Generation of neuronal diversity in the zebrafish spinal cord - How does the same combination of transcription factors lead to the differentiation of distinct neuronal subtypes?

• Applicant: Sepand Rastegar, Ph.D.
• Subject Area: Developmental Neurobiology ; Developmental Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517446433

Defining the mechanisms that regulate neuronal diversity is fundamental for understanding how the nervous system is built and how it functions. This knowledge will help to guide the formation of tailored nervous tissue in vitro and has bearings on the development of regenerative therapies. The cerebrospinal fluid-contacting neurons (CSF-cNs) in the ventral spinal cord of vertebrates, called Kolmer-Agduhr (KA) neurons, are evolutionarily conserved neurons that control locomotion and posture. Although KA´´ and KA´ neurons develop from two different progenitor domains of the spinal cord and differ in their location within the spinal cord, they are very similar in morphology - both contacting the cerebrospinal fluid. Additionally, they both express gad1b, a GABA-synthesizing enzyme, and are thus GABAergic inhibitory neurons. Furthermore, they express the transcription factors (TF) gata2a, gata3, tal1 and tal2. Astonishingly, however, gata2a and tal2 are required for the differentiation of gad1 expressing KA´´ cells, but they are dispensable for gad1 expression in KA´ cells, which instead depends on gata3 and tal1. Thus, the paradigm that simple combinatorial actions of transcription factors are key developmental determinants of neuronal diversification in the spinal cord does not seem to hold true for the specification of KA cells. Instead, regulatory mechanisms controlling the expression of specific genes can evolve rather flexibly by reshuffling common components of the regulatory circuits underlying further neuronal diversification into functional subtypes.We hypothesize that TFs are employed differently in KA´ and KA´´ cells because their target gene gad1b has acquired a different responsiveness to the combination of gata2a/tal2 and gata3/tal1, which finally defines the fate of KA´´ and KA´ cells. This may be because KA´ and KA´´ cells differentiate from different progenitors, namely the motoneuron (KA´) and the V3 (KA´´) progenitor domains, and/or be due to slight differences regarding the birthplace and birthdate of the two neuron types. Building on preliminary data and employing new genetic tools, such as single cell and ATAC sequencing and CRISPR/Cas9 gene editing, we want to investigate the following questions: What are the different roles of shared TFs in KA´ and KA´´ cells? For example, why are gata2a and tal2 dispensable for the control of gad1b expression in KA´´ cells, but not in KA’ cells? How do the transcriptomes differ between the two KA cell types? Are different cis-regulatory modules (CRMs) employed to drive gad1b expression and do they discriminate between the active and inactive factors? Are repressors present in one but not the other KA cell type?

• DFG Programme: Research Grants