The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development

Farnworth, Max S.; Eckermann, Kolja N.; Ahmed, Hassan M. M.; Mühlen, Dominik; He, Bicheng; Bucher, Gregor

doi:10.1007/978-1-4939-9732-9_11

Cited by 14 publications

(20 citation statements)

References 63 publications

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“…For Drosophila , we generated an imaging line using CRISPR/Cas9-mediated homology-directed repair ( S3 Fig ). We replaced the stop codon of the endogenous rx locus with a P2A peptide sequence (this viral peptide induces ribosomal skipping during translation such that two separate proteins are formed from one messenger RNA) followed by an EGFP coding sequence [ 39 , 61 , 62 ]. The resulting bicistronic mRNA led to translation of nonfused Dm-Rx and EGFP proteins ( Dm-rx -EGFP; S3 Fig ), and we found complete coexpression of Dm-Rx and EGFP.…”

Section: Resultsmentioning

confidence: 99%

“…(E) An analogous analysis in young pupal brains of cells surrounding the protocerebral bridge (E i ) revealed more EGFP-expressing cells (E ii ) with overlap to Tc-Rx cells (E iii ) than in the adult (D). [39]). Two gRNAs next to the endogenous STOP codon (guide A, brown dashed line) and downstream of the Dm-rx 3'UTR (guide B, blue dashed line) were used.…”

Section: S2 Fig Characterization and Validation Of Tribolium Rx-egfpmentioning

confidence: 99%

“…Actually, recent technical advances have opened the possibility to study the genetic and cellular basis of brain development not only in the classic model organism D. melanogaster but also in other insects in order to reveal conserved and divergent aspects. The red flour beetle T. castaneum is spearheading comparative functional genetic work in neurogenesis because of its welldeveloped genetic toolkit and advances in neurobiological methods [9,[37][38][39][40][41][42][43][44][45][46][47]. Recently, we suggested to compare homologous cells in different taxa by marking what we called genetic neural lineages, i.e., cells that express the same conserved transcription factor [9].…”

Section: Introductionmentioning

confidence: 99%

“…It should be noted, however, that the actual identity of a given neuroblast lineage is not determined by a single transcription factor but by a cocktail of several factors [ 48 ]. Genetic neural lineages can be labeled either by classic enhancer trapping or a targeted genome editing approach, both available in Tribolium [ 39 , 41 , 47 ].…”

Section: Introductionmentioning

confidence: 99%

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Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: A fly–beetle insight

2020

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Animal behavior is guided by the brain. Therefore, adaptations of brain structure and function are essential for animal survival, and each species differs in such adaptations. The brain of one individual may even differ between life stages, for instance, as adaptation to the divergent needs of larval and adult life of holometabolous insects. All such differences emerge during development, but the cellular mechanisms behind the diversification of brains between taxa and life stages remain enigmatic. In this study, we investigated holometabolous insects in which larvae differ dramatically from the adult in both behavior and morphology. As a consequence, the central complex, mainly responsible for spatial orientation, is conserved between species at the adult stage but differs between larvae and adults of one species as well as between larvae of different taxa. We used genome editing and established transgenic lines to visualize cells expressing the conserved transcription factor retinal homeobox, thereby marking homologous genetic neural lineages in both the fly Drosophila melanogaster and the beetle Tribolium castaneum. This approach allowed us for the first time to compare the development of homologous neural cells between taxa from embryo to the adult. We found complex heterochronic changes including shifts of developmental events between embryonic and pupal stages. Further, we provide, to our knowledge, the first example of sequence heterochrony in brain development, where certain developmental steps changed their position within the ontogenetic progression. We show that through this sequence heterochrony, an immature developmental stage of the central complex gains functionality in Tribolium larvae.

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Section: Resultsmentioning

confidence: 99%

Section: S2 Fig Characterization and Validation Of Tribolium Rx-egfpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: A fly–beetle insight

2020

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“…Unfortunately, foxQ2 enhancer trap lines were neither available for Tribolium nor Drosophila . Hence, we used CRISPR/Cas9 genome editing (Gilles et al, 2015) for a non-homologous end joining strategy to generate an enhancer trap in the Tc-foxQ2 locus that drives EGFP (see Materials and methods; Supplementary file 1-tables 2–4; Figure 3—figure supplement 1, (Farnworth et al, 2019). By double immunohistochemistry we confirmed that EGFP and Tc-FoxQ2 protein expression correlated quite well throughout embryogenesis (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium

Buescher

Farnworth

et al. 2019

eLife

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The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.

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Immunohistochemistry and Fluorescent Whole Mount RNA In Situ Hybridization in Larval and Adult Brains of Tribolium

Hunnekuhl

Siemanowski

Farnworth

et al. 2019

Methods in Molecular Biology

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The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development

Cited by 14 publications

References 63 publications

Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: A fly–beetle insight

Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: A fly–beetle insight

An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium

Immunohistochemistry and Fluorescent Whole Mount RNA In Situ Hybridization in Larval and Adult Brains of Tribolium

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