Drosophila microRNAs exhibit diverse spatial expression patterns during embryonic development

Abstract

MicroRNAs (miRNAs) are an extensive class of regulatory RNA whose specific functions in animals are generally unknown. Although computational methods have identified many potential targets of miRNAs, elucidating the spatial expression patterns of miRNAs is necessary to identify the sites of miRNA action. Here, we report the spatial patterns of miRNA transcription during Drosophila embryonic development, as revealed by in situ hybridization to nascent miRNA transcripts. We detect expression of 15 "stand-alone" miRNA loci and 9 intronic miRNA loci, which collectively represent 38 miRNA genes. We observe great variety in the spatial patterns of miRNA transcription, including preblastoderm stripes, in aspects of the central and peripheral nervous systems, and in cellular subsets of the mesoderm and endoderm. We also describe an intronic miRNA (miR-7) whose expression pattern is distinct from that of its host mRNA (bancal), which demonstrates that intronic miRNAs can be subject to independent cis-regulatory control. Intriguingly, the expression patterns of several fly miRNAs are analogous to those of their vertebrate counterparts, suggesting that these miRNAs may have ancient roles in animal patterning.

Fig. 1.

Detection of nascent miRNA transcripts with in situ hybridization. (A) Typical mRNA expression is detected in the cytoplasm; expression of CG10479 is shown here. (B) miRNA expression is detected as pairs of nuclear dots; shown here is expression of the miR-309/3/286/4/5/6 cluster.

Fig. 2.

Expression patterns of stand-alone (A–N) and intronic (O–X) miRNAs. Expression patterns of Drosophila miRNAs broadly divided into those that stand alone (i.e., reside in genomic regions between annotated mRNA-encoding genes, A–N) and those located within introns of annonated genes (O–X). Note that miR-274 (P) is actually oriented antisense to its “host” gene CG10415. miRNAs in clusters are labeled with the name with the most 5′ member followed by the suffix “clus.” Anterior is to the left and lateral views are shown, excepting dorsal (I and K) and ventral (J and N) views. Progressively older embryos are shown from top to bottom: blastoderm (A–C and O–Q), germband-extending (D–H, K, R–T, and V–X), and germband-retracted (I, J, L–N, and U). Note that in many cases the expression of a given locus is dynamic with time. (A–C) miR-309/3/286/4/5/6–1/6–2/6–3 cluster (A), miR-10 (B), and iab-4 (C) are expressed in subset regions along the anterior-posterior axis in blastoderm embryos. (D) miR-31a is expressed in a pair-rule pattern of 14 stripes and in the foregut, anterior endoderm, and hindgut. (E and F) miR-1 (E) and miR-316 (F) are expressed specifically in the mesoderm. (G) miR-184 displays expression in the mesoderm, anterior endoderm, and posterior endoderm of germband-extending embryos. (H) miR-8 is expressed in the salivary placode, mesoderm, and a segmented subset of the ectoderm. (I) miR-279 is expressed in the atrium, head sensory systems, anterior spiracles, and gonads. (J–L) miR-124 (J), miR-315 (K), and miR-13b-1/13b-2/2c (L) clusters are expressed in the brain and ventral nerve cord. (M) miR-14 is ubiquitous. (N) miR-375 is expressed in the salivary gland and hindgut. (O–Q) miR-11 (O), miR-274 (P), and miR-281–1/281–2 (Q) all are expressed in subset regions along the anterior-posterior axis. (R) The miR-283/12/304 cluster is expressed in the foregut, posterior midgut, hindgut, salivary gland, and a subset of peripheral nervous system cells. (S) The miR-2a-2/2a-1/2b-2 cluster is expressed in the epidermis and hindgut. (T) miR-92a is expressed in the brain primordium and a subset of the ventral nerve cord. (U) miR-13b-2 is expressed in somatic muscles and the gut. (V) miR-263b is expressed in sensory organ precursors of the peripheral nervous system. (W) miR-7 is expressed in a subset of the ventral nerve cord. (X) miR-307 is ubiquitous.

Fig. 3.

Examples of miRNAs with temporally and spatially dynamic expression. Anterior is to the left and lateral views are shown, except dorsal views are shown in D and T.(A–E) The miR-309/3/286/4/5/6–1/6–2/6–3 cluster. Expression is throughout the embryo at stage 4 (A), but is soon repressed at both poles and within an anterior domain (B, arrows). In later stage 5, its expression rapidly resolves into stripes (C and D) that become restricted dorsally (E, arrow). (F–J) miR-10. Expression is found at ≈50–80% of egg length at stage 5 (F) and quickly resolves into stripes (G). (H) By stage 7, miR-10 expression has mostly ceased. (I) At stage 11, miR-10 transcription reinitiates in the ventral nerve cord, posterior midgut (PMG) and hindgut (HG). (J) At stage 14, expression remains in the hindgut (HG) and anal pad (AP). (K–O) miR-1. (K) At stage 5, expression is found throughout the ventral side of the embryo. (L) At stage 6, a prominent gap at the site of the presumptive anterior endoderm is seen (arrow). (M) At stage 10, miR-1 expression is seen throughout the mesoderm. (N and O) By stage 12 (N) and stage 13 (O), it is visible in all differentiating muscles, including somatic muscles (SM) and pharyngeal muscles (PM). (P–T) miR-8. (P) At stage 10, miR-8 is expressed in yolk nuclei (YN). (Q) At stage 11, it is weakly expressed in a subset of trunk mesoderm (M), with stronger staining in a subset of the posterior mesoderm adjacent to the hindgut primordium, as well as in the anlage of the salivary gland (SG). (R) At stage 12, miR-8 expression is evident in the salivary gland, mesoderm (M), and in a metameric subset of the ectoderm (E). (S and T) At stage 13, miR-8 remains strongly expressed in the ectoderm and is also present in the posterior midgut (PMG).

Fig. 4.

Expression and function of intronic miR-7 is distinct from its host gene bancal. (A) Map of the bancal locus and exon structures of three full-length bancal isoforms (RA, RB, and RC) and a short isoform (RD) (31). miR-7 is indicated by the green box. One cluster of P-elements is located near the start of RA-RC, and a second cluster is located near the start of RD (31). (B–E) Expression patterns detected by a 600-bp probe surrounding miR-7 in stage 6–12 embryos. (F) Ubiquitous expression pattern of bancal. (G) ptc-Gal4 adult wing. (H) Activation of EP(2)0954 (see A) using ptc-Gal4 results in a notched wing and diminution of the L3–4 intervein region. * indicates wing notching. (I) Misexpression of a UAS-miR-7 transgene phenocopies the EP(2)0954 misexpression phenotype. * indicates wing notching.