Study of the interpeduncular nucleus and the trigeminal sensory complex as paradigms of neuronal migration and brain segmentation in the hindbrain

Abstract

This doctoral thesis is included in the field of Developmental Neurobiology. The main objective has been to deepen the knowledge on two fundamental processes for brain formation: neuronal migration and brain segmentation. To this end, I have focused on two structures respectively related to these processes: the interpeduncular nucleus (IPN), that previous studies have described as a model of neuronal migration in the rostral hindbrain; and the trigeminal sensory column, which spans through successive segmental units along the hindbrain. For the study of both structures, this work takes as basis the neuromeric model, that considers successive segmental or metameric units along the longitudinal axis of the brain. These neuromeres are called rhombomeres (r) in the hindbrain. They display specific expression of combinations of developmental genes and give rise to respective transverse domains of the adult hindbrain. Regarding the IPN, it is formed entirely by neuronal migration of various neuronal populations with different dorsoventral and rostrocaudal origins. This structure is subdivided into three rostrocaudal components, the prodromal (Pro) located in the isthmus (also called r0), and the interpeduncular rostral (IPR) and interpeduncular caudal (IPC), placed in rostral r1 (r1-r) and caudal r1 (r1-c), respectively. Some authors have proposed that r1-r and r1-c can be considered as independent segments, so that the three aforementioned IPN domains would correspond to respective neuromeres. Previous studies demonstrated that the cell populations of the IPN express several transcription factors (TF) (Nkx6.1, Pax7, Otp, Otx2, Irx2) during the development of this structure, thus allowing to determine their origin, migratory pathways, and final fate. In study 1 (García-Guillén et al., 2020) the objective was to study the IPN migration process in a mouse mutant lacking DCC (Deleted in Colorectal Cancer), which is a receptor for Netrin-1. This molecule participates in multiple migration processes during hindbrain development. The principal technique used was in situ hybridization with RNA probes for the forementioned TF in knockout DCC-/-versus wildtype brains. Next, in study 2 (García-Guillén et al., 2021) we focused on the regionalization within this nucleus, by searching for genes differentially expressed within the subregions of the IPN. We performed this screening in the Allen Developing Mouse Brain Atlas (ADMBA) (https://developingmouse.brain-map.org/). Studies 1 and 2 were performed at perinatal stages (mainly E18.5 and also P4) because the IPN is a late-formation nucleus, so that the screening of genes was thought to give rise genes that could participate both in the neuronal migration and in the regionalization of this nucleus. Concerning the trigeminal sensory column, it extends along most of the hindbrain, spanning thus several rhombomeres. This column is formed by the principal sensory nucleus (Pr5), located in rhombomeres r2 and r3, and the spinal trigeminal nucleus (Sp5), which covers from r4 down to the hindbrain/spinal cord boundary, according to previous studies in the mouse. The objective pursued in study 3 (García-Guillén et al., 2021b) was to study its segmental or neuromeric organization, by looking for genes differentially expressed within this structure. For this, we searched the databases ADMBA and the Allen Mouse Brain Atlas (AMBA) (https://mouse.brain-map.org/) at juvenile (P4, P14, P28) and adult (P56) stages, since we aimed to provide new molecular support for the possible rhombomeric subdivisions of this structure that would be not only transient in the embryo but also maintained in the adult brains. Results from study 1 show that the Netrin-1/DCC signaling pathway is one of the mechanisms involved in the migration of the IPN, since in the mutant there is a general but differential impairment of neuronal migration of all the populations analyzed. The absence of DCC mainly affects those of the Pro and IPR domains of this nucleus. In study 2 we retrieved 135 genes expressed in the IPN in a regionalized way. The functional analysis of these genes highlighted an overrepresentation of gene families related to neuron development, cell morphogenesis and axon guidance. In study 3, we characterized the expression pattern of 12 genes differentially expressed along the trigeminal column: Baiap3, Camk2a, Calb1, Calb2, Irx2, Kcng4, Fn1, Mafb, Tac1, Tac2, Pde1c and Zbtb16. Through the analysis of the pattern of these genes, we provided new genetic support of the segmentation of this column in the adult. On the whole, these works provide new knowledge on the developmental mechanisms of two hindbrain structures, the IPN and the trigeminal sensory column, as examples of cell migration and segmentation. This contribution can serve as basis for further studies on the function or pharmacology of both structures, or can be applied as well to the study of other neuronal populations in whose formation the migration and/or the segmentation are involved.