Introduction

Neural development of insects allows to study molecular and cellular principles in easy to manipulate invertebrate models. The fruit fly Drosophila melanogaster (Drosophila hereafter) has mostly been used for his purpose, making it one of the best understood models for neurogenesis [1,2]. However, it has remained a puzzle, how the huge ecological diversity of insects and the divergent neural anatomies adapted to different niches [3,4] evolved. Furthermore, Drosophila may in many instances not be a good representation of insect development and some processes are derived in the fly lineage [5–7]. For these reasons the beetle Tribolium castaneum (Tribolium hereafter) has been introduced as an additional model for insect neural development [8–11] and many molecular genetic manipulation and labelling methods have been established [12–16]. Specifically, labelling of neural cell types in Tribolium has been facilitated by the advent of CRISPR-Cas9 [12,15,17]. Another factor that makes Tribolium an informative model for the understanding of insect brain development is the more conserved development of the head neuroectoderm facilitating the identification of specific neurogenic domains and allowing cross-species comparisons [11,18,19].

The insect central complex is an anterior, midline spanning neuropile and constitutes an important brain centre for the processing of sensory input, coordination of movement and navigation [20,21]. Between the two insect models Drosophila and Tribolium a temporal shift in the emergence of central complex neuropile was observed with Tribolium developing a functional larval central complex during embryogenesis whereas it develops only at the onset of adult life in Drosophila [3,10]. It is however not known how these temporal differences are established during development on a cellular and molecular level. The entire insect central nervous system including the brain is produced by neural stem cells, the neuroblasts (NBs) [22]. While there are evolutionary modifications in the relative position of trunk NBs and their gene expression profiles between fly and beetle, the core determinants specifying their role as neural progenitors are conserved [8]. NBs undergo repeated divisions producing rows of ganglion mother cells (GMCs) which divide one more time to produce neurons and/or glia (see Fig. 1, top panel). This leads to neural cell lineages that include the neuroblast itself and its progeny [23,24]. Several NBs of the anterior-most part of the neuroectoderm contribute to the CX and compared to the ventral ganglia produced by the trunk segments, it is of distinctively greater complexity [25,26]. Intriguingly, a neuroblast subtype, type-II neuroblasts (type-II NBs), were found to prominently contribute to the formation of the central complex in Drosophila and the grasshopper Schistocerca gregaria [27,28]. These specific neuroblasts generate more offspring by producing another class of neural precursors, the intermediate progenitors (INPs), which also divide in a stem cell like fashion [29,30] (Fig. 1, lower panel). While type II NBs were found in both grasshopper and flies, only in Drosophila they have been characterized molecularly.

Figure 1

The dramatically increased number of neural cells that are produced by individual type-II lineages, and the fact that one lineage can produce different types of neurons, leads to the generation of increased neural complexity within the anterior insect brain when compared to the ventral nerve cord [27,30]. Type-II NBs have attracted a lot of attention because intermediate cycling progenitors have also been described in vertebrates, the radial glia cells, which also produce a variety of cell types [32,33]. In addition, in Drosophila brain tumours have been induced from type-II NBs lineages [34], opening up the possibility of modelling tumorigenesis in an invertebrate brain, thus making these lineages one of the most intriguing stem cell model in invertebrates [35,36].

Based on descriptions of large proliferative lineages in the grasshopper Schistocerca gregaria, a hemimetabolous insect distantly related to flies, it is widely believed that type-II NBs and INPs are conserved within insects [25,37]. However, molecular characterisation of such lineages in another insect but the fly and a thorough comparison of type-II NBs lineages and their sub-cell-types between fly and beetle are still lacking.

The characterization of type-II NBs in Drosophila has in large parts been based on a specific reporter line in which eGFP expression is driven by an earmuff (fez/erm) enhancer element and marks INP-lineages [30,38,39]. Tribolium earmuff has been first described as Tc-fez, referring to the vertebrate earmuff ortholog fez [19]. We will use Tc-fez/erm in the following. A defined sequence of gene expression has been described in Drosophila type-II NBs, INPs and GMCs. The ETS-transcription factor pointed (pnt) marks type-II NBs [40,41], which do not express the type-I NB marker asense (ase) but the pro-neural gene deadpan (dpn) (Fig. 1). Intermediate progenitors express fez/erm and ase and at a more mature stage also dpn, whereas prospero (pros) marks mature INPs and GMCs [30,42]. Six4 is another marker for Drosophila type-II NBs and INPs [43].

In the present work we characterize type-II neuroblasts in Tribolium with respect to their number, their location and the conservation of the molecular markers known from Drosophila. We also wanted to test, in how far the earlier emergence of the central complex in beetles would be reflected by a change of division activity of type-II NBs or intermediate progenitors. To that end, we created a Tc-fez/erm enhancer trap line to characterize embryonic Tc-fez/erm -expressing cells including INPs and GMCs of type-II NBs lineages and combined this with other labelling methods including multicolour hybridisation chain reaction (HCR) [44,45]. We found that the Tribolium embryo produces 9 Tc-pnt-expressing type-II NBs compared to only 8 type-II NB lineages in the Drosophila embryo [46]. We show that these lineages produce central complex cells and confirm a largely conserved molecular code that differentiates INPs of distinct maturation stages and GMCs. Intriguingly, we found that lineage sizes in Tribolium embryos are considerably larger and include more mature INPs than in the Drosophila embryo [46], thus providing the material for the embryonic early central complex formation in Tribolium. We also show that the placodal marker gene Tc-six4 characterizes the embryonic tissue that gives rise to the larger anterior group of type-II NBs, whereas Tc-six3 is marking only an anterior subset of these lineages. Interestingly there is a part of the central complex precursors expressing neither Tc-six3 nor Tc-otd, which is absent from all type-II NBs lineages.

Results

A CRISPR-Cas9-NHEJ generated enhancer trap lines marks Tc-fez/erm-expressing cells at the embryonic and larval stage

We created an enhancer trap line driving eGFP that reflects Tc-fez/erm gene expression in the embryo. We named the line fez magic mushrooms (fez-mm-eGFP) because it marks the mushroom bodies of the larval brain (Fig. 2 A-C). The reporter construct was inserted 160 bp upstream of the fez/erm-transcription start site using the non-homologous end joining (NHEJ) repair mechanism (see supplementary Fig. S1 for scheme). Analysis of eGFP co-expression with Tc-fez/erm-RNA at the embryonic stage revealed that fez-mm-eGFP co-localized with most Tc-fez/erm-expressing cells (Fig. 2, panel D). The fez-mm-eGFP line marked several progenitor cell types including cells that we characterize as type-II NB-derived intermediate progenitors (see below). Based on these analyses, we use the fez-mm eGFP expression as a reporter for Tc-fez/erm expression.

Figure 2

Tc-pointed marks a population of nine type-II NBs that are associated with fez-mm-eGFP marked INP-lineages

To identify type II NBs, we searched for cell groups that express the markers known from flies. The transcription factor pointed (pnt) marks type-II NBs in Drosophila and is required for their correct differentiation [40,41]. In both Tribolium and Drosophila, pnt is also expressed in other areas (this work/[47,48]). Therefore, we looked for Tc-pnt-expression in large cells with large nuclei (typical NB morphology) that were closely associated with Tc-fez/erm-expressing cells (marking putative INPs). We found a total of 9 such clusters instead of the 8 expected from flies (Fig. 3 A-C). We also found that the large Tc-pnt expressing cells as well as fez-mm-eGFP expressing cells are mitotically active (Fig. 3 C-I). Further molecular and cell size analysis corroborated our interpretation that these cells constitute type-II NBs, intermediate progenitors (INPs) at different maturation stages and ganglion mother cells (GMCs) (see details below).

Figure 3

Looking at a series of embryonic stages we determined the emergence and further development of Tc-pnt and fez-mm-eGFP expressing lineages as well as their arrangement with respect to the embryonic head and to one another. We first detected conspicuous adjacent expression of Tc-pnt and fez-mm-eGFP at stage NS7 (not shown). At stage NS11 seven Tc-pnt-clusters are present in a horse-shoe-shaped formation surrounding fez-mm-eGFP positive cells (white arrows in Fig. 3 A, star marks one cluster out of focus). At stage NS13 these groups are found in a position at the medial border of the head lobes and their number has increased to nine. The seven clusters that were already present at stage NS11 are arranged in an anterior group (white arrowheads in Fig. 3B) at stage NS13 while two additional clusters have emerged more posteriorly (yellow arrows in Fig. 3B-I). The clusters are in different depths (reflected by the three optical sections shown in Fig. 3 B-I to B-III, clusters out of focus are visualized by grey circles in Fig. 3 B’I-II, respectively). Fez-mm-eGFP expression not related to type II NB offspring was found in the lateral head lobe from NS13 onwards (blue arrowheads in Fig. 3). At stage NS14 six of the anterior-medial clusters are aligned in one plane along the medial rim of the developing brain (Fig. 3 C-I), whereas the most posterior cluster of the anterior group is located at a separate position and in a deeper plane (Fig. 3C-II). The posterior group is found at about the same focal plane as well (Fig. 3 C-II). The aligned six clusters produce their Tc-fez/erm-positive offspring towards laterally whereas the Tc-fez/erm-positive offspring of the more posterior cluster is found medially of it (Fig. 3 C-II). The two most posterior clusters produce Tc-fez/erm-cells in an anterior-lateral direction (Fig. 3 C-II). The apparent re-arrangement of the Tc-pnt-clusters from the anterior rim of the embryonic head to a medial position reflects not active migration but follows the overall tissue movements during head morphogenesis that was previously described in Tribolium [19] (Fig. 3 D). Based on their position by the end of embryogenesis (stage NS14) we assign the nine type-II NB clusters into two groups: one anterior median group consisting of seven type-II NBs and one posterior group consisting of two type-II NBs (see Fig. 3 C and D).

Characterization of cell- and nuclear size of pnt-positive type II NBs

We wanted to confirm that each of the Tc-pnt and Tc-fez/erm expressing lineages contains a neuroblast. Neuroblasts, including type-II NBs, are larger than other cells, have larger nuclei and are mitotically active [37]. Therefore, we determined the cell size of the largest cells of the Tc-pnt cluster and compared it to a random sample of cells of the embryonic head. Both samples were taken from stage NS13 and NS14 embryos. We found that the cells that we had assigned as Type-II NBs had a significantly larger diameter (av.: 12.98 µm; n=43) than cells of the reference sample (av.: 6.75 µm; n= 1579) (Fig. 4). We also found that the diameters of nuclei of type-II NBs were significantly larger (av.: 8.73 µm; n=17) than the ones of a control sample (av.: 5.21 µm; n=1080) (Fig. 4).

Figure 4

Conserved patterns of gene expression mark Tribolium type-II NBs, different stages of INPs and GMCs

Drosophila, type-II NBs, INPs and GMCs express a specific sequence of pro-neural genes including asense (ase), deadpan (dpn) and prospero (pros) (Fig. 1) [30]. However, these markers have up to now not been tested for expression in type-II lineages in other organisms. We tested if the respective lineages express these markers in a comparable sequence in the beetle and if they mark distinctive cell types within the lineages (Fig. 5 A-D and Fig. 6 A, B). We found that the large Tc-pnt-positive but fez-mm-eGFP negative cells (i.e. type-II NBs) express the pro-neural gene Tc-dpn, in line with expression of this gene in Drosophila neural precursors (Fig. 5, panel A). Like Drosophila type-II NBs, these cells do not express the type-I NB marker ase (yellow arrowhead in Fig. 5, panel D). We further found that, like the type-II NBs itself, the youngest Tc-pnt-positive but fez-mm-eGFP-negative INPs neither express Tc-ase (Fig. 5D, pink arrowheads). Expression of Tc-ase starts in slightly more mature (but still Tc-pnt-positive) INPs (Fig. 5 D; blue arrowhead) and is maintained in the Tc-pnt-negative INPs that are marked by fez-mm-eGFP (Fig. 5, panel D and E). Tc-dpn is absent from the youngest Tc-pnt-positive INPs (pink arrowheads) but is expressed in more mature INPs (blue arrowheads in Fig. 5, panel B). Cells that are located at the distal end of the lineages do not express Tc-dpn but are positive for fez-mm-eGFP and Tc-ase are classified as GMCs (Fig. 5, panels B, C and E). We found the pro-neural gene Tc-pros expressed in most fez-mm-eGFP expressing cells of a lineage (in Fig. 6, panel A-B) and infer from the extent of the expression that it is expressed in mature INPs (Tc-dpn+) and GMCs (Tc-dpn-). Fez-mm-eGFP positive cells at the base of the lineage that do not express Tc-pros are young INPs (Fig. 6 A, B; blue arrowheads). In summary, the expression dynamics found in the Tribolium type-II NBs lineages are very similar to the one found in Drosophila. Therefore these neural markers can be used for a classification of type II NBs (Tc-pnt+, Tc-ase-), young INPs (Tc-pnt+, Tc-fez/erm-, Tc-ase-), immature INPs (Tc-pnt+, Tc-fez/erm+, Tc-ase+), mature INPs (Tc-dpn+, Tc-ase+, Tc-fez/erm+, Tc-pros+), and GMCs (Tc-ase+, Tc-fez/erm+, Tc-pros+, Tc-dpn). This classification is summarized in Fig. 7 A-B.

Figure 5

Figure 6

Figure 7

Type-II NBs lineages produce central complex cells that are marked by shaking hands (skh)

To test if the Tribolium embryonic type-II NB lineages contribute to the beetle central complex like in the fly, we used the shaking hands- (skh-) reporter line that marks central complex neurons and their postmitotic embryonic precursors [49]. By co-staining for Tc-pnt and Tc-fez/erm RNA in the background of the skh-line we found that the skh-positive cells are located directly distally of the type-II NBs lineages in a position where the type-II NBs derived neural cells are expected (Fig. 8 panels A-F). Most skh-cells have switched off fez-expression, but at the transition between Tc-fez/erm and skh cells we found some cells that express both markers (Fig. 8 D, E), supporting the view that many skh-positive central complex cells stem from the fez-expressing GMCs of the type-II NBs lineages (Fig. 8 E). We found skh cells at the end of lineages of the anterior-median group (Fig. 8, panel A) and at the end the two posterior lineages (Fig. 8, B). We therefore conclude that both groups contribute to central complex neuropile (Fig. 8 F). We cannot say whether the lineage of type-II NB seven, which we assigned to the anterior-median group, but which is located with some distance to the other type-II NB of that group (Fig. 2 C, D, grey circle in Fig. 8 F), produces skh+ cells, but we assume that it does.

Figure 8

The Tribolium embryonic lineages of type-II NBs are larger and contain more mature INPs than those of Drosophila

In beetles, a single-unit functional central complex develops during embryogenesis while in flies, the structure is postembryonic [3,21]. As type-II NBs contribute to central complex development, we asked, in how far the embryonic division pattern of these lineages would reflect this heterochronic development. To assess the size of the embryonic type-II NBs lineages in beetles we counted the Tc-fez/erm positive (fez-mm-eGFP) cells (INPs and GMCs) associated with a Tc-pnt-expressing type-II NBs of the anterior medial group (type-II NBs lineages 1-7). As we were not always able to distinguish between cells belonging to neighbouring INP-lineages we quantified and averaged the cell number of all lineages of the anterior medial group. We also evaluated the proportion of dividing cells in that group based on anti-PH3 staining (table 1).

Table 1:

We found that at stage 13 each lineage consisted on average of 18,4 progenitor cells and at stage 14 of 16.8 progenitors. Between 5-10 % of the cells were marked by the PH3 antibody. As PH3 marks dividing cells for in a specific phase, the portion of dividing cells may be higher. We further quantified the number of young and mature Tc-dpn-positive INPs (table 2).

Table 2:

We used the numbers from Tribolium (table 1, 2) for comparison with type-II NBs lineages from Drosophila embryos (data from [29]). We compared our data for the anterior lineages of stages 13 and 14 to the anterior and median cluster in the Drosophila stage 15 and 16 embryos. In both species these stages are the two penultimate stages of embryogenesis when all type-II NBs lineages are present. We found that the beetle lineages were significantly larger than the corresponding ones in Drosophila (Fig. 9; Drosophila data taken from [29]). Due to a lower quality of the image data from this cluster which is in a deeper layer (see Fig. 3) we were not able to quantify cells of the Tribolium posterior group. In Drosophila, the posterior lineages are larger (av. 10 cells [29]) than the anterior and median ones, but they are still smaller than the anterior lineages of Tribolium at the corresponding stages. We also found that the embryonic type-II NBs lineages in Tribolium comprised significantly more mature INPs (Tc-dpn+/ Tc-fez+) than the corresponding lineages in Drosophila (Fig. 9). We conclude that the embryonic development of a functional central complex in beetles is associated with an increased activity of type-II NBs and INPs during embryogenesis.

Figure 9

Type-II NBs and their lineages are differentially marked by the head patterning transcription factors Tc-six4 and Tc-six3 but do not express Tc-otd

All type II lineages show the same developmental trajectory from NB via the INP to the GMC and express the respective marker genes. However, these lineages show at least partially diverging projection patterns [3,17,49]. Hence, we wondered, what genes might give type II NBs different spatial identities. We tested co-expression with previously characterized head patterning transcription factors Tc-otd, Tc-six4, Tc-six3, known to be active in the developing protocerebrum (Tc-otd) and in the anterior medial region of the neuroectoderm, where type-II NB emerge (Tc-six3, Tc-six4) [11,19,50,51]. We found no co-expression of fez-mm-eGFP marked cells of both the anterior medial and posterior lineages with Tc-otd. Rather, Tc-otd was expressed in the surrounding embryonic head tissue. Tc-otd was also absent from the type-II NBs themselves (Fig. 10 panel A-B). However, Tc-otd might be expressed in neural cells derived from these lineages as we can detect expression in the central area of the brain lobes where for instance skh cells are located (Fig. 9 panel A, F; 10 panel A-B). Interestingly, we found that Tc-six4 is expressed specifically in that area of the head lobe in which the type-II NBs clusters 1-6 of the anterior medial group first differentiate. At stage NS13 Tc-six4 marks both, type-II NBs and fez-mm-eGFP expressing INPs and some surrounding cells (Fig. 11, panel A). At the following differentiation stage (NS14) Tc-six4 still marks the type-II NBs clusters of the anterior medial group but is not expressed in the fez-mm-eGFP INPs (Fig. 11 panel B). It is also not expressed in the posterior group of type-II NBs but only marks type-II NBs of the anterior medial group. Finally, we found that Tc-six3 marks, and is restricted to, the anterior-most lineages 1-4 including both the type-II NBs and the fez-mm-eGFP marked INPs/GMCs (Fig. 11 panel C). These results reveal candidates for distinguishing type-II identity from type-I NBs in the brain. Only the latter is possibly marked by Tc-otd as Drosophila otd is expressed in type-I brain neuroblasts [52]. By contrast, Tc-six4 only marks type-II NB and appears to be an early determinant of the anterior medial group. Tc-six4 is also a candidate for distinguishing the anterior medial from the posterior cluster. The results also reveal a subdivision of the anterior cluster by Tc-six3, which marks the four most anterior NB-type II.

Figure 10

Figure 11

Discussion

A beetle enhancer trap lines reflects Tc-fez/earmuff expression

The discovery of the CRISPR-Cas9 system has brought the possibility of performing targeted genome editing to create mutants and knock-in lines to a wide range of less traditional model organisms [53]. The possibility of creating imaging lines that mark the expression of specific subsets of neural cells in the beetle greatly enhances its suitability as a model for developmental neurogenetics [11,15,17]. We used non-homologous end joining (NHEJ) to insert a reporter construct upstream of the Tc-fez/erm-gene and by these means created an enhancer trap [12,15]. Enhancer traps sometimes only reflect parts of a gene expression pattern, which can also make them more cell type specific. However, our line shows extensive co-expression with Tc-fez/erm RNA at the embryonic stage, suggesting that it reflects the entire expression of Tc-fez/erm at least at this stage.

Evolutionary divergence of number and grouping of embryonic type-II NBs lineages between beetle, fly and grasshopper

We have identified a total of 9 type-II NBs lineages on each side of the Tribolium embryonic head. This is surprising as in both, the hemimetabolan grasshopper Schistocerca gregaria and the fruit fly Drosophila melanogaster only 8 embryonic type-II NBs were identified [29,54]. Our finding of an additional type-II NB lineage at the embryonic stage represents a major evolutionary divergence between these insects. Further research is required to determine if this difference is associated with morphological differences of the brain. Although in the frame of this work we did not perform lineage tracing we can speculate about which NB might be lacking in fly embryos based on our data. In the hemimetabolan Schistocerca there is no obvious clustering of type-II NBs and they are all arranged in one row at the medial rim of the head lobes. In Tribolium we have observed an arrangement of the type-II NBs into two groups per side, one large anterior medial group containing seven type-II NBs clusters and one posterior group of two type-II NBs. The anterior six of the anterior medial group are arranged in one row like in the grasshopper. In Drosophila there are three described groups referred to as the anterior, the middle and the posterior cluster [29]. The posterior cluster consists of two type-II NBs and therefore most likely corresponds to the posterior two type-II NBs in Tribolium. The Drosophila middle and anterior cluster are most likely equivalent to the anterior medial group of Tribolium. However, the type-II NB 7, which is we assigned to the anterior medial group but which is a bit separated and produces offspring into the opposite direction (see Fig. 2 C-II, white arrow) might be the one that does not have a homologue in the fly embryo The identification of more specific spatial markers for type-II NBs (in addition to Tc-six3 and Tc-six4) or lineage tracing tools are required to identify the neuroblast, which is not present in fly embryos. Subsequently, it would be especially interesting to see what the role of the additional type-II NBs in the beetle is and which neuropile it contributes to.

Summing up, there is a tendency towards grouping of type-II NBs into subsets in the holometabolan models that was not observed in the only hemimetabolan studied so far. A ninth type-II NBs was only found in the Tribolium embryo represents a striking developmentally divergent feature, but its role and the homologisation of individual type-II NBs between the different insects requires further studies.

Gene expression identifies homologous cell types and suggests conservation of gene function between fly and beetle type-II NB lineages

The genes defining the different stages of differentiation in type-II NB lineages have been identified and intensively studied in Drosophila, but it had remained unclear, in how far the respective patterns and mechanisms were conserved. Our results show a large degree of conservation of expression and probably also function – at least in holometabola. We identified type-II NBs by their larger than average size [37] and the expression of the signature marker Tc-pnt adjacent to a group of Tc-fez/erm expressing cells (INPs and GMCs). In Drosophila both these factors have key functions in defining the developmental potential of type-II NBs and INPs: Pnt suppresses Ase in type-II NBs and promotes the formation of INPs [41]. Loss of pnt expression leads to a de-differentiation of INPs as fez/erm is no longer repressed in young INPs [40]. Fez/Erm controls proliferation of INPs by activating Pros and prevents dedifferentiation of INPs into type-II NBs [39]. We show that also in Tribolium INPs undergo a maturation process, during which expression of Tc-pnt stalls and expression of Tc-fez/erm is switched on. Despite both factors characterising different stages of INP maturation they are not completely mutual exclusive and we observed a small window of overlap in young INPs, highly suggestive of a conserved role of Pnt in the activation of fez/erm [40] (Fig. 12). Drosophila dpn is a neural marker expressed in type-II NBs and again in more mature INPs, leaving a gap of expression in immature INPs. Drosophila ase is repressed in type-II NBs but expressed together with fez/erm in INPs and GMCs where its expression overlaps with pros [55]. We found identical dynamics of gene expression in Tribolium (see Fig. 12) suggesting conservation of gene function within the lineages and a conserved process of INP maturation.

Figure 12

In conclusion, expression of the genes examined in this study in type-II NBs and their lineages is highly conserved in fly and beetle, which is implicit of conserved gene function and a conserved process of central complex formation.

Divergent timing of type-II NB activity and heterochronic development of the central complex

Previous work described a heterochronic shift in central complex formation between fly and beetle. In Tribolium parts of the central complex develop during embryogenesis. It becomes functional at the onset of the first larval stage, and is presumably required for the more complex movements of the beetle larvae using legs [3]. In both Drosophila and Tribolium the anterior groups of type-II NBs (anterior medial group of Tribolium and anterior and middle cluster of Drosophila) are fully present in the last 3^rd of embryogenesis, with the beginning of germ band retraction [8,29,56]. However, in the Drosophila embryo only a very limited number of INPs and GMCs are produced before they enter a resting stage prior to hatching [29]. By contrast, we show that Tribolium type-II NB lineages are much larger compared to Drosopohila [29]. They also include more mature, Tc-dpn expressing, cycling INPs which have the capability of driving the increase in lineage size. This increased activity very likely contributes to the embryonic development of a functional central body and protocerebral bridge [3]. The upstream factors responsible for the different timing of NB activity and the early formation of a functional central complex neuropile in the beetle remain to be identified.

A number of features remain to be studied in beetle type II lineages. We do not have any information whether Tribolium type-II NBs, or Tribolium neuroblasts in general, also enter a stage of quiescence at the end of embryogenesis as they do in Drosophila. After preformation in the embryo Drosophila type-II NBs have their main period of activity in the 3^rd larva and the lineages are much larger and contain more INPs than the Tribolium embryonic lineages [30]. It is another open question if Tribolium type-II NB lineages are active during larval development and how that relates to the described steps of central complex development. Adult like x, y, and w tracts as well as protocerebral bridge are present at the end of the embryonic stage but a bipartite central body containing both the ellipsoid and the fan shaped body is only differentiated in the pupa [3]. Therefore, it would be interesting to see if type-II NBs lineages are present and active in the late larval brain of Tribolium.

The placodal marker Tc-six4 may be responsible for the differentiation and the spatial identity of the anterior medial group of type-II NBs

The different type-II NB lineages contribute to different parts of the brain. For instance, only the anterior four type-II NB lineages form the w, x, y, and z tracts of the central complex in flies and grasshoppers. Hence, there must be signals that make them different despite the conserved and well-studied sequence of neural gene expression and functional interactions in Drosophila (Fig. 12) [30,39,40,57]. In the ventral nerve cord, the different identities of the NBs are specified by the combination of spatial patterning genes expressed in the neuroectoderm at the time of delamination [24]. However, little is known about these signals for type II NBs. In Tribolium the domains of head patterning genes in the brain neuroectoderm are well characterized and some highly conserved territories like the anterior-medial six3-positive domain were described and suggested to give respective NBs different spatial identities [11,19,50]. The Tc-six4 expression domain is also largely coincident with an embryonic structure termed insect head placode, a neurogenic, invaginating tissue [51,58] potentially homologous to the vertebrate adenohypophyseal placode marked by six4 [11,51]. A placodal origin of Drosophila type-II NB has been shown for one type-II NB, and it is assumed that the other type-II NB also stem from placodal tissue [29,46,59].

Tc-six4 is also a very interesting factor with regards to the specification of type-II NBs because it is expressed only in the anterior group of type-II NBs. Hence, it could contribute to distinguishing their developmental fate from the posterior group. This is however different in the Drosophila larva. Here Six4 is expressed in all eight lineages where it prevents the formation of supernumerary type-II NBs and a premature differentiation of INPs [43]. In the Tribolium embryo Six4 may have a similar rate limiting role within the anterior group type-II NBs. However, its early expression in a small part of the neuroectoderm [51] and the delamination of the anterior-medial type-II NBs group within this domain also hints at an instructive role of Tc-six4 in the formation of the anterior median group.

Tc-six3 marks a subset of type-II NBs whereas Tc-otd is absent from all lineages

Importantly, we found that in Tribolium late embryogenesis, Tc-six3 is expressed specifically within the lineages of type-II NBs 1-4 of the anterior medial group, but not in the other linages. In grasshopper and fly these anterior 4 lineages (DM1-4) give rise to the z, y, x and w tracts [25,54] and contribute crucially to the development of protocerebral bridge and central body. These tracts form a major midline crossing neuropile of the central body which serves as a scaffold in the development of this structure [3,25]. We conclude that the evolutionary ancient six3 territory gives rise to the neuropile of the z, y, x and w tracts. Indeed, the central body is also missing in weak Tribolium Tc-six3-RNAi phenotypes, suggesting that Tribolium six3 is required for the formation of this structure [19]. Given that central complex neuropile is marked by additional factors such as Tc-foxQ2 (Drosophila fd102c [60]) and Tc-rx-[3,17], it would be interesting to see the relationship of additional spatial patterning genes with regards to the type-II NB lineages in future studies.

The gene otd is a marker of the posterior protocerebrum and six3 and otd are believed to subdivide the embryonic anterior brain into two major domains [50]. This subdivision is also part of the recently revisited concept of an ancestral division of the insect protocerebrum into archicerebrum (six3-positive) and prosocerebrum (otd-positive) [11]. As discussed above, Tc-six3 is expressed in the anteriormost four type-II NBs lineages, but unexpectedly, we found that Tc-otd is specifically absent from all type-II NBs lineages, suggesting that its inhibition is required for the development of all these lineages. Interestingly, the lineages of type-II NBs 5 and 6 (and presumably of type-II NB 7) of the anterior cluster and the posterior type-II NBs lineages do neither express Tc-otd nor Tc-six3 showing that there are protocerebral structures expressing none of these conserved markers in development.

In summary, our findings are just the beginning of the quest for the genes required for the identity specification of type-II NB lineages. The genes known to be involved in head patterning are an excellent starting point for this purpose [11,18].

Methods

Creation of transgenic reporter line

Using CRISPR-Cas9 we have inserted a transgene into the Tc-fez/erm locus that includes egfp behind the Tribolium basal heat shock promoter (bhsp68), which is not active by itself but can be activated by nearby enhancer elements [61,62]. The transgene also contained the coding sequence of Cre-recombinase (not used in this work) transcribed from the same promoter, and the eye pigmentation gene Tc-vermilion behind the eye-specific 3xP3 enhancer element [63] (see Fig. S1 A for map of the transgene). On the repair plasmid the transgene was flanked by CRISPR target sequences derived from Dm-yellow and Dm-ebony, which were used for in vivo linearisation of the transgene.

Using CRISPR Optimal Target Finder [64] we designed three guide RNAs (gRNAs) targeting the genomic region 2.5 kb upstream of the Tc-fez/erm transcription start site (see table S1). We produced three plasmids where single gRNAs were transcribed from the Tribolium pU6b RNA promoter, as described in [12]. We co-injected the three gRNAs plasmids at 125µg/µl each, gRNAs targeting the Dm-yellow and Dm-ebony sequence at 125µg/µl each, Cas9 helper plasmid (see [12]) at 500 µg/µl and the repair plasmid at 500 µg/µl. Embryos of the white eyed Tribolium vermilion-white (vw) strain (G0) were injected, raised and then bred to vw-beetles in individual crosses. The offspring of these crosses (F1) were screened for black eyed heterozygous carriers of the transgene. These were again outcrossed to vw-beetles and the heterozygous offspring (F2) were further analysed genetically (see table S2) and with respect to fluorescent signal. Siblings of the F2 generation were then crossed to one another to generate the homozygous line (fez-mm-eGFP). Details of the insertion can be found in Fig. S1 A.

We also used the shaking hands (skh) enhancer trap line (G10011-GFP) which marks central complex cells [49].

Larval brain dissection, fixation, and staining

Larval brains of the newly generated Tc-fez/erm enhancer trap line fez-mm-eGFP were dissected and stained as described in [16]. Brains were mounted in Vectashield for microscopic inspection. See table S4 for a list of antibodies and staining reagent used.

Embryonic RNA in situ hybridisation, hybridisation chain reaction and antibody staining

Gene identifiers and primer sequences that were used for amplification can be found in table S3. The fragments were inserted into a pJet1.2 cloning vector. Standard RNA in situ probes were synthesised using the 5X Megascript T7 kit (Ambion) according to the manufacturers protocol. Embryo fixation and single colour RNA in situ chain reaction in combination with antibody staining to eGFP or phospo-histone H3 staining to mark mitoses was conducted as described in [65]. Embryos were also stained for DNA or cell membranes. A full list of antibodies and staining reagents can be found in table S4.

Probes for multicolour hybridisation chain reaction (HCR) binding Tc-fez/erm, Tc-dpn, Tc-ase and Tc-pnt were produced by Molecular Instruments (see table S5). Labelling reactions were performed as described in [66]. Antibody staining to eGFP was performed following the completion of the HCR staining (see [65]). All embryos were mounted in Vectashield for microscopic inspection (see table S4).

Image acquisition and analysis

Multichannel image stacks were recorded using a Zeiss LSM 980 confocal laser scanning microscope. Image stacks consisted of 100-300 slices, depending on the specimen. The resolution ranged from 1024 x 1024 to 2048 x 2048 pixels. Plane thickness was optimized according to width of the pinhole and ranged from 0.1 – 1 µm. We used FIJI [67] for 3 dimensional inspection, to export individual planes or to generate maximum intensity projections, as well as for quantification of cell size, cell number and number of mitoses. Statistics on the numerical data were performed using Microsoft Excel 2016. Cropping and adjustment of brightness and contrast was done with GIMP (version 2.10.32) or Adobe Photoshop CS5.

Acknowledgements

We would like to thank Claudia Hinners for technical support with molecular biology methods, and Elke Küster for help with screening and beetle stock keeping. We also thank Christoph Viehbahn for valuable feedback on the project. Author Simon Rethemeier was supported by a scholarship of the Göttingen Promotionskolleg für Medizinstudierende, funded by the Jacob-Henle-Programm.