The development of the pharyngeal pouches and clefts

The aim of this proposal is to investigate key early events in the development of the pharynx. This is an important and intricate region of our bodies but one that is much understudied. During embryogenesis, the pharynx has its origin in a series of bulges found on the surface the head called the pharyngeal arches. Although, the pharyngeal arches are transient structures that only exist during our early development, it is within these structures that the components the adult anatomy of the pharynx are organised; structures that later play roles in feeding, respiration, immune and hormonal activities. Furthermore, defects in the development of the pharyngeal arches lead to numerous birth defects, including branchial arch and cleft anomalies. The first steps in the formation of the pharyngeal arches involves the generation of the pharyngeal pouches which form at specific locations as the inner lining of the pharynx outpockets and contact the overlying tissue resulting in the formation of the pharyngeal clefts. The pouches and clefts are significant structures in organising the development of the pharynx as they act to define the limits of the each of the arches and to direct the development of these structures. The pouches and clefts will also later form important structures in our bodies. The first pharyngeal pouch/cleft contributes to the ear canal and its covering, the tympanic membrane. The second pharyngeal pouch gives rise to the tonsils. The third pouch will develop into the thymus and inferior parathyroid glands, and the fourth pouch gives rise to the superior parathyroid glands and the ultimobranchial bodies which are the source of the C-cells (Calcitonin producing) of the thyroid. The aims of this proposal are to explore key steps in the development of the pharyngeal pouches and clefts. Firstly, we will determine how the pharyngeal pouches are generated and define the cellular mechanisms and signalling pathways that underpin their formation. The next key event that we will scrutinise is the consequence of the contact between the pharyngeal pouches and the overlying ectoderm. Interestingly we have found that this results in a stereotypical series of events that initiates breakdown of the matrix that exists between these two layers and we we want to test the hypothesis that it is the pouches that trigger this process. This could be significant as it may give us insights into other developmental anomalies that occur when endoderm and ectoderm contact each other. The final event that we will analyse is the route through which the pharyngeal pouches and clefts become obscured during development. As mentioned, the pharyngeal arches are only evident for a period during early development and they become obscured as they are enclosed within a cavity, the cervical sinus, which is subsequently eradicated. Thus, our final aim is to identify how this cavity is eradicated and define the cellular and molecular events that underpin this. This is an important step as it is the failure at this paint that results in branchial cleft and sinus anomalies, which are the second most common congenital lesion of the head and neck in children.

The pharynx has its origin in a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches, and central to their organisation are the endodermal pharyngeal pouches and the ectodermal clefts. These define the limits of the arches, are key signalling centres and if their development goes awry there are serious consequences. The aims of this proposal are to explore key steps in the development of the pouches and clefts. In doing this we will use the chick as our model system. The preeminent reason for this is that it is the best system for this work. It is amenable at the relevant stages, it can be easily manipulated and pharyngeal pouch and cleft development in the chick is typical of amniotes. Firstly, we will determine how the pharyngeal pouches are generated. To do this we will fate map the origin of the pouches using the chick EC culture system, which allows us direct access to the endoderm. We will further define the cellular mechanisms and signalling pathways that underpin their formation by using inhibitory compounds to interfere with this process. We will then scrutinise the consequence of the contact between the pharyngeal pouches and the overlying ectoderm. This results in a stereotypical series of events that initiates breakdown of the basement membrane between these two layers and we will test the hypothesis that this driven by the endoderm. We will further test the role of metallopetidases in basement membrane dissolution. The final event that we will analyse is the route through which the pharyngeal pouches and clefts become obscured during development. This involves the pouches and clefts becoming enclosed within a cavity, the cervical sinus, which is subsequently eradicated. We will use fate mapping techniques to determine how the sinus is eradicated and use transcriptomic analysis to identify the cellular and molecular mechanisms at play here.

Academic impact - This project will have impact for those studying animal development and those analysing birth defects. They will benefit from this work as an outcome of our research will be an understanding of how this complex but much understudied region of the body develops. We will provide a clear analysis as to how pharyngeal development is directed and the lessons learned here will be applicable to many developing systems. Furthermore, this work will give us insights into abnormal development events affecting the pharynx and other regions of the body. In particular we anticipate that this work may shed light on the basis of anorectal malformation, another important but understudied area.

Academic beneficiaries will also extend to those analysing quite different biological systems as the genes and pathways that we are studying will undoubtedly be involved in other quite different biological contexts and the links and interactions that we find in our system will be important elsewhere. In particular our work will be of interest to Cancer Biologists as one of the events that we are studying, dissolution of the basement membrane is a key event in cancer progression and this is particularly true of cancers , such as pancreatic, which have an endodermal origin.

3Rs impact - The planned programme of work is to be conducted using chick embryos. These are the most amenable species for this type of study and they are very cost effective. It would be extremely difficult to conduct any of this work in the mouse, and we will be able to demonstrate the usefulness of using chick embryos to address basic biological problems of significance to the understanding of human syndromes and birth defects. This will lead to better science.

Economic and Societal impact - Our work will also have a number of societal beneficiaries. Branchial fistula and sinuses are the second most common anomalies of the head and neck in children and thus our work will provide a context, for affected individuals and their families, in which these can be understood. Our work will further contribute to the public understanding of science and we routinely engage in widening participation activities here at Kings in which we discuss our research with school pupils and their tecahers.