Signal integration and interpretation during neural development

An important question in biology is "how do cells know where they are within a tissue and how is this information translated so that they form the appropriate structures for their positions?" Our central nervous system (CNS) contains many different types of neural cells arranged and interconnected in a complex pattern. This is a consequence of early embryonic development when neural cells acquire their unique location and identity. Initially cells can form any neural cell type, but in response to chemical signals they make a decision to become a specific type. The various signals spread from different directions providing the coordinates in what can be thought of as a 3D map of the developing nervous system. Cells decide what type of nerve to become by interpreting the nature and amounts of signals to which they are exposed. While much has been learned about the activities of individual signals, how cells perceive multiple signals and integrate this information to make the appropriate neural cell types for the specific location is very poorly understood. Here we will tackle this problem by performing experiments in two parts of the CNS - the hindbrain and the spinal cord. These studies will provide insight into how different nerve types are made during embryonic development and may help explain how the ordered complexity of the CNS arises.

Nerve cells arise from pools of proliferating progenitors that are arrayed in a stereotypic order along the dorsal-ventral (DV) and anterior-posterior (AP) axis of the neural tube. Signals that act in a gradient along the DV axis as well as signals that act along the AP axis are very important for cell fate specification. These signals are called morphogens and are necessary for neural cell fate specification. A morphogen has two important characteristics: (a) it functions in a concentration dependent manner to induce different responses in a field of receiving cells and (b) it spreads though a tissue to act at a distance from its source. Shh is known to control the expression of specific genes that induce a cascade of events that give rise to specific neuronal cell types. However the types of neurons are generated in response to Shh depend also the region of the nervous system, for example cells located at similar positions in the hindbrain and in the spinal cord produce different types of neurons. To understand how this is achieved we will identify how genes that encode the AP position (Hox genes) interact with Shh signaling. We have established a system, based on the differentiation of embryonic stem (ES) cells, in which we can control and direct the differentiation of specific neural types in a dish. We will use this powerful system with the latest technologies, such as genome analysis to identify the molecular mechanisms. The findings from this research will improve our understanding of nervous system development as well as the differentiation protocols of pluripotent stem cells to specific neuronal cell types. The findings are likely to have important implications for the growing fields of stem cell and systems biology.

Neural progenitor cells (NPCs) at distinct positions along the antero-posterior (AP) and dorso-ventral (DV) axes acquire their fate by exposure to different types and concentrations of inductive signals. How DV and AP positional information is integrated to generate specific neuronal cell types remains unclear. This question is exemplified in the case of neural progenitor pools that are topographically related in the DV axis but nevertheless generate different neuronal subtypes along the AP axis. One example is the p3 progenitor domain. Despite occupying the same DV position and having similar molecular identities, hindbrain p3 NPCs generate visceral motor (VM) and serotonergic (5HT) neurons, whereas spinal cord p3 NPCs produce V3 interneurons. To understand the molecular basis of this difference we will take advantage of an in vitro model system we have established in which stable mouse ES cell lines inducibly express different Hox genes under the control of doxycycline. These will be used to generate NPCs of specific AP identity. We will ask whether the AP identity of NPCs affects the perception or interpretation of DV patterning signals. Genes differentially regulated in anterior (MNs, 5HT) and posterior (V3) p3 NPCs will be identified. The genomic targets of Nkx2.2, the transcription factor that confers DV identity to p3 progenitors, will be identified to determine whether these differ in cells with distinct AP identities. Genes differentially regulated along the AP axis will be investigated in vivo. These findings will provide new insight into the cellular process and genetic network that integrates positional information to confer identity to neural progenitors. This knowledge is of fundamental importance to mechanisms of tissue development and will facilitate the use of the directed differentiation of stem cells in basic and applied research and provide the basis for further systems level investigations.

The anticipated impact on the field will be to increase knowledge and the breadth of research skills in key techniques of post-genomic research, systems and stem cell biology. These are important fields, poised for a large expansion as genomic data and stem cells are exploited and applied. Increasing our knowledge base and training skilled individuals are key elements of success. One of the major challenges is to acquire the diverse techniques necessary to carry out the research work in this field. Most of these experimental approaches can only be learned with hands-on experience and teaching. Our proposed project takes full advantage of the skills of the fellow thus facilitating the acquisition of new techniques and knowledge. Developmental biology is an essential discipline of biological science in the post-genomic era, as researchers in the field are systematically elucidating gene function. Moreover the project will provide training and new resources in the fields of systems and computational biology.

The project is anticipated to impact on several intellectual and scientific fronts. A significant contribution will be the new knowledge on mechanisms of central nervous system development that are generated from this project. An important feature of the project we propose is the integration of currently disparate mechanisms and experimental approaches. The study of neural tube development, until now, been fragmentary such that related findings on the cellular and molecular mechanisms have not been assimilated into a whole. Our integrated approach will begin to address this deficit. Moreover the protocols and experimental approaches we will develop during the course of this work will benefit researchers studying similar problems in the spinal cord or other systems. The availability of these types of data will aid a better understanding of brain function and represent and are likely to be employed and extended by other researchers. The findings will be disseminated via scientific conferences and papers in appropriate journals.

These findings will have practical significance. Our project will contribute to fundamental knowledge that may be relevant to understanding the normal or abnormal function of the nervous system. Second, the ability to direct the differentiation of stem cells to specific cell types will be a major impact of this project. Given their unique properties, stem cells are promising candidates for tissue engineering and applied research. A significant problem, however, is generating pure populations of the desired cell type from the initial pluripotent stem cell line. We anticipate that based on the new knowledge of the basic mechanisms of spinal cord development uncovered in this project, improvements in the current state of the art approaches for stem cells will be forthcoming. This is likely to be widely applicable to those using these approaches in the field of systems biology.

The themes of this proposal - post-genomic research, developmental and stem cell biology - are medically and economically important fields for the UK and are expected to undergo a large expansion as genomic data and stem cells are exploited and therapeutic interventions begin to rely on this knowledge. Thus, trained researchers with experience in appropriate fields of research are necessary. Specialised genetic research is expanding at very high rate and requires highly trained personnel to support this. Personnel with skills in functional genomics, developmental biology and stem cell biology are likely to play a key role in the post-genomics era. This project will contribute to the training of such personnel and will allow the dissemination of this knowledge and skills.