baker and Bronner-Fraser, stem group (Gans and Northcutt, 1983; Northcutt and Gans, 1983). 2001; Schlosser, 2005). Thickened columnar epithelia are also found Hence, cranial placodes were long considered to be vertebrate specific in other structures called placodes, however, here, the term “placode” structures (Shimeld and Holland, 2000). However, based on molec- will be used as synonym for “cranial placode.” ular and morphological data from cephalochordates and ascidians (i.e. non-vertebrate chordates), it has been suggested that these taxa already possessed adenohypophyseal/olfactory- and otic/lateral line- placode-like structures which develop from six1/six4/eya positive ⁎ Corresponding author. Department of Dev. Biology, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany. Fax: +49 551 395416. tissue (Bassham and Postlethwait, 2005; Boorman and Shimeld, 2002; E-mail address: [email protected] (G. Bucher). Christiaen et al., 2002; Manni et al., 2004; Manni et al., 1999; Mazet

0012-1606/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2010.10.024 N. Posnien et al. / Developmental Biology 350 (2011) 208–216 209 et al., 2005). Schlosser however maintains that placodes as such wildtype and RNAi embryos with a mix of probes against Tc-caudal and evolved only in the vertebrate ancestor in the form of an adenohy- Tc-eya, Tc-six4 and Tc-six1 transcripts, respectively. The RNAi embryos pophyseal/olfactory protoplacode that subsequently split to form the showed normal Tc-cad staining but a strongly reduced Tc-eya, Tc-six4, other placodes. In this view, the “rostral neurosecretory organ” of and Tc-six1 expression levels (not shown). In a small fraction of each Amphioxus is no real placode but became integrated into the evolving control experiment we also found embryos without the respective protoplacode (Schlosser, 2005). Northcutt, in contrast, concedes that expression indicating efficient knockdown. Off-target effects are the cephalochordate/vertebrate ancestor did possibly have an unlikely as a blast-n analysis of the template sequences does not reveal adenohypophyseal placode homolog (Northcutt, 2005). It is impor- identical sequence stretches with N20 bases. For all RNAi experiments tant to note that recent phylogenetic data places the urochordates, where embryos were fixed and stained, a fraction of those embryos was instead of the cephalochordates, as sister group of vertebrates incubated until the first larval stage where gene specific cuticle (e.g. Bourlat et al., 2006; Delsuc et al., 2006). In the light of this new phenotypes were observed to confirm the efficiency of RNAi. Tc-eya phylogeny Holland's suggestion that the ancestor of vertebrates and RNAi induces sterility upon pupal and adult injection—hence we cephalochordates might have had an olfactory placode while the performed embryonic RNAi. Eggs were collected after 1 h and incubated adenohypophyseal placode could have evolved already in the last another hour at 25 °C, injected and incubated for a further 20–26 h (at common ancestor of vertebrates, urochordates and cephalochordates, 32 °C). The resulting embryos were fixed and the vitelline membrane suggests that both placodes might have been in place at the base of removed manually before staining. In total, the following number of chordates. He further concedes that the ability of the placodes to embryos have been analyzed: Tc-eya staining in Tc-six1 aRNAi: 24, differentiate many different cell types and to generate migratory cells Tc-eya staining in Tc-six4 pRNAi: 35, Tc-six4 staining in Tc-six1 aRNAi: 42, evolved only in the vertebrate ancestor, which supports Schlosser's Tc-six4 staining in Tc-eya eRNAi: 20, Tc-six1 staining in Tc-eya eRNAi: 32, view (Holland and Holland, 2001). In many studies in the deutero- Tc-six1 staining in Tc-six4 pRNAi: 50. Cuticle phenotypes of first larval stome clade, coexpression of a few upstream marker genes (six1, six4, cuticle heads were determined including scoring a comprehensive set of eya) and differentiation markers (e.g. pitx, six3) is used as one dorso-lateral bristles (Schinko et al., 2008). argument for homology. Moreover, all data indicate that the adenohypophyseal/olfactory placodes with their contribution to the Whole mount in situ hybridization neuroendocrine system evolved first (reviewed in Schlosser, 2006). Recently, a high degree of similarity has been revealed between Single (NBT/BCIP) and double in situ stainings (NBT/BCIP & protostome and deuterostome anterior CNS (e.g. Arendt and Nübler- FastRed or INT/BCIP) were performed and documented as described Jung, 1996; Denes et al., 2007; Reichert, 2005; Reichert and Simeone, (Schinko et al., 2009; Wohlfrom et al., 2006). 2001; Simeone et al., 1992a; Simeone et al., 1992b; Tomer et al., 2010; Urbach, 2007) as well as the respective neuroendocrine systems (de Epifluorescent and confocal imaging Velasco et al., 2007; De Velasco et al., 2004; Tessmar-Raible, 2007; Tessmar-Raible et al., 2007; Wang et al., 2007). These similarities open Cuticles were documented using a Zeiss Axioplan microscope to the possibility that the placodes did not evolve in the Chordate lineage record stacks of 40–80 planes. For deconvolution the “No Neighbour” but stem from a placode precursor in the last common ancestor of method of the ImagePro software (Version 6.2; MediaCybernetics) was bilaterian animals. In order to test this hypothesis, we analyzed the used. Z-projections were achieved by a “maximum intensity” projection expression and function of the panplacodal markers six1, six4 and eya using ImageJ (version 1.40 g). Embryos stained with the fluorescent along with specific markers for the adenohypophyseal/olfactory membrane dye FM1–43 (Molecular Probes) were prepared free from placodes in the red flour beetle Tribolium castaneum as a represen- the yolk and documented with the Zeiss LSM 510 and processed using tative of protostomes. the Zeiss LSM Image Browser (Version 4.2.0.121). We show that all three panplacodal marker genes are transiently co-expressed in an anterior head region and identify Tc-six4 as specific Results marker. This region is located at the anterior rim of the neuroectoderm in the embryo, is morphologically distinguishable from Tc-eya, Tc-six1 and Tc-six4 are co-expressed in an anterior-median head surrounding tissue, contributes to neural and non-neural tissue and region expresses genes that are specific for adenohypophyseal placodes. We conclude that a placode precursor was already present in the last In vertebrates, the PPE is marked by co-expression of eya, six1 and common ancestor of protostomes and deuterostomes. Furthermore, six4 (Ghanbari et al., 2001; Pandur and Moody, 2000; Schlosser and we identify the Tc-six1, Tc-six4 and Tc-eya regulatory gene network Ahrens, 2004). In Tribolium development, Tc-eya (Yang et al., 2009b) and show that all three genes are required for epidermal head is first expressed at blastodermal stages. In early germ bands, development. expression is highest in the lateral rim of the head lobes (arrow in Fig. 1A). Throughout embryonic development, Tc-eya is transcribed in Materials and methods the stomodeal region (stars in Fig. 1A–D). Expression in the head lobes increases during early elongation (arrow in Fig. 1B). In fully Used clones and RNAi experiments and controls elongated germ bands the rim-domain is split in two domains, one anterior-median adjacent to the labrum (black arrowhead in Fig. 1C) Genes were PCR amplified from cDNA by using the following and a more posterior one in the lateral eye lobe tissue (open primers: Tc-six4 (forw: 5′-AAGTCGGCGCGAAAGAAC-3′ and rev: 5′-CTA arrowhead in Fig. 1C). This distinction is more obvious in retracting AATTTATGGTACTTGATAATCCG-3′), Tc-eya (forw: 5′-TACCCCCAATC embryos, when the posterior domain marks the larval eye anlagen GTACTCAGC-3′ and rev: 5′-GAAAAACGCCTCCTAGACC-3′), Tc-lim3/lhx3 (Yang et al., 2009b) (open arrowhead in Fig. 1D) while the anterior (forw: 5′-TGCCTCGATGGGGACCAGC-3′ and rev: 5′-AGTGGGCACTGGAC domain remains in the anterior-median head lobe (black arrowhead GGCTCC-3′)andTc-ptx/pitx (forw: 5′-ACTCAAATGGAGCGGATGG-3′ and in Fig. 1D). In addition to the head domains, Tc-eya possesses dynamic rev: 5′-GATCAAACTCCGGGCGAT-3′). The Tc-six1 clone was provided by segmental expression (Fig. 1B–D). M. Friedrich. Tc-six1 (synonym: Tc-sine oculis, Tc-so) (Yang et al., 2009b) starts to RNAi was performed by the injection of dsRNA into pupae (Tc-six4), be expressed later than Tc-eya but in a largely overlapping domain adults (Tc-six1) or embryos (Tc-eya)(Bucher et al., 2002; Posnien et al., (Fig. 1E). However, at elongated germ band stages, the anterior 2009b; Schinko et al., 2008). As a control for efficiency, we stained domain fades and eventually disappears (black arrowhead in Fig. 1F) 210 N. Posnien et al. / Developmental Biology 350 (2011) 208–216

Fig. 1. Tc-six4 marks a morphologically visible unit which constitutes the only domain where all orthologs of vertebrate panplacodal markers are co-expressed. The orthologs of panplacodal markers and Tc-wg are stained in blue and red, respectively. The embryos are oriented with anterior to the top. The stage is schematically depicted on the left side. (A–D) Expression of Tc-eya.Atthe1–3 wg-stripe stage expression of Tc-eya covers the lateral rim of the future head (arrow in A) and a median domain (white star in A). The median domain becomes located to the stomodeal region (stars in B–D). Tc-eya expands to cover the entire rim of the head lobes at the 5–8 wg-stripe stage (arrow in B). At later stages the rim expression is subdivided into an anterior part (arrowhead in C, D) and a posterior ocular domain (open arrowhead in C, D). (E–G) The expression of Tc-six1 is similar to the one of Tc-eya but starts later and fades at later stages in the anterior rim region (black arrowhead in F), while the lateral ocular domain (open arrowhead in F) stays active. (H–J) Tc-six4 is expressed in the anterior rim region of the head lobes throughout embryonic development (e.g. arrowhead in I). A minor additional expression is found in cells of the thoracic segments (see J). (K–M) Morphology of the Tc-six4 marked region. The rim region in the Tribolium embryo becomes subdivided into an anterior and a posterior part. The anterior part (anterior and posterior boundaries marked by arrows and arrowheads, respectively in K–M) is further subdivided (K, L) and specifically marked by Tc-six4 (quenched fluorescent signal in M). L is a magnification of K. whereas the expression in the lateral eye lobe is retained (open throughout embryonic development (Fig. 1J). Importantly, it coincides arrowhead in Fig. 1F). In retracting embryos, the anterior head lobes with the anterior domain of Tc-eya expression (compare black arrow- lack Tc-six1 expression except for one single cell (black arrowhead in heads in Fig. 1C and I) and corresponds to those cells that switch off Fig. 1G). Tc-six1 (compare black arrowheads in Fig. 1F and I). In addition, the We cloned the Tribolium Tc-six4 ortholog and found that its median most part of the early expression does not overlap with Tc-eya or expression starts slightly later than Tc-six1 at the 5–8 wg-stripe stage in Tc-six1 (compare Fig. 1H with B, E). Hence, the only region where all three a restricted anterior domain (Fig. 1H) which expands subsequently panplacodal markers are expressed during embryogenesis is the tissue (black arrowhead in Fig. 1I). This expression remains detectable marked by Tc-six4 making it a candidate marker for a placode precursor. N. Posnien et al. / Developmental Biology 350 (2011) 208–216 211

Tc-six4 marks a morphological unit (bf1)/Foxg1 (Bourguignon et al., 1998; Toresson et al., 1998). Vertebrate pitx3 and lhx3 genes are more restricted to the anterior adenohypoph- In order to test whether Tc-six4 marks a morphological unit we yseal placode (Dutta et al., 2005; Pommereit et al., 2001; Taira et al., analyzed confocal images of embryonic heads stained with a 1993) while emx2 marks the olfactory placode (Pannese et al., 1998). membrane specific dye. Indeed, an incision becomes visible that Additionally, it has been shown that lhx3 is involved in the regulation of divides the rim region into an anterior and a lateral portion (incision adenohypophysis specific genes, suggesting a defined role in its indicated by white arrowheads in Fig. 1K–M). Medially, the tissue is cytodifferentiation (reviewed in Mullen et al., 2007). clearly separated from the adjacent labrum (white arrow in Fig. 1K–M We used advanced embryonic stages where the Tc-six4 positive indicates the border). A co-staining of Tc-six4 and the membrane region is readily identified morphologically (see black bars in Fig. 2I–O) marker shows that this morphological unit is marked by Tc-six4 in order to test whether this unit is marked by orthologs of these placode expression (Fig. 1M). genes. We find that Tc-optix/six3 marks a large anterior portion of the Tc-six4 positive tissue (Fig. 2J), while late Tc-otd1/otx (Li et al., 1996; The Tc-six4 positive region gives rise to neural and epidermal cells Schinko et al., 2008) expression covers the posterior part of it (Fig. 2K). Tc-ey/pax6 (Yang et al., 2009a)andTc-slp/bf1/Foxg1 (Choe and Brown, We next asked whether the Tc-six4 positive cells contribute to both 2007) are expressed in small cell clusters or individual cells within the neural and epidermal cells like the cranial placodes do. In contrast to Tc-six4 expression domain (Fig. 2L, M). In addition, small clusters of vertebrates, the arthropod neurogenic epithelium consists of a mixture Tc-six4 positive cells express Tc-ptx/pitx (Fig. 2N) and Tc-lim3/lhx3 of cells with neural and epidermal fates (Stollewerk and Simpson, 2005; (Fig. 2O), respectively. Tc-ems/emx is not expressed in this region Urbach and Technau, 2004). Double stainings of Tc-six4 and the neural (not shown and Schinko et al., 2008). Hence, adenohypophyseal related, stem cell marker Tc-asense (Tc-ase) (Dominguez and Campuzano, 1993; but not olfactory markers are expressed in the Tc-six4 region. Wheeler et al., 2003) shows that neural cells arise from this tissue (Fig. 2A). Next, we scrutinized Tc-six4 RNAi animals for epidermal Tc-eya, Tc-six1 and Tc-six4 interact genetically to establish the insect defects. Indeed, Tc-six4 RNAi leads to the formation of an impaired head placode cuticle in the head. Specifically, the region between the labrum (black arrowhead in Fig. 2C) and the antennae (black star in Fig. 2C) is affected In order to test genetic interactions, we knocked down each gene by (100%, n=8; arrow in Fig. 2C). The dorsal and lateral parts of the RNAi and subsequently analyzed the expression of the other two. Knock Tribolium head are marked by an invariant bristle pattern (Schinko et al., down of Tc-eya leads to strong reduction of Tc-six1 (compare Fig. 2P 2008). Detailed analysis of this pattern shows that the posterior vertex with P') or its complete absence. The same is true for Tc-six4 expression triplet bristle and the antenna basis bristle are affected in 15–20% and (Fig. 2Q/Q'). Tc-eya expression is unaltered after Tc-six1 orTc-six4 RNAi. N25% of all cuticles, respectively, after Tc-six4 knock down (see Fig. 2D This puts Tc-eya on top of the regulatory hierarchy in line with its early and Table S1). Hence, the Tc-six4 positive region–like cranial placode expression. Tc-six1 is expressed prior to Tc-six4 and could therefore be tissue–contributes to both neural and epidermal cell types. involved in its activation (compare Fig. 1E/H). Indeed, Tc-six4 expression is strongly reduced after Tc-six1 knock down (compare Fig. 2RwithR' All panplacodal genes are required for head development and S with S'). Later, the expression of Tc-six1 disappears in the prospective insect head placode while Tc-six4 expands suggesting a Motivated by the Tc-six4 phenotype, we analyzed the cuticles of repressive interaction (see Fig. 1F, I). Indeed, Tc-six4 knock down leads Tc-eya and Tc-six1 RNAi embryos. RNAi against Tc-eya leads to the loss to some ectopic anterior Tc-six1 expression at late embryonic stages of the complete dorsal head capsule, including the labrum, the (compare black arrowheads in Fig. 2T and T'). However, the strength of antennae and the setae that mark lateral and dorsal head in 46,2% of the ectopic expression was lower compared to the endogenous one (see the analyzed cuticles (n=13) (Fig. 2E). The ventral part of the head, Fig. 2U for summary of interactions). represented by the gnathal appendages, seems less affected. However, in some larvae the mouthparts are highly reduced (arrow in Fig. 2E). Discussion These could be secondary effects due to the loss of large portions of dorsal head tissue. In weaker phenotypes the dorsal head is still Identification of the insect head placode with similarities to vertebrate present. The lack of lateral setae in such heads reveals that the lateral placodes part of the head capsule is most sensitive to Tc-eya RNAi (dark grey area in Fig. 2F, see also Fig. S1). The knock down of Tc-six1 does We have identified a structure with similarities to cranial placodes not result in strong defects of the head capsule. However, the analysis and term it insect head placode in order to reflect both their common of the head bristles revealed that mainly the lateral gena triplet setae origin and the differences due to independent evolution. The insect and the antenna basis bristle are missing or misplaced (see head placode is a morphological unit marked by Tc-six4.Itisdefined incomplete green triangle in Fig. 2G and schematic representation of as a developmental unit by the fact that Tc-six4 depends on Tc-eya and deletion domain in H). These results show an involvement of Tc-six1 activity in the prospective insect head placode but not outside orthologs of all three panplacodal gene orthologs in head epidermis (see below). On the upstream regulatory level, it is the only region in formation in insects. the insect embryo that coexpresses all panplacodal markers (Tc-eya, Tc-six1 and Tc-six4) which–judged by their early and dynamic The Tc-six4 positive region expresses genes related to adenohypophyseal expression and interactions–act as upstream regulators like in placode development vertebrates. Moreover, the insect head placode expresses several downstream patterning markers that are, among others, typical for Each vertebrate cranial placode is marked by a unique gene early development of adenohypophyseal and the olfactory placodes, expression profile required for the differentiation of its cell types namely Tc-six3, Tc-otd1, Tc-ey/pax6, Tc-ptx/pitx and Tc-slp/bf1/Foxg1. (Baker and Bronner-Fraser, 1997; Brugmann and Moody, 2005; More strikingly, in late embryonic stages, when patterning processes Schlosser, 2006). In vertebrates, the early adenohypophyseal and are completed and differentiation likely starts, Tc-lim3/lhx3 is olfactory placodes are, although not exclusively, marked by six3/6 expressed in distinct cell clusters in the Tc-six4 positive domain. In (Ghanbari et al., 2001; Zhou et al., 2000; Zuber et al., 1999), otx2 (Kablar vertebrates this gene marks adenohypophysis/pituitary specific et al., 1996; Pannese et al., 1995),pax6( Hirsch and Harris, 1997; Li et al., cytodifferentiation. The very restricted expression of Tc-lim3/lhx3 1994; Schlosser and Ahrens, 2004; Zygar et al., 1998)andbrain factor1 makes a coincidental co-expression with Tc-six4 unlikely. The position 212 N. Posnien et al. / Developmental Biology 350 (2011) 208–216 of the insect head placode at the anterior most rim of the namely Tc-six3 (Fig. 2J) (Oliver et al., 1995; Posnien et al., 2009a), neuroectoderm corresponds to the vertebrate adenohypophyseal/ Tc-otd1 (Fig. 2K) (Li et al., 1996; Schinko et al., 2008; Simeone et al., olfactory placodes. The similar anterior location is further evidenced 1992a) and Tc-tll (Schröder et al., 2000; Yu et al., 1994). Moreover, it by the proximity of several marker genes that are specific for the has been shown previously that the anterior brain regions of anterior neural plate of vertebrates and the anterior head in Tribolium, vertebrates and protostomes are homologous (e.g. Tomer et al., N. Posnien et al. / Developmental Biology 350 (2011) 208–216 213

2010; Reichert, 2005; Reichert and Simeone, 2001; Tessmar-Raible, recent development of the Gal4/UAS system in Tribolium (Schinko 2007) and that the ocular parasegment boundary corresponds to the et al., 2010) will allow creation of such reporters in the future. mid hind brain boundary (Urbach, 2007). These boundaries are located posterior to the placode region in both taxa (compare The insect head placode regulatory gene network expression of Tc-eya relative to the posterior border of the anterior most Tc-wg stripe shown in red in Fig. 1A, B). Similar to most placodes Our data show how the panplacodal genes interact to pattern the (e.g. olfactory) the insect head placode gives rise to both non-neural insect head placode (summarized in Fig. 2V). Tc-eya is clearly on top of and neural cells. this cascade as it is expressed more early than Tc-six4 and Tc-six1,is required for the activation of both but does itself not depend on their Contribution of the insect head placode tissue to the neuroendocrine activity. Tc-six1 is acting on an intermediate level as it is expressed system earlier than Tc-six4 andisrequiredforitsexpression.Hence, activation of Tc-six4 by Tc-eya could be indirect via Tc-six1. The insect head placode–like the adenohypophyseal placode in Interestingly, Tc-six4 appears to repress its activator Tc-six1 to some vertebrates (Couly and Le Douarin, 1985)–appears to contribute to extent (Fig. 2V). As Tc-six4 is exclusively expressed in the insect head the neuroendocrine system. This statement is based on the finding placode, we hypothesize that it is the key component of insect head that the Tc-six4/Tc-six3 positive tissue is positioned in the anterior placode differentiation. Systematic testing of the interactions found in most part of the developing head, where also the neuroendocrine pars Tribolium in vertebrates may reveal conservation, as, for instance in intercerebralis/pars lateralis Anlagen in Drosophila are located mouse eya1 knock-out mutants, no six1 transcripts are detectable in (de Velasco et al., 2007; Hartenstein, 2006). In Drosophila the some placodal regions (Xu et al., 1999; Zheng et al., 2003). expression of Dm-optix/six3 in the region of the developing neuroen- The network depicted in Fig. 2V does not suffice to explain Tc-six4 docrine system has recently been shown (Steinmetz et al., submitted). activation. For instance, additional components are required to Although a comprehensive expression analysis of Dm-six4 in the restrict Tc-six4 activation to the insect placode despite Tc-eya and embryonic head is missing so far, the location of the published Tc-six1 coexpression in stomodeal and the eye regions. Based on expression domains indicates that also in Drosophila this is located expression timing and location, Tc-six3 (Posnien et al., 2009a) and where the neuroendocrine system is known to develop (Seo et al., Tc-otd1 (Li et al., 1996; Schinko et al., 2008) could be co-activators. 1999). We also find Tc-chx expressed in the insect head placode Tc-six4 could also be specifically repressed outside the insect placode (Posnien, Bucher, unpublished), which is an early marker for the pars e.g. by the Tc-Dll gene that is expressed in the rim of the ocular region intercerebralis of the neuroendocrine system in Drosophila (not shown and Beermann et al., 2001). (de Velasco et al., 2007; Hartenstein, 2006). Additionally, it is widely Based on anterior expression of Drosophila six4, a function in head believed that six3 is a conserved factor for bilaterian neuroendocrine and brain development has been suggested (Seo et al., 1999). system development since it is expressed in the Anlagen of the However, Dm-six4 phenotypes have so far only been described for adenohypophysis in vertebrates and in the developing neuroendo- muscle and gonad development (Kirby et al., 2001). Our finding adds crine system in Drosophila and annelids (Tessmar-Raible et al., 2007 the requirement for head epidermis and neural development to insect and Steinmetz et al., submitted; reviewed in Hartenstein, 2006; six4 functions. Tessmar-Raible, 2007). Another marker is pitx (Fig. 2N) which in vertebrates is important for the formation of the neuroendocrine Several co-factor switches of Tc-Eya may be required for insect placode pituitary (Pommereit et al., 2001; Szeto et al., 1999; Tremblay et al., specification 1998). Of note, the very restricted expression of Tc-pitx in only one small domain in the anterior insect head makes it a very reliable Eya proteins lack DNA-binding domains and, hence, require marker. co-factors for transactivation like So/Six1, Dac (dachshund) (Chen Together, these data indicate the formation of neuroendocrine cell et al., 1997; Pignoni et al., 1997) and Six4 proteins (Ohto et al., 1999). types in the insect head placode. This claim could be further tested by In the insect head placode, the activation of Tc-six4 could depend on a exact molecular fingerprints of individual cells (Arendt, 2005) and a physical interaction of Tc-Eya and Tc-Six1. However, none of the comprehensive description of cells and tissues built by the insect head known co-factors (e.g. Dac, Six4) are active at the right time and place placode. A prerequisite for such investigations is the development of to cooperate with Tc-Eya to activate Tc-six1 in the insect head placode. tools to permanently mark and trace the cells of the insect head Tc-Optix/Six3 may be a so far unidentified co-factor of Eya as it placode. However, we are not aware of Drosophila driver lines that belongs to the Six protein family and is expressed in anterior median would allow the tracing of Dm-six4 positive cells. Our finding that regions from early stages on (Posnien et al., 2009a). Interestingly, Tc-six4 is a very specific marker for the insect head placode and the several switches of Tc-Eya co-factors appear to occur during insect

Fig. 2. Functional analysis of panplacodal markers in Tribolium and evolution of the placode precursor. (A) Co-staining of Tc-six4 (brown) and Tc-ase (blue) shows that Tc-six4 marks several neural stem cells indicating contribution to brain development. (B–H) Epidermal function of panplacodal genes. Orange and red dots in the schematic larval heads in D, F and H refer to individual bristles that are affected in 15–24% or N25% of all analyzed larvae, respectively. The dark grey area represents the potential region of cuticle effects besides the affected bristles. (B) Wildtype larval head in a lateral view. The labrum (black arrowhead in this and other panels) is a lobe projecting between the antennae (marked by a * in this and other panels). The lateral and dorsal head capsule is marked by an invariant bristle pattern (Schinko et al., 2008). Green colored lines connect the bases of long setae marking the lateral head while purple lines connect dorsal setae (in this and other panels, see complete pattern in panel D and a detailed description in (Schinko et al., 2008)). (C) Tc-six4 RNAi results in a disturbed cuticle between labrum and antennae (arrow). (D) In addition we observe the loss of the antenna basis bristle (red dot) and the vertex basis bristle (orange dot) in a portion of the cuticles. In the cuticle shown in (C), the antenna basis bristle is present (red dot) while the vertex basis bristle is absent. (E) In strongly affected Tc-eya RNAi larvae the entire dorsal head is missing (arrow points at the remaining mouthparts). (F) Less strongly affected Tc-eya knock down larvae lack several bristles of the lateral head capsule. (G/H) Knock down of Tc-six1 results in the loss of dorso-lateral bristles. The green arrows in G and H mark the remaining but misplaced posterior gena triplet seta. The dorsal setae are not affected (purple lines in G) (I–O) The Tc-six4 positive region co-expresses several genes that are involved in vertebrate adenohypophyseal placode development. The Tc-six4 positive region is identified by the morphological subdivision and by relative expression to Tc-wg (in red). (P–T) The regulatory network of insect head placode development. (P–T) are wildtype stainings, (P'–T') are RNAi embryos at the same stage. (P/P') Tc-eya RNAi results in a strong reduction of Tc-six1. (Q/Q') Tc-eya RNAi also leads to reduction of Tc-six4 expression. Shown is an embryo with intermediate knock-down where Tc-six4 is asymmetrically reduced. (R, R' and S, S') In Tc-six1 RNAi embryos the expression of Tc-six4 is highly reduced (R represents a younger stage than S). (T–T') The knock down of Tc-six4 results in ectopic anterior-median expression of Tc-six1 (compare arrows in T and T'). (U) Summary of the analyzed genetic interactions of the panplacodal markers in Tribolium. (V) Hypothetical switches of Tc-Eya co-factors during insect head placode specification— see discussion for details. (W) Previously, the cranial placodes were suggested to have evolved in the Urochordate (3), the Chephalochordate (2) or the Vertebrate ancestor (1) (a=adenohypophyseal placode; o=olfactory placode). We suggest that a placode precursor was already in place at the base of bilaterian evolution. This structure evolved to the cranial placodes in the vertebrate lineage and into the insect head placode in insects. See text for details. The phylogeny given here is based on (Bourlat et al., 2006; Delsuc et al., 2006). 214 N. Posnien et al. / Developmental Biology 350 (2011) 208–216 head placode specification: First an unknown co-factor (maybe show for the first time the involvement of Tc-six4, Tc-eya and Tc-six1 in Tc-Six3) is required for Tc-six1 activation which in turn is likely to head epidermis development. Finally, we identify the gene regulatory act as co-factor for Tc-six4 activation. Finally, Tc-Six4 probably network between the orthologs of the panplacodal genes in insects. replaces Tc-Six1 as co-factor—this last complex may be responsible for switching on the differentiation gene cascade (Fig. 2V). Appendix A. Supplementary data Evolution of cranial placodes Supplementary data to this article can be found online at doi:10.1016/ j.ydbio.2010.10.024. The “New Head” theory of Northcutt and Gans poses that the cranial placodes and the neural crest evolved in vertebrates and contributed to the evolution of vertebrate head structures (Gans and Acknowledgments Northcutt, 1983). However, molecular data from ascidians and amphioxus indicate that olfactory and adenohypophyseal placodes N.P. wrote the paper and did all experimental work except for the homologs may already have been present in non-vertebrate chordates repetition of Tc-eya injection which was done by N.K. G.B. supervised (Bassham and Postlethwait, 2005; Begbie and Graham, 2001; Graham the project and wrote the paper. We thank Inga Schild and Julia and Begbie, 2000; Holland and Holland, 2001; Manni et al., 1999; Lauterbach for help and Markus Friedrich for clones. Gregor Gilfillan Mazet et al., 2005; Northcutt, 2005; Schlosser, 2005). Based on a critically read the manuscript. We are very thankful for valuable comparison of vertebrate, cephalochordate and urochordate data, comments from two anonymous reviewers. This work was supported Schlosser proposes that ancestrally, expression of the six and eya by the DFG research center “Center of Molecular Brain Physiology” genes was restricted to openings of the pharynx. Only late in (CMPB) and the grant DFG BU/1443-2 to GB. evolution, in the chordate ancestor, they became recruited to the interface of neurogenic and non-neurogenic ectoderm where they References became integrated with other genes to form the placode gene network (Schlosser, 2005). Arendt, D., 2005. Genes and homology in nervous system evolution: comparing gene Our data, in contrast, show that the respective regulatory network functions, expression patterns, and cell type molecular fingerprints. Theory Biosci. – was already in place in the last common ancestor of all bilaterian 124, 185 197. Arendt, D., Nübler-Jung, K., 1996. Common ground plans in early brain development in animals (see Fig. 2W). We find many similarities of the molecular mice and flies. Bioessays 18, 255–259. code of the insect head placode and the adenohypophyseal placode, Baker, C.V., Bronner-Fraser, M., 1997. The origins of the neural crest. Part I: embryonic – indicating a common evolutionary origin. 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