Roles of the transcription factor Pax6 in regulating embryonic development of the prethalamus

Abstract

The transcription factor Pax6 is a pleiotropic player during neural development. In the central nervous system, Pax6 is mostly expressed by neural progenitors, where its functions have been most extensively studied. However, in the anterior diencephalon, the prethalamus, Pax6 is expressed in both neural progenitors and post-mitotic neurons. This distinctive expression pattern of Pax6 makes the prethalamus a unique place in which to explore the functions of Pax6 and its mechanisms of action during development.

I have found that in post-mitotic prethalamic neurons, Pax6 seems to regulate the process of neuronal morphogenesis. Gene ontology analysis on the RNAseq data, which showed significant transcriptional changes of genes in the prethalamus when Pax6 is lost, revealed that genes involved in neuritogenesis, establishment of neuronal polarity, axon elongation and axon initial segment (AIS) were significantly differentially expressed. To further explore this, I performed dissociated cell cultures of the prethalamus at embryonic day 13.5. Various aspects of neuronal morphogenesis were analysed in these primary neurons cultured for 1-9 days in vitro (DIV). I found that Pax6-null prethalamic neurons constantly displayed fewer neurites and a disturbed rate of neurite elongation. Additionally, I discovered that the AISs of these neurons were located further away from the soma. The AIS is where the neurons generate action potentials, and its location and molecular composition can determine the amplitudes and firing frequencies of the neurons. I found that the components of the AISs seemed to have been altered as increased amount of voltage-gated sodium channels and AnkyrinG was found in the AISs of the Pax6-null prethalamic neurons. AnkyrinG is a cytoskeletal protein known to be the master regulator of AIS formation. Therefore, my analysis suggested that the Pax6-null prethalamic neurons might display different electrophysiological properties. Indeed, whole-cell patch clamping on dissociated prethalamic neurons showed that the Pax6-null neurons required a much lower amplitude of current stimuli to initiate an action potential. In the adult brain, derivative neurons of the prethalamus are organized into nuclei, which innervate with the thalamus in a reciprocal manner and can modulate the activities of the thalamus. The thalamus is the relay station where all sensory inputs (except olfactory) are received, processed and further projected to the cortex. Consequently, the loss of Pax6 in the embryonic prethalamus might impact the functionality of entire nervous system.

Additionally, I have also found that Pax6 removal from the prethalamus significantly deregulates the activity of various genes involved in canonical and non-canonical Wnt-signaling pathways. My data indicate that in the prethalamic ventricular zone, where neural progenitors reside, Pax6 seems to suppress the expression of various canonical Wnt-signalling pathway effectors by promoting the expression of antagonists of Wnt-signaling pathways, such as Sfrp2 and Dkk3.

By utilising the RNAseq data and comparing the transcriptome profiles of the thalamus and prethalamus, I found that in the absence of Pax6, the prethalamus develops a thalamus-like expression profile of the voltage-gated ion channels and AIS component genes. During embryonic development, the thalamus and prethalamus reside adjacent to each other, and receive the same amounts of morphogens. However, the thalamus and prethalamus give rise to neurons with completely different morphologies and functions. Pax6 is expressed simultaneously by the thalamus and prethalamus. However, in the thalamus, Pax6 is only expressed in the progenitors, whereas in the prethalamus, Pax6 is expressed by both the progenitors and post-mitotic cells. My results thus indicated that such differential expression of Pax6 helps to explain the inter-regional diverse development of the thalamus and prethalamus.

My study revealed novel and distinct functions of Pax6 in the developing prethalamus of the mouse embryos- suppressing the activity of canonical Wnt signalling pathway in the progenitor cells and regulating neuronal morphogenesis and functionality in a cell autonomous manner in the post-mitotic cells. Additionally, Pax6’s expression in the prethalamus also influences the prethalamic electrophysiological identities from the neighbouring thalamus, thus contributing to the differential development of these two tissues. My findings hence provide new insight into the temporal and spatial regulation governed by master regulators such as Pax6 during brain development.