Elsevier

Gene Expression Patterns

Volume 11, Issues 1–2, January–February 2011, Pages 105-111
Gene Expression Patterns

Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme

https://doi.org/10.1016/j.gep.2010.10.001Get rights and content

Abstract

Epithelial–mesenchymal interactions are critical for normal pancreas development. Fibroblast growth factor (Fgf)-10 is expressed in the pancreatic mesenchyme and its signalling is required for normal growth and regulation of gene expression in the pancreatic epithelium. However, little is known about putative Fgf signalling to the mesenchyme. Here we have examined the embryonic pancreas expression of differentially spliced Fgf receptor isoforms and their targets; the Sprouty (Spry) and Spred family genes which are induced by Fgf signalling. Using qPCR to quantify mRNA levels in microdissected pancreatic epithelium and mesenchyme as well as in FACS isolated Pdx1-GFP+ and -GFP cell populations we demonstrate that several members of the Spred and Sprouty families are expressed in embryonic mouse pancreas and find Spred1 and -2 as well as Spry2 and -4 to be predominantly expressed in pancreatic mesenchyme. Using embryonic pancreas explant cultures we demonstrate that Spred1/2 and Spry2/4 expression is regulated by Fgf receptor signalling and is increased by treatment with Fgf9, but not by Fgf7 or Fgf10. We extend previous work showing that Fgf9 is expressed in pancreatic mesenchyme, and since Fgf9 is known to activate the mesenchyme-specific “c”-splice forms of Fgf receptors, while Fgf7 and -10 both activate the epithelium-specific “b”-splice forms of Fgf receptors, these results suggest that Fgf signalling is active in the pancreatic mesenchyme, where expression of Spred1/2 and Spry2/4 appear downstream of Fgf9 signalling.

Section snippets

Embryonic mouse pancreas mesenchyme express FGF receptor IIIc splice forms and Fgf9

To begin to understand the potential for mesenchymal Fgf signalling we first analyzed the expression of FGFR splice forms and candidate Fgf ligands in embryonic mouse pancreas epithelium and mesenchyme by qPCR. We separated E11.5 dorsal pancreatic epithelia from their surrounding mesenchyme and isolated RNA from both fractions, which was subsequently processed for qPCR analysis. To determine the purity of each fraction we analyzed the expression of epithelium-specific markers (Cdh1

Discussion

To investigate the possible involvement of Spred and Sprouty members in murine pancreas development, we undertook a PCR screen for all known members of this family. Among them, Spry2 and Spred1 were the most highly expressed as measured by qPCR and the only ones detectable at the protein level. By two independent methods of separation, we found their expression to be preferentially localized to the pancreatic mesenchyme. The same pattern of expression has been described for Spred genes in the

Mice

Pdx1-eGFP transgenic mice were generated by pronuclear microinjection of a 6.3 kb-long transgene, which was cloned by replacing the Cre coding region of Pdx1-Cre (Herrera, 2000) by the coding sequence of eGFP. Two different families were established from separate founder mice, displaying the same phenotype. Only one was used in these studies.

Culture of pancreatic buds

Dorsal pancreatic rudiments were dissected from embryonic day (E)11.5 NMRI mice, and cultivated in 40 μl hanging drops. Culture medium was DMEM/F12 + Glutamax

Acknowledgements

We thank Heidi Ingemann Jensen and Søren Refsgaard Lindskog for excellent technical support, R. Scott Heller and members of Department of Developmental Biology for critical reading of the manuscript. We want to thank K. Schuh and A. Yoshimura for sharing of antibodies and plasmids. This work was made possible by support from the EU 6th Framework Program (to P.S.).

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