The role of the Lin28 regulatory pathway in early development

Animal development begins with a single cell, the fertilized egg. This cell contains all the information necessary to generate the complex body of the adult organism, containing many diverse cell types such as muscle, blood, nerve cells etc. The various cells of the embryo all contain the same genetic information. However, not all of this genetic information is used in each cell-type. Each cell-type is characterized by a different pattern of gene activity which is necessary for them to fulfill their various functions within the body. As developmental biologists we are interested in understanding the mechanisms involved in generating these diverse patterns of gene activity. Work in recent decades has shown that the cells of the developing embryo communicate with each other via secreted proteins known as growth factors. Signals passing from cell to cell play a key role in directing which cell types will eventually be formed. During development such signals act to change cell fate by activating or inhibiting groups of genes within a responding cell. These genes act as internal switches directing development of a particular cell types. The fibroblast growth factors (FGFs) are a related group of signaling proteins which are present in all animals. Our laboratory is interested in understanding the role of the FGFs in regulating gene activity in early animal development. The animal of choice for these studies is the frog Xenopus. The embryos of this organism are ideally suited for these studies for a number of reasons. The embryos are large, can be obtained in large numbers and develop rapidly outside the mother. Furthermore, there are excellent techniques available for analyzing and manipulating gene activity in the developing Xenopus embryo. We have recently undertaken a study which has enabled us to identify many of the genes which are regulated by FGF signaling in the cells of the early embryo. One of the genes that we identified as being a target of FGF signaling is Lin28. Our preliminary experiments, which have led to this proposal, indicate that Lin28 has a critical role in early frog development. If we inhibit lin28 function the head-to-tail pattern of the embryo is severely disrupted. Interestingly our experiments suggest that part of Lin28 function is associated with modulating the response of embryonic cells to growth factor signaling. Recently there has been considerable interest in Lin28 because it appears to be important in regulating how stem cells behave in culture. We believe that investigating how Lin28 functions in early development will have great benefits in understanding Lin28 function in stem cells. We plan to investigate in detail the role of Lin28 in frog development and have designed experiments which will allow us to determine how Lin28 regulates growth factor signaling. Lin28 codes for a protein which binds to and regulates the activity of various RNA molecules in the cell. These RNAs fall into two broad classes 1) protein coding messenger RNAs and 2) regulatory microRNAs, which regulate the activity of other protein coding messenger RNAs. The identification of these targets will greatly increase our understanding of how Lin28 functions during early development. We plan to share our observations with our collaborators working on human stem cells in culture. This will eventually allow us to test whether conclusions made about Lin28 activities during early development also apply to stem cells in culture.

The fibroblast growth factor (FGF) family of signalling molecules plays multiple roles in animal development and has been shown to regulate embryonic stem (ES) cell behaviour in culture. The target genes which mediate the biological activities of the FGF pathway are of considerable interest. We have undertaken a microarray based screen in Xenopus which has indentified a well validated set of candidate FGF targets. Amongst these genes is Lin28a, which was originally identified as a regulator of developmental timing in C.elegans. Recently there has been considerable interest in the role of Lin28 as a regulator of stem cell properties. Two closely related genes, Lin28a and Lin28b, are present in the Xenopus genome. Initial zygotic expression of both genes is within the early mesoderm, a known site of FGF activity. Our preliminary data indicate that morpholino oligo knockdown of the Xenopus Lin28 genes leads to derangement of normal development, characterized by loss of axial structures. Our experiments suggest that this phenotype arises from disruption to the response of embryonic cells to growth factor signalling during germ layer specification. We will undertake a detailed investigation Lin28a and Lin28b function in early development, with particular reference to their role as regulators of the response to growth factor signalling. We will determine at what level in growth factor regulated pathways the Lin28 proteisn act. Lin28 genes code for RNA binding proteins which have previously been shown to potentiate the translation of target mRNAs and negatively regulate the biogenesis of mature microRNAs (miRNA). A major aim of this project is to identify both mRNA and miRNA targets of the Xenopus Lin28 homologues and begin to investigate the role of these target RNAs in development.