The insect central complex as model for heterochronic brain development-background, concepts, and tools

Abstract

The adult insect brain is composed of neuropils present in most taxa. However, the relative size, shape, and developmental timing differ between species. This diversity of adult insect brain morphology has been extensively described while the genetic mechanisms of brain development are studied predominantly in Drosophila melanogaster. However, it has remained enigmatic what cellular and genetic mechanisms underlie the evolution of neuropil diversity or heterochronic development. In this perspective paper, we propose a novel approach to study these questions. We suggest using genome editing to mark homologous neural cells in the fly D. melanogaster, the beetle Tribolium castaneum, and the Mediterranean field cricket Gryllus bimaculatus to investigate developmental differences leading to brain diversification. One interesting aspect is the heterochrony observed in central complex development. Ancestrally, the central complex is formed during embryogenesis (as in Gryllus) but in Drosophila, it arises during late larval and metamorphic stages. In Tribolium, it forms partially during embryogenesis. Finally, we present tools for brain research in Tribolium including 3D reconstruction and immunohistochemistry data of first instar brains and the generation of transgenic brain imaging lines. Further, we characterize reporter lines labeling the mushroom bodies and reflecting the expression of the neuroblast marker gene Tc-asense, respectively.

Keywords: Brain; Central complex; Drosophila; Evolution; Heterochrony; Tribolium.

Fig. 1

Diversity of adult insect brains. Shown are illustrations of the brains of the vinegar fly Drosophila melanogaster (a), the red flour beetle Tribolium castaneum (b), the bee Apis mellifera (c), and the desert locust Schistocerca gregaria (d). Based on (Rein et al. ; Kurylas et al. ; Dreyer et al. ; Rybak et al. 2010). All brains were sized to the same width and the respective neuropils have the same color code: blue antennal lobes, red mushroom bodies, yellow lamina of optic lobes, orange lobula of optic lobes, green central complex

Fig. 2

Heterochronic development of the central complex. a–d The central complex of adult specimen of the vinegar fly Drosophila melanogaster (a), the red flour beetle Tribolium castaneum (b), and the desert locust Schistocerca gregaria (c) are shown sized to the same width. Note that the overall architecture of the CX components is similar as is their basic connectivity (not depicted). Central body with fan-shaped body (FB) and ellipsoid body (EB). No noduli; PB protocerebral bridge. d Heterochronic development of the CB is depicted schematically for the species (a–c). This neuropil is fully developed in desert locust hatchlings, representing the ancestral condition. In the beetle, only the FB (dark green) is present while the EB (light green) is added postembryonically. In the vinegar fly, the neuropil becomes apparent only at late larval and metamorphic stages. Light colors and hatched outlines mark neuropils that are developing but not yet functional while white indicates lack of detectable neuropil structure. Note that neuropil shapes are unified and other CX neuropils have been omitted for simplicity. a–d Redrawn from (Hanesch et al. ; Dreyer et al. ; Kaiser 2014)

Fig. 3

The first instar larval brain of Tribolium castaneum and expression of neuromodulators in the CB. a–d Dorsal view of a first instar larval brain stained with an antibody detecting synapsin. The level of the sections is displayed in the reconstruction (f). e–g 3D reconstruction of neuropils based on the synapsin staining (a–d). Color code: blue antennal lobes (AL), yellow (anlagen of the) optic lobes (OL), and red mushroom bodies (MB) with calyx (CA); PE pedunculus, vL vertical lobe, and mL medial lobe. The cortex layer containing most cell bodies is shown in light gray, while the entire neuropilar mass of the brain is shown in dark gray (Ne). h–q Optical sections through the CB in adults (h, l) and first instar larvae (m, q) stained against serotonin (5HT), myoinhibitory protein (MIP), allatotropin (AT), tachykinin-related peptide (TKRP), and periviscerokinin (PVK). Neural anterior (n-anterior; NA) is up in all panels. FB fan-shaped body, EB ellipsoid body. The staining in the larval CB resembles staining of the FB in adult brains, corroborating the previous assumption that only the FB develops during embryogenesis. The anterior rim of the adult FB lacks MIP and AT expression (white arrowheads in iʹ and jʹ), which is not the case in larval CB. An n-anterior lack of TKRP reactivity, in contrast, is found in both larval and adult brains (white arrowhead in kʹ and pʹ). Scale- and orientation bars (a) account for (b–d), bars (h, m) account for i–l, n–q, hʹ–lʹ, and mʹ–qʹ

Fig. 4

Transgenic in vivo imaging reporters for brain research in Tribolium castaneum. a, b The regulatory region of Tc-asense drives expression of Gal4 in neuroblasts. The overlap of expression was confirmed with double in situ hybridization detecting Tc-ase (red) and gal4 (green). a–a″ mid-embryogenesis; b–b″ late embryogenesis. c–e Developmental series of the fluorescence signal of the brainy line. The ECFP signal marking glia is shown in white while the DsRed-Express signal in neurons is depicted in red. Embryos are oriented n-anterior to the top. White arrowhead marks the developing central body; stars indicate the antennal lobes while open arrows mark the median lobes of the mushroom bodies. d, e The glial sheath of the central body is first detected in 60–64-h-old embryos and is clearly visible in brains of hatchlings (e). See Online Fig. S2 in Resource 2 for earlier stages