RNAseq experimental analysis

To study the molecular mechanisms involved in fate decision for liver and pancreas, which originate from a common progenitor, we profiled gene expression in liver and pancreas progenitors from two time points using RNAseq [1]. The non-canonical Wnt pathway was identified and found to be able to induce the pancreatic lineage. One of the genes observed to be differentially expressed in pancreatic or hepatic lineage commited cells was the TALE homeodomain-containing transcription factor TGIF2. As a follow up of this study, we were able to use the TGIF2 protein (using a lentiviral expression vector) to reprogram adult mouse hepatocytes into pancreatic progenitors in vitro and in vivo [2], characterized using RNAseq of the transformed cells.

The sea urchin is a non-chordate deuterostome that is used as a model for embryonic development. We measured gene expression at eleven time points during embryonic and larval development of Lytechinus variegatus. the sea urchin Atlantic species, which is currently not so well studied as Strongylocentrotus purpuratus, the Pacific species. Expression changes are observed not to be gradual but rather separated by three abrupt transitions (hatching, gastrulation and morphogenesis) in four stages during which expression is rather stable [3]. We propose to investigate if punctuated changes in expression are an organizational feature of the development of all animal and plant embryos.

Huntington's disease is a neurodegenerative disease caused by a mutation in the huntingtin gene that causes neuronal death in the brains of older people. To search for potential marker genes in blood, we produced and analysed the transcriptome of peripheral blood from patients and matched controls [4]. We found a number of genes significantly dysregulated, prominently, the upregulation of genes related to inflammatory and immune response, indicating that the progression of the disease in the brain can indeed be detected in the blood. This result implies that the effect of therapies could potentially be assessed using blood tests.

The gene USH1C (encoding the Harmonin protein) is particularly expressed in multiple splice isoforms in the human retina under the regulation of retina-specific splicing factors. Using RNA-seq of samples from human retina, we identified four major splice variants with different domain organizations, with the most frequent variant (harm_a1) constituting 50% of transcripts [5]. The cell types expressing Harmonin, subcellular localization and interactions, suggest the involvement of this protein in USH1, the most severe variant of the Usher syndrome (USH), a complex genetic disorder that is the most common form of hereditary deafness-blindness. This work suggests that delivery of the harm_a1 transcript could be a promising gene therapy.

Following work in Drosophila melanogaster implicating Yki in the activation of quiescent neural stem cells (NSCs), we studied if the the mammalian ortholog Yap1 could have a similar function. In mammals, there is an adult population of NSCs in the hippocampus, which remains in a quiescent state (not doing cell cycle) waiting to be activated. We found that during activation of NSCs Yap1 moves from the cytoplasm to the nucleus. In vivo deletion of Yap1 in mouse NSCs resulted in long-term (after 60 days) reduction on NSC activation. While overexpression of Yap1 on NSCs had no effect, overexpressing a constitutively active form of Yap1 (Yap1-5SA, that lacks five serines that can be phosphorylated) resulted in adult hippocampal NSC activation. Single cell RNAseq (scRNAseq) analysis of in vivo NSCs activated by lentiviral induction of Yap1-5SA at various time points, indicated cell populations of activated NSCs but also groups of cells with aberrant gene expression at later time points, which could reflect processes leading to glioblastoma [6].

To explore the role of miRNAs in venous thromboembolism (VTE), we studied transcriptomics of miRNAs present in the plasma (circulating miRNAs) of 181 persons with acute VTE (discovery cohort) followed by validation of results in similar data obtained from 315 persons with VTE (independent validation cohort) from a population-based cohort. We obtained a signature of 52 miRNAs associated with recurrent VTE or death in the discovery cohort (maximum follow-up of 6 years) [7]. Of those miRNAs, 19 were validated in the second cohort, 5 of them with publications reporting their association with VTE, recurrent VTE or thrombus resolution (the natural dissolution of a clot): miR-150, miR-424, miR-490, miR-183 and miR-379.


[1] Rodriguez-Seguel, E., N. Mah, H. Naumann, I.M. Pongrac, N. Cerdá-Esteban, J.F. Fontaine, Y. Wang, W. Chen, M.A. Andrade-Navarro, F.M. Spagnoli. 2013. Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineages divergence. Genes and Development27, 1932-1946. Seguel13_cover

[2] Cerdá-Esteban, N., H. Naumann, S. Ruzittu, N. Mah, I.M. Pongrac, C. Cozzicorto, A. Hommel, M.A. Andrade-Navarro, E. Bonifacio, F.M. Spagnoli. 2017. Stepwise reprogramming of liver cells to a pancreas progenitor state by the transcriptional regulator Tgif2. Nature Communications. 8, 14127.

[3] Hogan, J.D., J.L. Keenan, L. Luo, J. Ibn-Salem, A. Lamba, D. Schatzberg, M.L. Piacentino, D.T. Zuch, A.B. Core, C. Blumberg, B. Timmermann, J.H. Grau, E. Speranza, M.A. Andrade-Navarro, N. Irie, A.J. Poustka and C.A. Bradham. 2020. The developmental transcriptome for Lytechinus variegatus exhibits temporally punctuated gene expression changes. Dev. Biol. 460, 139-154.

[4] Andrade-Navarro, M.A., K, Muehlenberg, E. Spruth, N. Mah, A. González-López, T. Andreani, J. Russ, M.R. Huska, E.M. Muro, J.F. Fontaine, V. Amstislavskiy, A. Soldatov, W. Nietfeld, E.E. Wanker, J. Priller. 2020. RNA sequencing of human peripheral blood cells indicates upregulation of immune-related genes in Huntington’s disease. Frontiers in Neurology. 11, 573560.

[5] Nagel-Wolfrum, K., B.R. Fadl, M.M. Becker, K.A. Wunderlich, J. Schäfer, D. Sturm, J. Fritze, B. Gür, L. Kaplan, T. Andreani, T. Goldmann, M. Brooks, M.R. Starostik, A. Lokhande, M. Apel, K.R. Fath, K. Stingl, S. Kohl, M.M. DeAngelis, U. Schlötzer-Schrehardt, I.K. Kim, L.A. Owen, J.M. Vetter, N. Pfeiffer, M.A. Andrade-Navarro, A. Grosche, A. Swaroop and U. Wolfrum. 2023. Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy. Hum. Mol. Gen. 32, 431-449.

[6] Fan, W., J. Jurado-Arjona, G. Alanis-Lobato, S. Péron, C. Berger, M.A. Andrade-Navarro, S. Falk and B. Berninger. 2023. The transcriptional co-activator Yap1 promotes adult hippocampal neural stem cell activation. EMBO J. In press.

[7] ten Cate, V., S. Rapp, A. Schulz, A. Pallares Robles, K. Jurk, T. Koeck, C. Espinola-Klein, M. Halank, H.J. Seyfarth, M.E. Beutel, A.K. Schuster, F. Marini, L. Hobohm, M. Lankeit, K.J. Lackner, W. Ruf, T. Münzel, M.A. Andrade-Navarro, J.H. Prochaska, S.V. Konstantinides, P.S. Wild. 2023. Circulating microRNAs predict recurrence and death following venous thromboembolism. J. Thromb. Haemost. 21, 2797-2810.