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The physical linking of sister chromatids after S-phase is known as sister chromatid cohesion (SCC) and is largely provided by the cohesin complex. The coordinated loss of SCC at anaphase onset is essential for correct chromosome segregation, a process mediated by proteolysis of the kleisin subunit of cohesin. In addition, during mitotic prophase of most organisms a large portion of cohesin is removed from chromosomes by a non-proteolytic pathway that depends on the conserved protein WAPL. Cohesin is also known to reload and SCC is re-established in response to DSBs after mitotic S-phase. During meiosis, SCC is similarly established during S-phase, but is then released in two steps during the sequential meiotic divisions. Also, unlike mitosis, multiple cohesin complexes with divergent functions exist in meiosis. Whether cohesin is dynamically associated with chromosomes during meiotic prophase and how this may be regulated was not known. Thus, the key aims of this project were to determine if WAPL mediates cohesin removal during meiotic prophase, and to find out if meiotic cohesin complexes display turnover on prophase chromosomes of the nematode C. elegans. Alternative meiosis-specific kleisins (REC-8, COH-3, and COH-4) define the different complexes present during worm meiosis. I show here that WAPL-1 limits the association of COH-3/4 complexes with meiotic prophase chromosomes, which severely limits their cohesive function. REC-8 complexes on the other hand are not affected much by WAPL-1. I show that loss of WAPL-1 affects the structure of axial elements and disrupts chromosome segregation and DSB repair. I also demonstrate by FRAP live imaging that there is significant turnover of cohesin on meiotic prophase chromosomes. Dynamic turnover of COH-3 is much greater that REC-8, as predicted by the different sensitivity to WAPL-1 of REC-8 and COH-3 cohesin complexes. These findings demonstrate that cohesin is actively removed and reloaded during meiotic prophase. Dysregulation of these processes could be relevant for human fertility, since SCC exhaustion over time is thought to contribute to the decline in fertility with increased maternal age. Open Access

Máster Universitario en Neurociencia: de la Investigación a la Clínica., [EN] NMDA receptors (NMDARs) are glutamate-gated excitatory ion channels with central roles in synapse plasticity, learning and memory. NMDAR complexes containing the GluN3A subunit (GluN3A-NMDARs) possess distinct biological properties. GluN3A expression is largely restricted to short windows of postnatal development, having established roles in sensorydependent postsynaptic refinement. Ongoing work in the host lab has begun to demonstrate likely involvement of GluN3A in the refinement of axonal structures. Building from these findings, this project tested the influence of GluN3A on callosal axon development, setting up CTB retrograde tracing in two different injection, studying different aspects of callosal development. Both types of CTB injections were successfully established and optimized in the host lab. Cortical injections in young (P13) mice revealed a decrease in axon targeting to S1/S2 border region of the somatosensory cortex (SSCx) in Grin3a KOs. This indicates increased promiscuity for these SSCx CPNs, consistent with prior findings from In Utero Electroporation (IUE) labelling. Other injections into the Corpus Callosum (CC) of mature mice did not identify permanent differences in CPN distribution patterns. This may be due to a transient nature of phenotypes, as indicated in prior work. Additionally, RT-qPCRs on P11 SSCx confirmed a significant upregulation of Kirrel2 mRNA in Grin3a KO mice. Kirrel2 is an adhesion molecule known to control axon guidance, being an interesting candidate. Future experiments with a larger sample numbers, as well as injections into other regions of the SSCx and at different ages, will allow a clearer understanding of the role of GluN3A in circuit development., [ES] Los receptores NMDA (NMDAR) son canales iónicos excitatorios activados por glutamato que desempeñan un papel fundamental en la plasticidad de sinapsis aprendizaje y memoria. Los complejos NMDAR que contienen la subunidad GluN3A (GluN3A-NMDARs) poseen propiedades biológicas distintas. Su se restringe temporalmente durante el desarrollo postnatal, con funciones de refinamiento postsináptico dependiente de experiencia. Trabajos realizados en este laboratorio han comenzado a demostrar la probable participación de GluN3A en el refinamiento de estructuras axonales. Así, este proyecto probó la influencia de GluN3A en el desarrollo de los axones callosos, a través del marcaje retrógrado de CTB en dos inyecciones diferentes, estudiando distintos aspectos del desarrollo calloso. Ambos tipos de inyecciones de CTB se establecieron con éxito y se optimizaron en el laboratorio. Inyecciones corticales en ratones jóvenes (P13) revelaron una disminución de la orientación de los axones hacia la región del borde S1/S2 de la corteza somatosensorial (SSCx) en los Grin3a KO. Esto indica un aumento de la promiscuidad de estos CPNs de la SSCx, lo que concuerda con los hallazgos obtenidos por IUEs. Otras inyecciones en el cuerpo calloso (CC) de ratones adultos no identificaron diferencias permanentes en los patrones de distribución de los CPNs. Esto puede deberse a la naturaleza transitoria de los fenotipos. Además, RT-qPCRs en P11 SSCx confirmaron un aumento significativo del mRNA de Kirrel2 en los ratones Grin3a KO. Kirrel2 es una molécula de adhesión que controla la guía axonal, siendo un candidato interesante. Futuros experimentos con más muestras, así como inyecciones en otras regiones de la SSCx y a diferentes edades, permitirán una comprensión más clara del papel de GluN3A en el desarrollo del circuito.