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The role of the Lin28 regulatory pathway in early development

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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.

Layman's description

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.

Key findings

BBSRC Final Report Questionnaire

1. What were the most significant achievements from this grant?
(up to three, no more than 150 words in total).

1. Determining that lin28 function is required in the pluripotent cells of the early amphibian embryo to allow them to respond appropriately to growth factor signals involved in specifying the early mesodermal cell lineages. Our data suggest that lin28 function regulates the timing of responses to these signals.
2. Determining that lin28 function the early stages of germ layer specification is independent of the lin28’s recognised role of regulating the biogenesis of mature let-7 family miRNAs. However, during later development lin28 function does appear to be involved in regulating the abundance of at least some let-7 miRNAs.
3. Identifying that the expression levels of miRNAs from the 17~92 and 106~363 miRNA cluster are significantly down regulated in response to lin28 inhibition. Moreover, we show that lin28 proteins physically interact with mir-363 precursors, suggesting a novel mode of action for lin28 in regulating the abundance of mir17~92 family of miRNAs.

2. Indicate whether or not the main objectives of this grant were met and, for any which were not met, provide a brief explanation (no more than an average of 50 words for each, no more than 300 words in total).

Aim 1 Determine where and when Lin28 proteins are found in the developing Xenopus embryo
This has been successfully achieved. We have determined a detailed mRNA and protein expression patterns of both lin28a and lin28b
Aim 2 Determine the role of Lin28 in the response to growth factor signalling in vitro
This has successfully achieved and we have extended this work to highlight the role of lin28 proteins in regulating the timing of reponses to growth factor signalling in vivo.
Aim 3 Determine the requirement for Lin28 function in early development
This has successfully been achieved. We have carried an in depth characterisation of the effects resulting from single and compound knockdown of lin28a and lin28b function.
Aim 4 Determine the mode of action of Lin28 proteins in early development
We have had considerable success with this aim, having shown that the very earliest roles of lin28 in the amphibian embryo are independent of the lin28 function in regulating biogenesis of let-7 miRNAs that had previously been characterised in mammalian stem cells in culture. We have also identified a novel putative role for lin28 proteins in binding to and regulating the abundance of mir17~92 family miRNAs. However, at present our evidence falls short of proving a role for 17~92 miRNAs in mediating lin28 function in the embryo.
Science in Society and Outreach
One area of activity that I have been disappointed with during the course of this project is my normal level of engagement with outreach activities and dissemination of this research to the wider non-scientific community. At the start of this project I was appointed to be chair of the York Biology examinations committee. This is considerable administrative burden and has seriously impacted on the amount of time that I have been able to devote to these key activities.
August 2008
Effective start/end date1/09/0931/08/12

Award relations

The role of the Lin28 regulatory pathway in early development

Isaacs, H. V.

BBSRC: £415,892.00


Award date: 28/07/09

Award: UK Research CouncilsAward


  • BBSRC: £415,892.00

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