Research focus

The cerebral cortex underwent rapid expansion and increased complexity during recent hominid evolution, with significant impact on the acquisition of higher functions in the human species.

On the one hand, the enlargement of the surface and thickness of the cortex is mainly due to increased number of cortical neurons, which is thought to belinked to differences in the mechanisms underlying the generation of cortical neurons, or cortical neurogenesis.

On the other hand, the human cortex is characterized by larger neuronal diversity, increased relative size of specific layers and areas, increased neuronal connectivity, and prolonged developmental plasticity, which are linked to neuronal specification and differentiation.

Our projects are focused on the developmental mechanisms that control some of these key, species-specific, features of the human cortex, and how they relate to human evolution. We also study how the mechanisms of (mal)formation of human cortical neurons and circuits have direct relevance to neurodevelopmental diseases, in particular cognitive and psychiatric disorders. Finally we explore how basic principles of corticogenesis can be applied to the development of novel cellular models and therapies for neurological conditions such as neurodegeneration or stroke.

Our primary tools include classical tools such as mouse genetics and embryology, as well as innovative models of corticogenesis from human pluripotent stem cells, which we pioneered in the lab, and that recapitulate species-specific features of corticogenesis (Gaspard et al. Nature 2008Espuny-Camacho et al. Neuron 2013).

Microcephaly genes

Linking neurogenesis and evolution of brain size

Novel insights on human cortical development and evolution may come from diseases where species-specific developmental mechanisms have been implicated. Particularly striking among these is non-syndromic primary microcephaly (MCPH). MCPH is a neurodevelopmental disorder of genetic origin characterized by smaller brain size, particularly the cerebral cortex, while the rest of the body is essentially normal.

Interestingly, many MCPH genes, including the two most frequently mutated genes, ASPM and WDR62, encode proteins found at the level of the mitotic spindle and centrosomes, but the mechanisms underlying MCPH brain-specific defects remain poorly known. Morever similar mutations generated in the orthologous genes in the mouse do not lead to similarly prominent defects, suggesting that some key aspects of MCPH are linked to species-specific control of brain size and patterning.

In order to understand the significance of MCPH genes in relation to human brain evolution and microcephaly, we study their function in human cortical progenitors displaying specific mutations causing MCPH. We combine technologies of induced pluripotent stem cells (iPSC) generated from patients' skin cells, together with genomic editing, and several in vitro cellular models of corticogenesis, including 2D cultures and 3D models (organoids) developed in our and other labs (reviewed in van den Ameele et al. Trends Neurosci. 2014; Astick and Vanderhaeghen Curr Top Dev Biol 2018). This way, we aim to determine what the cellular and molecular consequences of MCPH gene mutations in human corticogenesis are, in order to define the pathophysiology mechanisms of MCPH, and how they may relate with species-specific mechanisms of human cortical neurogenesis.

Human-specific genes, cortical development & brain evolution

One of the drivers of evolution is the emergence of novel genes through duplication: an ancestral gene is duplicated and the copy evolves into a related, so-called "paralog" gene. Hominid-specific  (HS) gene duplications represent a potentially major driving force of evolution, but their impact on human brain evolution remains unclear.

Using tailored RNA sequencing (RNAseq), we recently identified a repertoire of 35 HS genes displaying robust and dynamic patterns during human fetal corticogenesis (Suzuki Cell 2018). Among these we found that NOTCH2NL genes, HS paralogs of NOTCH2, promote the clonal expansion of human cortical progenitors, ultimately leading to higher neuronal production (Suzuki et al. Cell 2018). These data identify NOTCH2NL as human-specific modifiers of neurogenesis that may have played an important role in human brain evolutionary expansion.

But what about the many other HS genes that we identifed during corticogenesis? Using a combination of gain of function in the mouse, and gain/loss of function in human and non-human primate pluripotent stem cell-based models of corticogenesis, we explore HS gene function from early stages of neurogenesis, to later aspects of neuronal migration, morphogenesis, and synaptogenesis.

Mechanisms controlling the timing of human neuronal maturation

The cellular basis of human brain neoteny

One important distinctive feature of human brain development is its unusually protracted rate of maturation, or "neoteny" (retention of juvenile features in a mature organism).

Interestingly, neoteny is particularly striking for human cortical neurons, even compared with non-human primates. Despite its crucial importance surprisingly little is known about the mechanisms underlying the timing of brain maturation and neuronal differentiation.

In this frame we made the surprising observation that protracted maturation of human cortical neurons can also be observed even following their transplantation into the mouse developing brain (Espuny-Camacho et al. Neuron 2013; Linaro et al. Neuron 2019), suggesting that intrinsic mechanisms play an important role in the regulation of the timing of neuronal differentiation.

Using gain of function in the mouse and loss/gain of function in human neurons, we now test several classes of candidates, including human-specific genes expressed in maturing neurons and genes that display human-specific patterns of neuronal gene expression. Moreover we combine single RNAseq and crisp/cas9 (CROPSEQ) to perform unbiased functional screens in human neurons for novel genes involved in neuronal development and connectivity, in close collaboration with the Stein Aerts Lab.

Modelling of neurodevelopmental disorders in human neurons in vivo

Neurodevelopmental disorders, in particular intellectual deficiency (ID) and autism spectrum disorders (ASD) constitute a major class of brain disorders with a heavy societal burden and very few therapeutic options to this day, mainly because of our lack of knowledge of the underlying mechanisms at the levels of the affected neuronal circuits.

In line with this gap of knowledge it has remained almost impossible to study experimentally human defects at the neuronal level, given the relative inaccessibility of live human neuronal material.

Our team has pioneered in vivo models of human cortical neuron development, whereby human pluripotent stem cells can be differentiated efficiently into pyramidal projection neurons, followed by xenotransplantation in the mouse cortex, where they display functional integration in the host neural circuits (Espuny-Camacho et al. Neuron 2013; Neuron 2017; Cell Reports 2018).

We are now using this technology to study the formation and plasticity of cortical circuits involving human neurons affected by specific mutations leading to human neurodevelopmental disorders.

The affected genes encode proteins thought to be important for the development of human synapses, as well as more general regulators of neuronal specification. The genes are first targeted by Crispr/Cas9 technology in human pluripotent stem cells or neural cells, followed by cortical differentiation in vitro and in vivo following xenotransplantation in the mouse. The neurons are then characterized morphologically and functionally, using combinations of single cell morphology, electrophysiology, and transcriptomics. Moreover, thanks to a close collaboration with the Bonin lab at the nearby NERF institute, we use in vivo multiphoton imaging to determine the developmental dynamics and function of the transplanted human neurons in awake animals.

Function and plasticity of transplanted human neurons

From circuit development to brain repair

A number of prominent neurological diseases, such as stroke, trauma, and specific degenerative conditions (such as ALS) could benefit in principle from therapies enabling to replace lost or dysfunctional cortical neurons.

We have recently tested this directly using mouse and human ESC-derived cortical neurons in an experimental model of neurotoxic lesioning in the adult mouse cortex : we found that transplanted neurons displayed robust integration and can contribute to reestablishment of cortical projections (Michelsen et al. Neuron 2015; Espuny-Camacho et al. Cell Reports 2018).

However it remains unclear whether transplanted neurons derived from pluripotent stem cells can integrate in a complex circuit and contribute to genuine restoration of lost function. Indeed, the functional characterization of transplanted neurons in the context of brain repair, as well as their impact on host circuitry function and plasticity, appears as a major, yet unreached milestone to the road of effective and reliable cell replacement therapies of the adult brain.

To address this key question we test whether and how the transplanted neurons display connectivity and functional properties in the host circuits that indicate physiological integration. The neurons are characterized molecularly, morphologically and functionally using single cell moprhology, electrophysiology and transcriptome analysis. Moreover, thanks to a close collaboration with the Bonin Lab at the nearby NERF institute, we use in vivo multiphoton imaging to determine the developmental dynamics and  function of the transplanted human neurons in the transplanted animals. This approach already led to the discovery that transplanted human neurons can display surprisingly precise patterns of connectivity enabling to respond to visual stimuli in a highly tuned fashion, like the mouse host neurons (Linaro et al. Neuron 2019).