Cell sorting and lineage specification at the midbrain-hindbrain boundary

• Applicant: Professor Dr. Michael Brand
• Subject Area: Developmental Biology ; Developmental Neurobiology ; Evolutionary Cell and Developmental Biology (Zoology) ; Cell Biology
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 251970880

During development, embryonic cells progressively organize into tissues and organs, with a highly characteristic complement and arrangement of cells. In developing vertebrates, this occurs in a stepwise fashion, under the control of cell groups known as organizing centers that use extracellular signals to instruct neighbouring cells about their cell identity. The midbrain-hindbrain boundary (MHB) contains a paradigmatic organizer cell population located at the interface between the forming midbrain and hindbrain territories. Embryological studies showed previously that it is of crucial importance to arrange and maintain both the organizer cell population itself, and the surrounding cells, in proper spatial order, but how this is achieved mechanistically is not well understood. Specifically, we and others have shown that the MHB is a lineage restriction boundary that prevents the intermingling of prospective midbrain and hindbrain cells during embryonic development, but the molecular mechanisms underlying this are unclear. In this project, we have used a combination of multiple, in-house-generated CRISPR-based knock-in reporter lines for genes expressed at the MHB, atomic force microscopy of single cells, and molecular force sensors in vivo, to show that prospective midbrain and hindbrain progenitors display differential adhesive and tensile properties during the early developmental stages. Further, disruption of Eph-ephrin signalling resulted in mis-sorting of cells across the MHB. These observations imply that, Eph-ephrin signalling may contribute to the molecular mechanisms leading to differential adhesion and tension. Building on these results, the proposed project aims to determine if the observed differential adhesion and tension are essential for cell sorting and restriction during MHB formation and maintenance. Specifically, we propose to (i) use genetic and optogenetic tools, in combination with transgenic reporter and Cre-recombinase driver lines, to target actomyosin and cytoskeletal components specifically in prospective midbrain and hindbrain cells, and to visualize the effects of their disruption on MHB formation. (ii) Second, we will address the nature and cell-fate potential of the boundary cells, using cell type-specific ablation and live imaging to track early Otx-Gbx overlapping boundary cells marking midbrain and hindbrain development, respectively, and carry out lineage tracing experiments for these cells, as well as organizer cells. Finally, we will (iii) determine the function of Eph-ephrin signalling for establishing differential adhesion and tension across the MHB, by evaluating the phenotype of mutants and after pharmacological blockade of Eph-ephrin signalling, in combination with molecular force sensors. The results will allow better understanding of ordered tissue formation, boundary formation and patterning which occur with such marvelous precision during embryonic development.

• DFG Programme: Research Grants