Defining the mechanism of the dual spindle assembly and alignment in the mammalian zygote

Descripción del proyecto

Comprensión del fenómeno del ensamblaje de doble huso en los cigotos de mamíferos

Durante la primera división embrionaria, los cigotos de mamíferos inicialmente forman dos husos bipolares de los cromosomas maternos y paternos. Todavía se desconocen los numerosos aspectos de la función y el ensamblaje de doble huso, incluidas cuestiones sobre la involucración de la nucleación de microtúbulos, tanto citoplasmática como cromosómica, en la formación de husos y los casos de alineación propensa a errores. El proyecto financiado con fondos europeos DualSpindleAssembly se propone descubrir el mecanismo y la función del ensamblaje de doble huso utilizando microscopía de lámina de luz, análisis de imágenes computacionales y perturbaciones moleculares. La imagenología en 4D de alta resolución de cigotos de roedores vivos permitirá el rastreo de los puntos de nucleación individual y las puntas de los microtúbulos. Esto generará el primer mapa espacial de la nucleación de microtúbulos y contribuirá a descifrar la función de las dos poblaciones de microtúbulos en el ensamblaje de doble huso.

Objetivo

The first embryonic division after fertilization is essential for development of the organism and has to promote the union of the parental genomes. My host lab recently showed that in mammalian zygotes two bipolar spindles form, which first independently congress the maternal and paternal chromosomes and then must be aligned in parallel for a faithful division. The novel dual spindle assembly provided a likely rationale for erroneous divisions into more than two blastomeric nuclei observed in human fertility treatment. Indeed, preventing the alignment of the two spindles gives rise to multi-nucleated two-cell embryos also in mice. Due to its recent discovery and the difficulty of imaging in the light sensitive zygote, dual spindle assembly and function remains elusive. It is for example unclear what the contribution of cytoplasmic versus chromosomal microtubule (MT) nucleation is for forming two spindles and why spindle alignment is error-prone and variable between different mammals. Recent advances in microscopy in my host lab now enable me to address these questions. In my project, I will dissect the mechanism of dual spindle assembly and function by combining light sheet microscopy, computational image analysis, and molecular perturbation. To achieve this, I will perform 4D imaging of live mouse zygotes at high resolution that allows me to track individual nucleation sites as well as MT tips. This will allow me to generate the first spatial map of MT nucleation and assess what the contribution of the two MT populations for dual spindle assembly is. Moreover, I will test which MT nucleation pathway is essential for dual spindle assembly by molecular perturbations. Finally, I will check if errors in spindle alignment are the cause of parental genome loss, by identifying the key alignment factors and validate them in model organisms with different alignment fidelities. Thus, my studies will improve our understanding of cell division in mammals and human infertility.