Pitx2 transcription factor: expression in the Central Nervous System and role in the development of spinal interneurons

Abstract

Pitx2 is a paired-like homeodomain transcription factor, mutations in which have been associated with the pathogenesis of Axenfeld-Rieger syndrome, an autosomal dominant condition that affects the eyes, teeth, heart and the umbilicus of the patients. Extensive studies in the mouse show that Pitx2 plays an important role in the development of the aforementioned tissues and more. In the spinal cord, Pitx2 marks a small Dbx1-derived ventral interneuron population that is divided in a cholinergic (V0c) and a glutamatergic (V0g) subset. V0c neurons give rise to all C boutons, namely large, cholinergic synapses on spinal motor neurons. Genetic ablation of the cholinergic transmission through the C bouton synapse leads to task-dependent inability to increase muscle-specific output. In my thesis, the use of molecular genetics enabled us to probe the origin of the C bouton synapse in the cranial motor neurons and map Pitx2 neurons throughout the postnatal mouse brain, with an emphasis on the cholinergic ones that are putative sources of the cranial C boutons. We showed that all C boutons on brainstem motor nuclei are Pitx2 derived, contrary to a previous publication stating that cranial C boutons have a mixed Pitx2+ and Pitx2- origin. New Pitx2 populations, including a small cholinergic one in the mammillary area, were identified for the first time in the postnatal mouse brain. Apart from using Pitx2 as a neuronal marker, we delved into the study of the role of Pitx2 protein on the development of spinal Pitx2 interneurons and discovered important parallelisms with known functions of Pitx2 in the subthalamic nucleus and the superior colliculus. For this study two Pitx2 knockout mouse models were studied: an embryonic lethal Pitx2 null and a conditional knockout in which Pitx2 expression is perturbed in Dbx1-derived neurons. In both models the homeodomain was eliminated leading to the generation of a truncated and non-functional Pitx2 peptide, which we were able to detect with immunofluorescence or in situ hybridization, thus visualizing the Pitx2 deficient neurons. The most prominent observation, common in both models, is that Pitx2 is required for the correct localization of the spinal Pitx2 interneurons, indicating a role for Pitx2 in the migration process. Moreover, loss of Pitx2 results in lack of cholinergic specification of the V0c subset and therefore in absence of the C boutons from the spinal motor neuron somata. Whether these effects can be attributed to Pitx2 loss directly, if Pitx2 is upstream of genes that regulate the cholinergic character or these effects are secondary to the mislocalization of Pitx2 deficient neurons and arise due to loss of correct positional cues is a matter that will need further examination. Pitx2 loss seems to affect the number of Pitx2 deficient neuron, as well. The results of this study with dual focus on the anatomy and connectivity of brain and brainstem Pitx2 circuitry and on the role of Pitx2 transcription factor in the spinal Pitx2 interneuron development contribute to the endeavor of bridging molecular identity with the charting and function of neural circuits, as a basis for the comprehension of physiology and pathology of the Central Nervous System.