The development of a multicellular organism requires the precise control of gene expression in space and time so that cells adopt their correct identity. However, genetic mutations can alter this complex process. Recently, a transcriptional adaptation (TA) has been uncovered as one of the mechanisms underlying genetic compensation in zebrafish, mouse cells in culture, and C. elegans1,2. TA refers to the phenomenon by which mutated genes (with mRNA-destabilizing mutations) trigger the transcriptional up-regulation of other genes, called adapting genes. Mutant mRNA degradation, e.g., via Nonsense-Mediated Decay (NMD), has been shown to be required for TA3,4. However, little is known about the spatial and temporal characteristics of adapting gene regulation. Yet, TA would be required to be fast and regulated at all the different steps of gene expression in order for the embryo to develop correctly. Recent technological advances have made it possible to access the dynamics of transcription but only a few have been implemented in vertebrates thus far. This project aims to decipher when and where the TA occurs in the context of the developing zebrafish embryo. Aim 1 will test the hypothesis that TA is initiated during the zygotic genome activation and that there are several possible modes of transcriptional upregulation of the adapting genes. To test this hypothesis, I will use mutant alleles of early expressed genes that have been shown to exhibit TA including vcla, aldh2 and alcama. These genes and their corresponding adapting genes will be tagged with different live reporter arrays (e.g. MS2, PP7 loops) using standard genome editing tools in order to monitor transcription in live embryos with high-end microscopy followed by quantitative image analysis. This approach will allow us to determine the time-scale at which TA occurs and also help us understand the different modes of transcriptional upregulation of the adapting genes. Aim 2 will test the hypothesis that mutant mRNA degradation occurs in specialized organelles that are critical for TA. To test this hypothesis, I will use high resolution microscopy to look at the localization of NMD components as well as membrane-less organelles, such as P-bodies granules, together with the mutated and the wild-type mRNA. To test the importance of these granules, I will use mutants defective in P-granule formation and look at the effects on TA in terms of transcriptional output. Until now, TA has been mostly investigated on pooled populations of cells, therefore we lack the understanding of this phenomenon at a cellular level. This project will be important to fill this gap and get a better understanding of the spacio-temporal characteristics of genetic compensation which help the robustness of vertebrate development. 1. Rossi, A. et al. Nature 524, 230–233 (2015). 2. Ma, Z. et al. Nature 568, 259–263 (2019). 3. El-Brolosy, M. A. et al. Nature 568, 193–197(2019). 4. Serobyan, V. et al. Elife 9, e50014(2020).

Pathogenic RNA viruses pose a serious health concern. Influenza A virus (IAV) is a single-stranded negative-sense RNA virus with a segmented genome that causes seasonal flu epidemics and circumvents vaccination strategies as a result of genetic reassortment and antigenic drift. Thus, developing effective therapies requires understanding and targeting the conserved biology of the virus. IAV is an obligate intracellular parasite that depends heavily on host machinery for its replication and propagation. While it encodes a polymerase for RNA synthesis, it must make effective use of host RNA-binding proteins and ribonucleoprotein (RNP) complexes to coordinate splicing, nuclear import and export, and translation of each viral RNA segment in space and time. I hypothesize that the RNA lifecycle nuclear localized negative-sense viral RNA genomes (vRNA) and the cytoplasmic positive-sense viral mRNAs (vmRNA) are governed by entirely different sets of host RNPs that are recruited to their client RNAs in a spatiotemporally regulated manner. This study will identify the molecular architecture of the specific RNPs that assemble on each viral RNA segment in a strand-resolved manner to shape the fate of viral RNA. To address this challenge, I will leverage my graduate training in RNA biology and mass spectrometry (MS) to systematically characterise the dynamics of RNP formation on each IAV RNA segment using state-of-the-art MS methodologies. I will utilise RNA antisense purification mass spectrometry (RAP-MS) to capture each viral RNA segment in its positive and negative-sense orientation along with its direct protein interactors. These directly bound proteins will constitute the first layer of the IAV vRNA interactome. Next, I will identify proteins linked to direct RNA binders via protein-protein interactions by combining RAP with in situ cross-linking and mass spectrometry (in situ CLMS). In situ CLMS utilises cell-permeable cross-linking reagents to introduce a covalent bond between nearby residues on proteins in the native context of a virus-infected cell. This enables the identification of RNP complex members and also maps protein-protein interactions at the peptide level by MS-based identification of cross-linked peptide pairs. Hence, combining RAP with CLMS will provide not only the composition of vRNA-associated RNPs, but also reveal information on their 3D conformation and stoichiometry. Upon identifying key proteins and amino acid residues, I will dissect the structure-function relationship of identified RNPs using genetic means. I will perturb proteins and the interacting residues to assess their impact on viral RNA transport/localisation and vRNA replication. This project will yield fundamentally new insights into the host dependency of viral RNA biology and reveal the composition and structure of regulatory relevant vRNA-associated RNPs, which will lay the foundation for rationally designing novel antivirals.

To flexibly navigate their environments, animals create internal maps of the world. Such internal maps have been proposed to be crucial not only to spatial navigation, but also to abstract higher order cognitive inferences in humans. Seminal works in rodents have uncovered diverse representations of space in the brain, which likely implement these internal maps. However, the sheer complexity of mammalian brains has hampered our understanding of how microscopic circuits of neurons construct these internal maps. Larval zebrafish, with its small size, optical accessibility, and genetic tractability, is an ideal vertebrate model to study circuit mechanisms underlying neural representations of space. A recent work in larval zebrafish has discovered a group of neurons innervating a highly conserved brain structure called interpeduncular nucleus (IPN) whose population activity keeps track of the animal's heading by integrating its intended turns in an angular coordinate. However, it still remains unclear how this heading representation is used for flexible behaviors, as well as how the circuitry surrounding these compass-like neurons implement angular integration. My postdoctoral project will address these two key questions. First, to explore potential behavioral functions of the compass-like neurons, I will establish novel place learning assays that exploit ethologically relevant, naturalistic behaviors of larval zebrafish, such as positional homeostasis or hiding in the presence of visual threats. Using targeted laser ablation and optogenetic manipulation, I will then aim to demonstrate that the heading representation in the compass-like neurons is involved in such spatial behaviors. Second, I will attempt to identify the sources of turn-related motor signals provided into the compass neuron network. To this end, I will first morphologically identify neuron populations that innervate both the compass neurons and the IPN using light-inducible gene expression systems and/or photoactivatable fluorescent proteins. I will then functionally characterize the activity of these identified neurons with two-photon calcium imaging to see if any of them encode intended turning of fish. In parallel, I will perform electron micrograph reconstruction of IPN-projecting neurons to find circuit motives that can support angular integration. Overall, these experiments will uncover the functions and mechanisms of a conserved circuit that computes heading directions, shedding a new light to how internal maps of the world are constructed in vertebrate brains, including ours.