Decoding the lamination, cytoarchitecture and cellular composition of the entorhinal cortex

PhD Student: Tobias Borgtoft Bergmann email:

Thesis defended on 21 April 2021. 

The project

The entorhinal cortex, as part of the parahippocampal region, is involved in unique functions related to spatial navigation and consolidating episodic memory. The entorhinal cortex and the entorhinal cells that are responsible for its unique tasks have mainly been investigated by electrophysiological and histological studies.

Understanding the molecular profile of these unique neurons will allow for a better understanding of their functions. The entorhinal cortex is also unique, as it is amongst the first area to be affected by Alzheimer’s disease pathology.

A particular type of principal neuron, the stellate cells, has been demonstrated to be the very first cell type to be affected in mice by Alzheimer’s disease pathology, such as amyloid-beta accumulation, with neuron death as a consequence.

Understanding the molecular profile of these neurons will provide a better understanding of the initial mechanisms in the disease and the possibility of studying these specific entorhinal cortical cells isolated would provide new means to study and understand Alzheimer’s disease.

The purpose  

The overall purpose was to investigate the molecular profile of different cell types in the developing entorhinal cortex, in order to establish an in vitro protocol for production of entorhinal cortical-specific neurons. To achieve this, the projected aimed to elucidate the anatomy and cytoarchitecture of the developing porcine entorhinal cortex to asses its temporal development and anatomical position in the porcine brain, which was to be used as main model animal. Furthermore, the lamination pattern of the entorhinal cortex was to be investigated using birthdate labeling of newborn neurons. The development and heterogeneity of cells in the developing entorhinal cortex was to be investigated by single-cell sequencing to identify transcription factors that specify certain entorhinal cortical neuron identities, which were to be used to reprogram induced pluripotent stem cells into entorhinal cortical neurons.

The results

In this study, we implement Bromodeoxyuridine birthdate labeling and single-cell RNA sequencing of the developing EC. We demonstrate that the medial entorhinal cortex develops by a distinct lamination pattern that diverges from the canonical inside-out model of the neocortex. The deep layers (L)V and LVI emerges in parallel first. Stellate cells, one of the two major populations of principle neurons in the LII of the medial entorhinal cortex, subsequently emerge together with LIII neurons and the two layers also form in parallel, after which a late-born neuron population finalizes the LII. We term this distinct lamination pattern in the medial entorhinal cortex, parallel lamination.

We identified unique populations of glia cells and neurons, including 1 population of intermediate progenitors, 7 populations of pyramidal cells, and 4 populations of stellate cells. We provide novel cell-type specific markers for these together with transcription factors enriched in one stellate cell population. Based on these stellate cells specific transcription factors we developed a novel protocol to reprogram induced pluripotent stem cells which resulted in the production of stellate cell-like cell in vitro. Our findings provide new insights into the cellular heterogeneity and transcriptional identity of the entorhinal cortex and demonstrates that this unique structure is developed and laminated in a non-canonical fashion. By establishing a new protocol for developing specific entorhinal cells in vitro we potentially provide a novel system to study these entorhinal cortical-specific cells.