Development of the Layer 5 Pyramidal Neuron Subgroup Expressing Er81

In the developing cerebral cortex, pyramidal neurons are generated in the germinal zones and migrate into the developing cortical plate, with the earliest generated occupying the deepest layers, and the last generated the more superficial layers. Within the germinal zones the type of pyramidal neuron generated is controlled by differential regulation of gene expression, some of which have been identified. We do not yet know how expression of particular genes leads to particular patterns of axon outgrowth, target selection, or physiological properties. Er81 is expressed at early stages of cortical development in the ventricular zone in dividing cells and then later in post-mitotic L5 neurons in the cortical plate. In both Pax6 mutants and Ngn2 mutants the expression of Er81 is disrupted, implying Er81 is regulated by both of these genes. This suggests an early role for Er81 in neuronal differentiation and cortical lamination/migration, similar to its role in olfactory bulb development. At later stages, most L5 Er81 expressing neurons also express Ctip2 but not Otx1. Ctip2 controls the formation of corticospinal axon outgrowth, whilst Otx1 controls outgrowth of collicular/pontine projections. Whilst some studies have shown ER81 to be down-stream of Ctip2, our preliminary microarray and qPCR data show that Er81 controls Ctip2, which is confirmed by the up-regulation of Ctip2 seen in the Er81 KO. Loss of Ctip2 results in a loss of CST axons, whilst reduced expression results in mis-specification of cortical pyramidal neurons, suggesting that Ctip2 and Er81 control the processes of axonal specification and collateral withdrawal of L5 projections. Outside the cortex, Er81 is expressed specifically in target nuclei of L5 neurons, such as the superior colliculus and inferior olive, offering an exciting avenue for investigation of specification of cortical connectivity. A similar role has been shown for Er81 in formation of circuitry in the spinal cord where ER81 is expressed by specific motor and sensory neurons and there are defects in the formation of these interconnections in mutants. Er81 and Ctip2 are both regulated by Fezf2. In Fezf2 mutants there is a mis-specification of the L5 Ctip2 expressing cells, which instead express Tbr1 and Satb2, migrate to L6 and send axons through the anterior commissure/corpus callosum. Satb2 is expressed in L5 callosal projection neurons and represses the expression of Ctip2. In the Er81 KO mouse we showed an increase in the number of Ctip2-expressing neurons with callosal projections suggesting that Er81 controls Ctip2 by suppressing Satb2. Er81 can be seen to play a central role in the establishment of cortical connectivity. In the last few years our understanding of the molecular control of cortical neuronal specification has improved and we have produced some very exciting preliminary data underpinning the proposed experiments. Whilst this complex network of genes regulates the development of particular pyramidal cells, we have very little idea about how changes in these networks allow for specific regional connectivity which is a fundamental process of brain wiring. As clinically related studies of brain development identify new gene associations with developmental disorders that result in altered brain function, it is essential to understand the details of the normal transcriptional networks. Whist it is perhaps premature to suggest that knowledge of how specific genes influence cortical wiring could enable future therapies, this will be essential if we are ever to use cell based therapies in brain repair. Current research has focused almost exclusively on the generation of identifiable neuronal cell classes from both innate and experimentally induced stem cells, no understanding of how such cells might be controlled in terms of their connectivity has yet been made.

Characterisation of L5 Er81 neurons Using the Er81-GFP mouse the expression pattern of Er81 during the developmental stages of neuronal genesis and migration, dendritic elaboration, axonal outgrowth, and refinement of connections will be described. This will corroborate previous studies but also allow the correlation of Er81 expression with other L5 marker genes, using in situ hybridisation (ISH) and immuno-histochemistry (IHC). Combining these studies with axon tracing, (viral eGFP will optimise details of cell morphology), we will characterise the different molecular subclasses of pyramidal cells with axonal projection targets. Role of Er81 in the transcriptional network Using microarrays on Er81 KO mice cortex at specific stages of development, we will look for genes regulated by Er81. Er81 linked genes in cortex will be compared with those previously identified in DRGs by our collaborator Silvia Arber. These target genes will be validated by ISH, qPCR and IHC. Using slice cultures candidate genes will be screened to show linked changes in expression following Er81 over-expression or knockdown. Further analysis will be made using ChIP-seq to discover Er81 interacting partners. Using ChIP-PCR the link between Er81 and Ctip2 and other candidate genes, such as the recently identified L5 gene Tshz2. For Tshz2 we can further characterise L5 neurons in Tshz2-GFP and Tshz2 KO mice. Role of Er81 in L5 neuron identity Using the Er81-LacZ KO mice, the expression of Er81 target genes, and the changes in dendritic morphology/axonal projections of null-Er81 cells will be determined. Er81 will be locally over-expressed in cortex using in utero electroporation and changes in pyramidal cell connectivity, somatodendritic morphology and gene expression will be detailed.

This research addresses fundamental questions about normal brain development. There are several important levels at which this work will be of major benefit in the short and long term. Recently our understanding of CNS development has improved dramatically, in particular molecular techniques and the ability to manipulate genes has revolutionized our understanding of the controls of brain development. At a scientific level, no one would doubt the fascination in understanding how the brain develops, in particular how the myriad of different neurons classes are generated in specific places and ratios and then link in a highly specific fashion to form the amazing connectivity which underpins brain function. Our experience of discussion with scientific and clinical colleagues, students and school children, and with lay audiences testifies to the widespread interest in this area of research. A deeper and intriguing aspect of this research is how changes in these regulatory patterns must underpin subtle changes in circuit formation, which form the basis of all differences in personality, intelligence, and performance. Whilst the brains of all species are remarkably similar at a gross level, the subtle changes in development of the precise patterns of circuitry underpin individual differences. In man this accounts for the different abilities of the mathematical, genius, the concert pianist or professional sportsman, and even world leaders. Tentative steps towards an understanding of the development of the brain and how this accounts for such differences are clearly of huge public interest. The subtle changes in the process of brain wiring which generate positive differences in abilities can also have tragic negative outcomes. With the advent of new molecular techniques and clinical screening programmes, many candidate genes and genomic interactions have been identified which are linked to brain abnormalities. In some cases these are severe malformations, resulting in major functional impairment, whilst in others they are subtle changes which affect only certain functions, such as dyslexia. As candidate genes are identified, finding out how they fit into the regulatory networks, such as those highlighted in this proposal, will be the obvious progression. This will certainly be of interest to clinicians, who diagnose and define the potential outcomes associated with such abnormalities. Patients and patient groups will naturally have a keen interest in understanding such conditions. Indeed the specific charities and support groups commonly focus on such research as the key to understanding the nature of the disability and the implications for sufferers. Commercial concerns will also be interested in the development of diagnostics and in the longer term for therapies. Increased longevity and concomitant increase in the number of patients diagnosed with CNS degenerative diseases, is a major global health problem. Stem cell research is currently one of the best hopes for curing such disorders. The ability to harness innate stem cells from the brain, or from elsewhere in the body, to generate neurons is a key strategy for the potential repair of CNS disorders. Similarly the use of embryonic of other donor stem cells to form specific neuronal subtypes is a major area of translational research. In stroke or CNS injury the promotion of regeneration and plasticity, or generation of new neurons from intrinsic stem cells, is key to functional repair. Unless we understand the normal controls on neuronal development and in particular connectivity, it is unclear how any realistic therapy could be achieved. Indeed mis-wiring the CNS from grafted or regenerating neurons could have even more problematic consequences. This work will inform the vast field of basic and clinically related research in this area and again be of enormous interest to patient groups, funding agencies and pharmaceutical and biotechnology companies in this area.