This study is the first description of the miRNA transcriptome during the critical period of hepatobiliary development that spans the end of gestation to the early neonatal period. We have identified a core set of 38 miRNAs whose expression levels change during the period of hepatobiliary specification. When we examined the spatial expression patterns of selected miRNAs we found additional evidence for complex, independent regulation of their expression. We have also demonstrated regionally restricted expression of miRNAs within subsets of cells of both parenchymal and non-parenchymal origin within the liver. As is to be expected given the hematopoietic nature of the fetal liver, several of the miRNAs identified in our study were found to be restricted to developing blood cells. However, both hepatocyte and cholangiocyte-specific miRNAs were also identified.
The finding that cells expressing the granulocyte-specific miRNA miR-223 are interposed between the developing ductal plate and the portal vein was unexpected. Previous studies have implicated the Notch signaling pathway in normal biliary development,36, 37
and it has been hypothesized that direct signaling between the portal vein or periportal mesenchyme and the ductal plate is a critical component of biliary differentiation.38
Our work indicates that during the period between E18.5 and the neonatal period, these populations of cells are not in direct intercellular contact, as would be required for Notch signaling.
Any attempt to identify cholangiocyte-specific gene products is complicated by the fact that these cells comprise only a small proportion of the total mass of the liver. Since the appearance and remodeling of the ductal plate involves only a small number of cells and occurs in a relatively brief developmental period, the search for miRNA transcripts specific to these cells by large-scale cloning strategies would have been unlikely to be successful. Our discovery of ductal plate-specific miRNA transcripts validates the approach of investigating the subset of miRNAs that are dynamically changing in abundance in late gestation. In the case of the mouse miR-30a and miR-30c transcripts, the cholangiocyte-specific expression is temporally restricted to the period between E18.5 and the early neonatal period, suggesting that these miRNAs function in the final differentiation of cholangiocytes.
Given the highly similar pattern of the miR-30a and miR-30c positive cells, and given the genomic location of the genes encoding miR-30a and miR-30c2 (less than 20kb apart), it seems likely that the two transcripts are co-regulated and that miR-30c2 rather than miR-30c1 is the source of the mature miR-30c transcript; however, we have not formally tested this hypothesis.
Since functional studies of miR-30a and miR-30c in mice will require the generation of conditional knockout strains, we have used ASO technology to reduce expression of the zebrafish ortholog of miR-30a as a rapid, preliminary assay. While bile duct development in the zebrafish does not occur through a ductal plate intermediate, the regulatory molecules which function in mammals (the Onecut factors, HNF1β, Jagged/Notch) play analogous roles in the fish.26, 27
Our findings in the zebrafish model provide the first evidence that miRNA plays a critical role in the development of the biliary system.
At the inception of this study, none of the computationally predicted targets of miR-30a or miR-30c had been validated. A recent study has identified 100 targets of miR-30a using proteomic methods by transfection of a miR-30a mimic into HeLa cells39
. Of these, only two (Slc12a4
) were also identified in our screen. There are several potential explanations for this small overlap. The use of miR-30a over-expression carries the risk of false positives due to supra-physiologic levels of the miRNA. Furthermore, since the regulatory function of miRNA involves binding of miRNAs to target mRNAs, a particular miRNA may modulate expression of distinct targets in different cell types. Our study was performed by decreasing the levels of endogenous miR-30a in BMELs, a hepatoblast model cell line, and is thus more likely to identify liver-specific target mRNAs.
Our study provides the first links between miR-30a and two well-described regulators of liver development: Inhba
(a component of the TGFβ agonist, Activin) and Egfr
(the epidermal growth factor receptor). The expression of both of these in the fetal liver has already been described40, 41
. Clotman and colleagues have shown that a gradient of TGFβ activity is essential for the normal differentiation of hepatoblasts into cholangiocytes and hepatocytes.42
Our study suggests that miR-30a expression in the ductal plate promotes the formation of this gradient by inhibiting Activin production. Egfr is a growth factor receptor expressed in the ductal plate which is down-regulated in the mature cholangiocyte; conversely, over-expression is Egfr
is frequently observed in cholangiocarcinoma. Our findings suggest that miR-30a participates in the control of Egfr
expression in normal cholangiocytes and loss of miR-30a may promote excess Egfr
expression in cancer. A third miR-30a target identified in this study is the canalicular marker Abcb1b.
In the context of embryonic biliary development, miR-30a may inhibit Abcb1b
expression in nascent cholangiocytes in the ductal plate as they undergo terminal differentiation from hepatoblasts.
Neither the Inhba nor the Egfr 3’-UTR is targeted by miR-30a in our reporter assay, although both are predicted to contain miR-30a binding sites by Targetscan. This may be because the luciferase gene is expressed at high levels under the control of the SV40 promoter; this strong expression may mask the effects of targeting by miR-30a. It is also possible that the regulation of Inhba and Egfr requires both the 3’-UTR and other elements within the mature transcript. Finally, the regulation of Inhba and Egfr by miR-30a may be indirect.
In summary, these results provide the first links between miRNA and hepatogenesis, particularly biliary development. Future studies will be directed to testing the function of miR-30a and miR-30c in the mouse by genetic means and to further establishing links between these regulators and other pathways relevant to liver ontology and function.