Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

Abstract

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.

Figures

Figure 1

miR-214 participates in somitogenesis. (a,b) Morphology of embryos injected with 214MO at the 14-somite stage. (c,d) Somite morphology in uninjected controls (UIC) (c) or in embryos injected with antisense morpholino oligonucleotides complementary to miR-214 (214MO) (d). (e) Expression of miR-214, as determined by RNA blotting. Embryonic stages are listed above, and ethidium bromide–stained rRNA is shown as a loading control. (f,g) Expression of miR-214 in somites at 1 dpf as determined by in situ hybridization in whole-mount embryos (f) and in a somite cross-section through the trunk region. Section in g was obtained from the region in f indicated by the black line.

Figure 2

Modulation of miR-214 expression alters Hedgehog-mediated cell fate specification. (a,b) Loss of muscle pioneers shown by eng2a in situ hybridization in 214MO morphants (b) compared with uninjected controls (UIC) (a). (c,d) Aberrant ptc1 expression in 214MO-injected embryos (d) compared with UIC (c). (e,f) Pixel intensities of ptc1 stain in three embryos. From left to right, plot show the intensity from the bottom to the top of images in c and d (that is, from left to right in the embryo). e shows the UIC and f the 214MO-injected embryo. (g–i) Eng-positive nuclei in 214MO-injected embryos (g), in embryos injected with 214MO and shh (h) and in embryos injected with shh mRNA (i). (j–m) Expression of nkx2.2a and olig2 in sections of spinal cord from UIC embryos (j,l) and in embryos injected with miR-214 (k,m).

Figure 3

su(fu) is a target of miR-214. (a) su(fu) 3′ UTR sequence elements complementary to miR-214. (b–g) Fluorescence in embryos injected with synthetic mRNAs encoding GFP with or without coinjected miR-214 RNA. b, d and f show embryos injected with GFP without the UTR sequence (–UTR; b), GFP fused to the su(fu) 3′ UTR (su(fu)UTR; d) or GFP fused to two perfect miR-214 sites (2XMRE; f) alone. c, e and g show embryos coinjected with the GFP reporters as in b, d and f, respectively, along with miR-214 RNA. (h) Protein blot analysis of lysates prepared from embryos injected with GFP without the UTR (–UTR), su(fu)UTR or 2XMRE GFP with or with out miR-214 RNA coinjection. As a control, blots were performed on the same lysates with α-tubulin antibodies.

Figure 4

Rescue of the miR-214MO phenotype by simultaneous inhibition of su(fu) expression. (a–h) Expression of Eng (green) and Prox1 (red) in 20-somite embryos (a,c,e,g) or expression of Prox1 (red) and slow-muscle myosin (green) in 1-dpf embryos (b,d,f,h). Embryos in a and b were injected with su(fu) mismatched morpholinos (su(fu) mmMO), and embryos in g and h were injected with two morpholinos targeted to su(fu) (su(fu)MO1,2). Embryos in c and d were coinjected with su(fu) mmMO and 214MO, and embryos in e and f were injected with su(fu)MO1,2 and 214MO.

Figure 5

Modulation of Hedgehog (Hh) signaling in somite cells by miR-214. Su(fu) acts on both activator (Gliact) and repressor (Glirep) forms of Gli, inhibiting nuclear trafficking. miR-214 downregulates Su(fu), allowing both maximal activation in the presence of Hedgehog and complete repression when Hedgehog signaling is minimal.