Research Overview
Neuroendocrine cells have an ancient evolutionary origin and predate the emergence of neurons. The integration of this system into bilaterian centralized nervous systems and the routes of developmental and functional divergence are subject of ongoing research.The lab studies the evolution and development of the insect neuroendocrine system with special focus on cell groups located in the brain and suboesophageal ganglion. We aim to functionally characterize these cells and elucidate their evolutionary and developmental origin. Further we ask which transcriptional networks are involved in the specification of these cells and how their development is interconnected with other processes in brain and ventral nerve cord development.
Our model is the beetle Tribolium castaneum, which is amenable to transgenesis and suppression of gene function. While most functional genetic data on insect CNS development and neurosecretory cell specification comes from the fly Drosophila melanogaster, we believe that insights from Tribolium are extremely valuable to add a comparative basis to understand which features of neuroendocrine development are conserved within insects and which ones might be species-specific adaptations.
There is reason to believe that the neuroendocrine system of the fly is of a derived state, which is for example reflected in the genomic loss of neuroendocrine pathway genes (see below). There is also growing evidence that many developmental processes described in the fly represent a mode specific to this lineage only. Therefore, we believe that Tribolium neuroendocrine system development and function are more representative for insects in general and will facilitate comparisons to more distantly related organisms.
Our toolkit involves cell and tissue labelling methods and methods to manipulate gene function.
Genome editing using CRISPR-Cas9 allows the generation of fluorescent reporter lines for neural and neuroendocrine development. One approach is to induce non-homologous end joining (NHEJ) repair and by this means create enhance trap lines. For this method we achieve a high transformation rate. Depending on the enhancer structure of the targeted gene only a subset of the expressing cells might be labelled which can be advantageous in some contexts. To precisely mimic the expression of a gene with a fluorescent reporter we use CRISPR-Cas9 induced homology directed repair (HDR). Currently this method is more laborious but allows an exact base-by-base editing and thus the in-frame fusion of a fluorescent reporter to an open reading frame.
To study the role of specific genes in neural- and neuroendocrine development we make use of the strong systemic RNAi response of this beetle. We can perform embryonic, larval, pupal or adult RNAi. If injecting female pupae or adults the parental RNAi effect is transmitted to the first-generation offspring (parental RNAi).
Inducing RNAi by injecting double stranded RNA into the whole body is a powerful method but it may induce pleiotropic or sterility effects, especially when applied in the neuroendocrine context. Therefore, we are working towards developing a conditional system that restricts the interruption of gene function to individual tissues or cell groups. For this we use the Cre-Lox-system in combination with CRISPR-Cas9. We are developing transgenic lines in which the expression of a Cas9-gRNA cassette is only active in the nervous system.
Projects
Neurosecretory pathway genesThe regulated neurosecretory pathway using genetically encoded neuropeptides is an evolutionary ancient signalling system used by all bilaterians. Neuropeptide precursors undergo posttranslational processing by a conserved cassette of enzymes including prohormone convertases (PCs) and carboxypeptidases (CPs). Most animals use two orthologues of both: PC1/3 and PC2 for internal cleavage and CPD and CPE for carboxyterminal processing. Notably, flies including the model Drosophila have suffered genomic loss of PC1/3 and CPE, whereas most other insect groups have retained a complete gene inventory of neurosecretory pathway genes.
Using RNAi in Tribolium we found that neither PC1/3 nor CPE shares functional redundancy with its respective paralogue. Interestingly, we found thatCPE is non-dispensable in embryonic development and that PC1/3 has an essential individual function in larval growth.
The Tribolium PC2 gene (Drosophila: amontillado) is expressed in many cells of the embryonic neuroectoderm and of the larval and adult nervous system. PC1/3 is specific to few cells in the suboesophageal ganglion, and potentially is additionally expressed in the digestive system. We aim to further analyse the neuropeptide targets of PC1/3 and to delineate the function in the nervous system from a function in the gut by applying a nervous system specific knockout system (see above).
Development of the central neuropeptidergic brain
The anterior insect brain contains the pars intercerebralis, a neurosecretory brain centre which is associated with the central complex. Using a combination of marker genes visualised by GFP-reporter constructs, antibody labelling and RNA-in situ hybridization we have characterized embryonic cell types that contribute to the Tribolium central complex and characterized a population of type-II-neuroblasts and intermediate progenitors. When compared to Drosophila these intermediate progenitors undergo an increased phase of mitotic activity at the embryonic stage which leads to the early formation of functional central complex neuropile.
We also found that an anterior medial subset of 3 type-II-neuroblasts is marked by the transcription factors six3 and foxQ2, both of which are markers for a conserved apical brain region that gives rise to neurosecretory brain centres in diverse animals. Further, another six-gene, which is marker for invaginating placodes that give rise to the vertebrate neurohypophysis, marks the tissue from which the anterior-medial group of NB-type-II delaminate.
Using our reporter lines, we are dissecting molecularly different subpopulations of the pars intercerebralis and tracing their embryonic progenitor cells. We are further interested in the question how identified transcriptions factors confer a specific neuropeptidergic profile to individual cell groups.
Transcription factors in neuroendocrine development and regulation
We are interested in how transcription factors coordinate the specification of neurosecretory cells and how they are involved in neurosecretory activity and control. We are RNAi-screening transcription factors and assess the treated animals for defects in neural and/or neuroendocrine development, neuroendocrine marker gene expression, and for typical neuroendocrine phenotypes that affect fertility or the moult cycle.
We have identified a homeodomain transcription factor that is expressed in the brain and that negatively regulates egg production: if this factor is knocked down the number of eggs laid by female beetles increases, and so does the expression of two Tribolium vitellogenin (yolk protein-) genes.
We are further elucidating the regulatory networks and the putative involvement of secreted factors affected in this phenotype by conducting mRNA-sequencing and follow-up functional experiments in combination with cytological stainings.