The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings

Article (PDF Available)inGenes & Development 19(24):2947-52 · January 2006with55 Reads
DOI: 10.1101/gad.1372505 · Source: PubMed
The Drosophila Bithorax Complex encodes three well-characterized homeodomain proteins that direct segment identity, as well as several noncoding RNAs of unknown function. Here, we analyze the iab-4 locus, which produces the microRNAs iab-4-5p and iab-4-3p. iab-4 is analogous to miR-196 in vertebrate Hox clusters. Previous studies demonstrate that miR-196 interacts with the Hoxb8 3' untranslated region. Evidence is presented that miR-iab-4-5p directly inhibits Ubx activity in vivo. Ectopic expression of mir-iab-4-5p attenuates endogenous Ubx protein accumulation and induces a classical homeotic mutant phenotype: the transformation of halteres into wings. These findings provide the first evidence for a noncoding homeotic gene and raise the possibility that other such genes occur within the Bithorax complex. We also discuss the regulation of mir-iab-4 expression during development.
The Drosophila microRNA
iab-4 causes a dominant
homeotic transformation of
halteres to wings
Matthew Ronshaugen,
Frédéric Biemar,
Jessica Piel,
Mike Levine,
and Eric C. Lai
Department of Molecular and Cell Biology, Division of
Genetics, Center for Integrative Genomics, University of
California, Berkeley, California 94720, USA;
Department of
Developmental Biology, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021, USA
The Drosophila Bithorax Complex encodes three well-
characterized homeodomain proteins that direct seg-
ment identity, as well as several noncoding RNAs of un-
known function. Here, we analyze the iab-4 locus,
which produces the microRNAs iab-4–5p and iab-4–3p.
iab-4 is analogous to miR-196 in vertebrate Hox clusters.
Previous studies demonstrate that miR-196 interacts
with the Hoxb8 3 untranslated region. Evidence is pre-
sented that miR–iab-4–5p directly inhibits Ubx activity
in vivo. Ectopic expression of mir–iab-4–5p attenuates
endogenous Ubx protein accumulation and induces a
classical homeotic mutant phenotype: the transforma-
tion of halteres into wings. These findings provide the
first evidence for a noncoding homeotic gene and raise
the possibility that other such genes occur within the
Bithorax complex. We also discuss the regulation of mir–
iab-4 expression during development.
Received September 6, 2005; revised version accepted October
31, 2005.
Classical genetic studies suggest that the Bithorax Com-
plex (BX-C) contains as many as nine homeotic genes
(Lewis 1978). However, only three encode Hox proteins,
Ultrabithorax (Ubx), abdominal-A (abd-A), and Ab-
dominal-B (Abd-B) (Martin et al. 1995). The bulk of ge-
nomic DNA comprising the BX-C is thought to function
as cis-regulatory DNA that controls the timing and site of
Hox expression (Sanchez-Herrero et al. 1985). Neverthe-
less, it has been known for more than 15 years that in-
tergenic regions of the BX-C are extensively transcribed
(Cumberledge et al. 1990; Bae et al. 2002; Drewell et al.
2002). The possible functional activities of the noncod-
ing RNAs have received little attention, despite the fact
that these transcripts, including iab-4, are expressed in
restricted domains along the anterior–posterior axis, like
conventional Hox genes (Cumberledge et al. 1990).
Hox gene clusters contain conserved miRNAs. For ex-
ample, miR-10 is located within the Drosophila Anten-
napedia gene complex (ANT-C) between the Hox genes
Deformed and Sex combs reduced (Lagos-Quintana et al.
2001). The sequence and location of miR-10 are con-
served in vertebrate Hox complexes (Tanzer et al. 2005).
Sex combs reduced has been proposed as a direct miR-10
target in insects (Brennecke et al. 2005). A second group
of Drosophila miRNAs map to a hairpin located at the
distal end of the iab-4 locus (Aravin et al. 2003), which
resides between abd-A and Abd-B (Fig. 1A). miRNAs
were cloned from both arms of this hairpin and are
termed iab-4–5p and iab-4–3p (Aravin et al. 2003). miR–
iab-4–5p was recently predicted to regulate Ubx activity
(Stark et al. 2003; Grun et al. 2005). Although vertebrates
lack an iab-4 ortholog, as defined by sequence identity, a
different miRNA, miR-196, resides at an analogous po-
sition adjacent to the posterior-most HOX 9–13 paralogs.
Tissue culture assays, in vivo cleavage products, and
transgenic lacZ “sensors” indicate that miR-196 inhibits
Hoxb8 activity (Mansfield et al. 2004; Yekta et al. 2004).
Despite these provocative target relationships, no phe-
notypes have been associated with any Hox miRNA.
miRNAs are short, 21–24-nt RNAs that attenuate pro-
tein synthesis by binding complementary sites in target
mRNAs (Lai 2003; Bartel 2004). An unexpectedly modest
amount of base-pairing appears to underlie target recog-
nition. Experimental and computational studies have
converged on the principle of “seed-pairing,” whereby 7
continuous Watson-Crick base pairs at the 5-end of the
miRNA mediate target recognition (Lai 2002; Brennecke
et al. 2005; Lewis et al. 2005). The limited sequence re-
quirement for miRNA–mRNA interactions has fueled
current proposals that a third or more of all mRNAs may
be regulated by miRNAs (Lewis et al. 2005; Xie et al.
2005). As tallies of miRNA loci continue to grow (with
current estimates for humans ranging from 800 to 1000)
(Bentwich et al. 2005; Berezikov et al. 2005), the network
of possible miRNA:target interactions will expand.
Only a small number of miRNA:target interactions
have been studied in vivo. Here we present evidence that
iab-4 microRNAs selectively attenuate Ubx activity in
vivo. The Ubx 3 untranslated region (3 UTR) contains
predicted target sites for miR–iab-4–5p and expression of
a GFP-Ubx-3 UTR “sensor” transgene is repressed by
ectopic expression of a mir–iab-4 minigene. This mini-
gene also reduces Ubx protein levels in haltere imaginal
discs, thereby inducing a classical homeotic transforma-
tion of halteres into wings. Taken together, these results
suggest that the iab-4 transcription unit encodes an au-
thentic homeotic regulatory gene. We suggest that addi-
tional noncoding RNAs correspond to “missing” homeo-
tic genes in the Bithorax complex, and discuss novel
mechanisms of iab-4 regulation during development.
Results and Discussion
Complementary patterns of iab-4 and Ubx expression
Among the sequenced Drosophilids, there are only a few
highly conserved sequences in the 120-kb region sepa-
rating abd-A and Abd-B in the BX-C (Fig. 1A). Three are
located in regions that flank known insulator elements
(Karch et al. 1994; Zhou et al. 1996; Barges et al. 2000). A
fourth conserved region is located within the 3 region of the
[Keywords: microRNA; iab-4; Ultrabithorax; homeotic gene]
Corresponding authors.
E-MAIL; FAX (212) 717-3604.
E-MAIL; FAX (510) 643-5780.
Article and publication are at
GENES & DEVELOPMENT 19:2947–2952 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; 2947
iab-4 transcription unit (Cumberledge et al. 1990; sum-
marized in Fig. 1A). The iab-4 locus contains regulatory
DNAs that control abd-A expression (Karch et al. 1990).
It also produces two spliced, polyadenylated transcripts
that differ by the presence or absence of the highly con-
served 3 sequences (Fig. 1A). This region contains a
single 100-nucleotide (nt) pre-miRNA hairpin structure
that encodes two stable miRNAs: iab-45p and iab-43p
(Aravin et al. 2003).
The pre-miriab-4 hairpin is conserved both in se-
quence and genomic location in the mosquitoes Anoph-
eles gambiae and Aedes aegypti, the honeybee Apis mel-
lifera, and the flour beetle Tribolium castaneum (Fig.
1B), species that last shared a common ancestor roughly
400 million years ago. In fact, it is by far the best con-
served sequence in the abd-A/Abd-B interval among
Drosophilid and non-Drosophilid genomes (Fig. 1A). It is
notable that the sequences corresponding to the mature
iab-45p and iab-43p miRNAs (each forming one arm of
the hairpin) are perfectly conserved among this broad
spectrum of insects.
Double-label RNA FISH and antibody staining was
used to determine the relative expression patterns of
iab-4 RNA and Ubx RNA/protein accumulation during
embryonic development (Fig. 1CE). The iab-4 primary
transcript is strongly expressed in the presumptive abdo-
men, mainly in the progenitors of the second (A2)
through seventh (A7) segments (see Cumberledge et al.
1990). There is also a weak transient stripe of expression
in anterior regions. Ubx RNA is distributed in a strong
stripe in parasegment 6, but only low levels are seen in
regions of the presumptive abdomen containing high lev-
els of iab-4 transcript. No Ubx protein is detected at this
early stage, possibly due to the time required to tran-
scribe the entire 80-kb locus (Shermoen and OFarrell
During the rapid phase of germband elongation, Ubx
protein becomes detectable in the abdomen (Fig. 1D). At
the conclusion of germband elongation, the Ubx and
iab-4 patterns are largely complementary in the dorsal
ectoderm (Fig. 1E). During segmentation of the germ-
band, the Ubx protein shows complex modulation in the
dorsal ectoderm (Fig. 1FH). There are three apparent
levels of Ubx protein distribution in these regions: high,
low, and none (Fig. 1F). We note an inverse correlation
between the levels of Ubx protein and the sites of iab-4
expression (Fig. 1G,H). The strongest expression of iab-4
occurs in regions having the lowest accumulation of Ubx
protein (Fig. 1FH, arrows), whereas intermediate and
low levels coincide with sites of diminished Ubx accu-
mulation (Fig. 1FH, asterisks); there is little or no iab-4
expression in those cells containing the highest levels of
Ubx protein (Fig. 1FH, arrowheads). These observations
are consistent with the possibility that Ubx protein syn-
thesis might be modulated by one or both iab-4 miRNAs.
Direct support for this possibility stems from the analy-
sis of a GFP-Ubx transgene containing the 3 UTR se-
quence from Ubx. This transgene displays slightly di-
minished expression in abdominal regions containing
high levels of iab-4 transcripts (data not shown; see be-
The Ubx 3 UTR is directly targeted by miRiab-45p
Computational studies have identified the Ubx 3 UTR
as a likely target of regulation by iab-45p (Stark et al.
2003; Grun et al. 2005). Of the seven potential sites iden-
tified by Stark and colleagues (Fig. 2A), five exhibit con-
served and canonical seed pairing of six or more nucleo-
tides (Fig. 2B). Of these, sites #3 and #6 are perfectly
conserved among sequenced Drosophilids and have
seeds of at least 7 nt, a length sufficient for efficient in
vivo recognition by miRNAs (Brennecke et al. 2005; Lai
et al. 2005); site #7 also has a 7-mer seed match that is
conserved in some species (see below).
In current target-finding approaches, greater confi-
dence is usually ascribed to those miRNA-binding sites
that are conserved in the greatest number of analyzed
species. Curiously, the putative iab-45p target sites
with the lowest free energy are not necessarily the best
conserved. Instead, there appear to be compensatory
changes among different iab-45p-binding sites in indi-
vidual Ubx 3 UTRs. For example, site #4 exhibits ca-
nonical 6-mer seed pairing in four species of Drosophila,
but contains a G:U base pair in Drosophila virilis and a
seed mismatch in Drosophila mojavenesis and is likely
nonfunctional in these two species. Conversely, site #7
is mispaired in D. melanogaster and Drosophila yakuba,
Figure 1. Structure, conservation, and expression of the iab-4
miRNA. (A) A 120-kb interval of the D. melanogaster Bithorax
Complex that includes the homeobox genes abd-A and Abd-B, the
noncoding RNA gene iab-4, and three transcribed boundary ele-
ments (MCP, Fab-7, and Fab-8). Shown are VISTA plots depicting
highly conserved sequences in the distantly related Drosophilid
Drosophila mojaviensis (regions of >80% identity in a window of 50
nt) and the honeybee A. mellifera (regions of >80% identity in a
window of 150 nt). Note that all of these conserved regions are
associated with transcribed noncoding elements. Depicted at higher
magnification are two iab-4 splice forms, an 2.1-kb transcript con-
taining the iab-4 miRNA hairpin and a shorter transcript that ter-
minates just 5 of the miRNAs. The two iab-4 probes used in this
study (iab-4 probe1 and iab-4 probe2) are shown as black bars below
the schematics in A.(B) Perfect conservation of the iab-45p and
iab-43p miRNAs in diverse arthropods: D. melanogaster (D. mel),
D. pseudoobscura (D. pse), Bombyx mori (B. mor), A. mellifera (A.
mel), A. aegypti (A. egy), A. gambiae (A. gam), and T. castaneum (T.
cas). (CE) Time course of iab-4 (red) and Ubx (green) nascent tran-
scription: stage 5 (C), stage 8 (D), stage 11 (E). Nascent iab-4 tran-
scription was detected with an intronic probe marked as probe 1 in
A.(FH) Complementary expression of iab-4 RNA (red) and Ubx
protein (green) in a stage 12 embryo. Shown is a maximum projec-
tion through a sagittal section revealing the ectoderm and underly-
ing mesoderm (approximately five cell layers). Domains of highest
Ubx protein levels do not express iab-4 (arrowheads), whereas cells
with highest levels of iab-4 do not accumulate Ubx (arrows). Other
regions show low levels of coexpression (asterisk).
Ronshaugen et al.
but is conserved as a strong 7-mer seed-paired site in D.
mojavenesis, Drosophila pseudoobscura, Drosophila
ananassae, and D. virilis. These observations suggest
that individual target sites may be evolutionarily labile,
and in vivo regulation depends on the net complement of
both high- and low-affinity sites contained in the target
mRNA. These compensatory changes in strong and
weak target sites are reminiscent of the evolution of in-
dividual Bicoid-binding sites in the eve stripe 2 enhanc-
ers present in divergent Drosophilids (Ludwig et al.
Direct evidence for iab-4:Ubx miRNA interactions
was obtained using a tubGFP-Ubx 3 UTR transgene
(the Ubx sensor). This construct directs ubiquitous ex-
pression of the GFP coding sequence fused to the Ubx 3
UTR, and wing imaginal discs bearing the Ubx sensor
display relatively uniform expression of GFP. Ectopic ex-
pression of UAS-DsRed under the control of ptc-Gal4,
which directs expression along the anteriorposterior
border of the disc, has little or no effect on the distribu-
tion of GFP staining (Brennecke et al. 2005).
We next assayed the expression of the Ubx sensor in
the presence of ectopic iab-4 miRNAs. For this purpose,
a transgene was created that contains DsRed and 400
base pairs (bp) from iab-4 encompassing the entire 100-
bp 3 hairpin sequence (UAS-DsRed-iab-4). As shown
previously, transgenes of this type direct the expression
of biologically active miRNAs in cells that are labeled by
expression of DsRed (Stark et al. 2003). When driven by
ptc-Gal4 in wing imaginal discs, Ubx sensor levels were
specifically diminished in those cells expressing the
iab-4 transgene (Fig. 2CE). Detailed analysis of the
DsRed-iab-4 and GFP-Ubx expression profiles suggests
that repression of the Ubx sensor by ectopic iab-4
miRNA is dose-sensitive (Fig. 2FH). These data consti-
tute in vivo evidence that iab-4 miRNAs specifically rec-
ognize target sequences in the Ubx 3 UTR and thereby
attenuate Ubx protein synthesis.
Ectopic iab-4 inhibits endogenous Ubx and induces
a Ubx-like homeotic transformation
Ubx protein is broadly distributed throughout the hal-
tere imaginal disc, where it imposes haltere identity by
repressing the expression of many genes that otherwise
direct wing development (e.g., Weatherbee et al. 1998).
This repression is very sensitive to Ubx levels, and con-
sequently, even partial loss of Ubx function can trans-
form halteres into wings (i.e., Fig. 4B, below). Haltere
discs were examined for the accumulation of Ubx pro-
tein in the absence or presence of ectopic iab-4 miRNAs.
Ubx is detected at high levels in most of the cells of the
presumptive pouch. Expression of DsRed alone using bx-
Gal4, which is active in the presumptive dorsal region of
the pouch, did not affect Ubx accumulation (Fig. 3AE).
In contrast, haltere discs expressing UAS-DsRed-iab-4
under the control of bx-Gal4 displayed strongly reduced
levels of Ubx protein (Fig. 3FJ). Thus, as seen for the
Ubx sensor in wing discs, ectopic iab-4 miRNA inhibits
accumulation of endogenous Ubx protein.
The effect of iab-4 miRNA misexpression on adult hal-
tere development was examined. The wild-type haltere
contains small lightly pigmented sensilla but lacks the
triple row of sensory bristles at the leading margin seen
in wings (Fig. 4A). In contrast, halteres that developed
from discs expressing UAS-DsRed-iab-4 under the con-
trol of bx-Gal4 or scalloped-Gal4 are flattened and elon-
gated in the proximaldistal axis, and exhibit an exten-
sive row of sensory bristles at the leading margin (Fig.
4C,D). All of these phenotypes are strongly indicative of
a classic haltere-to-wing homeotic transformation (Roch
and Akam 2000).
The demonstration that miRiab-4 represses the ante-
rior Hox gene Ubx might be relevant to the phenomenon
of posterior prevalence (Duboule and Dolle 1989).
Polycomb mutant embryos have previously been ob-
served to derepress Hox gene expression, resulting in
broad misexpression of all Hox genes (e.g., Pirrotta 1997).
Ultimately, ectopic expression of posterior Hox genes
(e.g., Abd-B or Hox913) leads to the transcriptional re-
pression of anterior Hox genes (e.g., Ubx or Hox8 para-
logs) (Choi et al. 2000). We observe that Polycomb mu-
Figure 2. Identification and validation of iab-45p target sites in
the Ubx 3 UTR. (A) Conserved regions of the Ubx 3 UTR revealed
by VISTA plots between D. melanogaster and other Drosophilids
(dark blue, Drosophila simulans; light blue, D. ananassae; white, D.
mojaviensis). All but one of seven putative iab-45p target sites
(Stark et al. 2003) correspond to islands of conserved sequence (num-
bered arrows); free energies of predicted iab-45p:target duplexes are
noted below. (B) Alignments and free energies of likely iab-45p
target sites from six divergent Drosophilids: D. melanogaster (D.
mel), D. yakuba (D. yak), D. ananassae (D. ana), D. pseudoobscura
(D. pse), D. virilis (D. vir), and D. mojaviensis (D. moj). Conserved
Ubx 3 UTR sequences are shaded gray; nucleotides that pair with
the iab-45p seed are shaded blue. Note apparent compensatory mu-
tations between strong/weak sites in different species (in red, ar-
rows). For example, site #4 is likely active in D. melanogaster but
inactive in D. mojaviensis, while site #7 probably inactive in D.
melanogaster but active in D. mojaviensis.(CH) Evidence for di-
rect recognition of the Ubx 3 UTR by iab-45p. Shown is expression
of a tubGFP-Ubx 3 UTR sensor transgene (green) in a background
where a UAS-DsRed-iab-4 transgene has been activated using ptc-
Gal4 (red). The Ubx sensor is specifically down-regulated in mir
iab-4-misexpressing cells marked by expression of DsRed. (FH)
Close-ups of the boxed region in E.
miRiab-4 represses Ultrabithorax
tant embryos also derepress iab-4 expression throughout
the embryo (Fig. 5, cf. B and A). Therefore, misexpression
of iab-4 miRNAs may contribute to the repression of
Ubx function observed in the Polycomb mutant back-
ground. Thus, posterior prevalence may arise from the
dual utilization of protein-based/transcriptional mecha-
nisms and miRNA-based/post-transcriptional mecha-
nisms (Fig. 5C).
Regulation and function of iab-4 miRNAs
The detailed analysis of intergenic transcripts in the
abd-A/Abd-B interval suggests a strand exclusion
model for iab-4 regulation (summarized in Fig. 5F). The
iab-4 locus is unusual in that both strands are tran-
scribed (Bae et al. 2002). However, each strand displays a
distinct pattern of expression. The strand producing the
iab-4 pri-miRNA is broadly expressed in the A2A7 re-
gion of the germband, while the other strand is expressed
in A8 and A9 (e.g., Fig. 5D,E). Double RNA FISH assays
suggest that the miR strand is initially expressed in A2
A8, but expression is lost in A8 as transcription of the
other strand progresses from the iab-8 domain. Perhaps
transcription from one strand diminishes transcription
from the other (Fig. 5F). Although the detailed mecha-
nism may be different, these observations are evocative
of the mutually exclusive expression of Xist and Tsix
RNAs on mammalian X chromosomes (e.g., Rougeulle
and Avner 2004). Additional target predictions for miR
iab-4 imply that exclusion of iab-4 expression from A8
might be important for stable accumulation of other po-
tential iab-4 target mRNAs, such as Abd-B (Enright et al.
2003; Stark et al. 2003).
Traditionally, recessive loss-of-function mutations are
used to assess the in vivo activities of patterning genes.
The principal argument for iab-4:Ubx interactions in de-
velopment rests with the analysis of dominant gain-of-
function phenotypes arising from the misexpression of
miRiab-4 in the haltere imaginal disks (see Figs. 3, 4).
The specificity of the resulting haltere-to-wing homeotic
transformation correlates with reduced levels of Ubx
protein accumulation specifically in the regions where
miRiab-4 products are misexpressed. It is likely that
there is redundancy in the transcriptional repression of
Ubx by Abd-A and Abd-B products, and the inhibition of
Ubx protein synthesis by iab-4 miRNAs. Indeed, noncod-
ing genes in the BX-C were mainly identified by domi-
nant mutations such as chromosomal rearrangements
(e.g., Lewis 1978). Therefore, misexpression assays may
prove effective in analyzing the function of other Hox
noncoding genes.
This study provides evidence that iab-4 encodes a
novel homeotic regulatory activity, which functions, at
least in part, by producing miRNAs inhibiting Ubx.
iab-4 miRNAs may regulate additional target mRNAs.
For example, computational analyses identify homotho-
rax as another potential target of interest (Grun et al.
2005). Homothorax works in parallel with the Hox co-
factor Extradenticle and various Hox proteins to control
Figure 4. Directed expression of miriab-4 induces a homeotic
transformation. (A) Wild-type haltere, which contains small lightly
pigmented sensilla but lacks the triple row of sensory bristles seen
in wings. (B) Haltere-to-wing transformation in a mild Ubx loss-of-
function mutant background. Misexpression of iab-4 miRNA hair-
pin in bx-Gal4/Y; UAS-DsRed-miriab-4 (C) and sd-Gal4, UAS-
DsRed-miriab-4 (D) animals induces a similar haltere-to-wing
Figure 3. Expression of miriab-4 in the haltere disc reduces en-
dogenous Ubx protein accumulation. (AE) Normal expression of
Ubx protein (green) in bx-Gal4/Y; UAS-DsRed/+ haltere discs. (A)
Ubx is detected throughout the pouch region of the haltere disc. (B)
DsRed (red) marks bx-Gal4 activity in the dorsal compartment of
the presumptive haltere pouch. (C) Merge. (D,E) Close-ups of the
region boxed in C.(FH) bx-Gal4/Y; UAS-DsRed-miriab-4 haltere
disc exhibits reduced accumulation of Ubx protein (green) in the
presence of ectopic miriab-4 (red). (I,J) Close-ups of the region
boxed in H.
Ronshaugen et al.
the patterning of legs and antennae (for review, see Mann
and Morata 2000). It is also possible that downstream
transcriptional targets of Ubx (realizators) might be
modulated by iab-4 miRNAs. Additional noncoding
RNAs in the BX-C, such as cbx, pbx and bxd (e.g., Lip-
shitz et al. 1987), might also possess homeotic regulatory
activities and account for the remaining genes identified
by Lewis (1978).
Materials and methods
Immunohistochemistry and in situ hybridization
Embryo fixation, probe labeling, and nascent transcript RNA FISH were
performed according to Kosman et al. (2004) with the following modifi-
cations. The sequence of the iab-4 probes were previously described (Bae
et al. 2002). RNA probes labeled with FITC-, DIG-, and Biotin-conjugated
UTP were detected as follows: DIG, sheep DIG (Roche); Biotin, mouse
Biotin (Roche); FITC, rabbit FITC (Molecular Probes). The secondary
detection was as follows: donkey sheep Alexa Fluor 555 (Molecular
Probes), donkey mouse Alexa Fluor 488 (Molecular Probes), and
chicken rabbit Alexa Fluor 647 (Molecular Probes). Ubx protein was
detected using mouse -Ubx (FP3.83) (White and Wilcox 1984). For de-
tection of GFP and Ubx, we fixed discs in 4% paraformaldehyde and
probed them with rabbit -GFP (1:1000; Molecular Probes) or mouse
-Ubx FP3.83 (1:10).
Drosophila strains
The UAS-miRiab4 construct was generated by cloning a 400-bp frag-
ment centered around the miriab-4 hairpin downstream of pUAST-
DSred2 (Stark et al. 2003). The Ubx sensor was cloned by inserting ge-
nomic DNA including the Ubx 3 UTR and 400 bp of downstream ge-
nomic DNA into tubGFP (Brennecke et al. 2003). These constructs were
injected into w
embryos, and multiple transgenic lines were estab-
lished. Other Drosophila lines were obtained from the Bloomington
Stock Center, including scallopedGAL4, bx
-GAL4, and various
Ubx and Pc alleles.
We acknowledge Nipam Patel for antibodies, David Bilder for fly stocks,
and Gerald Rubin for his support during this project. We also thank
Robert Zinzen and Kimberly Mace for discussions and helpful comments
on the manuscript, and Oliver Zill who provided important help during
the initial phases of this work. This study was funded by grants from the
NIH (GM34431 to M.L. and GM72395 to M.R.), and grants from the
Leukemia and Lymphoma Society (LLS #3096-05) and the Burroughs
Wellcome Foundation (CABS 1004721) to E.C.L.
Aravin, A.A., Lagos-Quintana, M., Yalcin, A., Zavolan, M., Marks, D.,
Snyder, B., Gaasterland, T., Meyer, J., and Tuschl, T. 2003. The small
RNA profile during Drosophila melanogaster development. Dev.
Cell 5: 337350.
Bae, E., Calhoun, V.C., Levine, M., Lewis, E.B., and Drewell, R.A. 2002.
Characterization of the intergenic RNA profile at Abdominal-A and
Abdominal-B in the Drosophila bithorax complex. Proc. Natl. Acad.
Sci. 99: 1684716852.
Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Muller, M., Shanower,
G., Schedl, P., Gyurkovics, H., and Karch, F. 2000. The Fab-8 bound-
ary defines the distal limit of the bithorax complex iab-7 domain and
insulates iab-7 from initiation elements and a PRE in the adjacent
iab-8 domain. Development 127: 779790.
Bartel, D.P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and
function. Cell 116: 281297.
Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O.,
Barzilai, A., Einat, P., Einav, U., Meiri, E., et al. 2005. Identification of
hundreds of conserved and nonconserved human microRNAs. Nat.
Genet. 37: 766770.
Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R.H.,
and Cuppen, E. 2005. Phylogenetic shadowing and computational
identification of human microRNA genes. Cell 120: 2124.
Brennecke, J., Hipfner, D.R., Stark, A., Russell, R.B., and Cohen, S.M.
2003. bantam encodes a developmentally regulated microRNA that
controls cell proliferation and regulates the proapoptotic gene hid in
Drosophila. Cell 113: 2536.
Brennecke, J., Stark, A., Russell, R.B., and Cohen, S.M. 2005. Principles
of microRNA-target recognition. PLoS Biol. 3: e85.
Choi, S.H., Oh, C.T., Kim, S.H., Kim, Y.T., and Jeon, S.H. 2000. Effects of
Polycomb group mutations on the expression of Ultrabithorax in the
Drosophila visceral mesoderm. Mol. Cells 10: 156161.
Cumberledge, S., Zaratzian, A., and Sakonju, S. 1990. Characterization of
two RNAs transcribed from the cis-regulatory region of the abd-A
domain within the Drosophila bithorax complex. Proc. Natl. Acad.
Sci. 87: 32593263.
Drewell, R.A., Bae, E., Burr, J., and Lewis, E.B. 2002. Transcription de-
fines the embryonic domains of cis-regulatory activity at the Dro-
sophila bithorax complex. Proc. Natl. Acad. Sci. 99: 1685316858.
Duboule, D. and Dolle, P. 1989. The structural and functional organiza-
tion of the murine HOX gene family resembles that of Drosophila
homeotic genes. EMBO J. 8: 14971505.
Enright, A.J., John, B., Gaul, U., Tuschl, T., Sander, C., and Marks, D.S.
2003. MicroRNA targets in Drosophila. Genome Biol. 5: R1.
Grun, D., Wang, Y.L., Langenberger, D., Gunsalus, K.C., and Rajewsky,
Figure 5. Regulation and function of iab-4.(A,B) Expression of
iab-4 in wild-type (A) and Pc
(B) stage 12 embryos. The domain of
iab-4 transcription is expanded anteriorly in the Pc mutant, which
correlates with suppression of Ubx activity in this mutant back-
ground. (C) Similar regulatory and functional arrangement of the
BX-C and the vertebrate HOX clusters. Posteriorly expressed Hox
proteins repress the transcription of anterior Hox genes; posteriorly
expressed microRNAs (iab-45p and iab-43p in insects, and miR-
196 in vertebrates) repress anterior Hox genes at a post-transcrip-
tional level (either by transcript cleavage or inhibition of productive
translation). (D,E) Double RNA FISH staining to localize iab-4 tran-
scripts (red) and a noncoding RNA produced from the opposite
strand (blue). The opposite transcript is not detected with iab-4
probes until the onset of germband elongation, presumably due to
the time it takes for Pol II to progress from the iab-8 promoter
located 75 kb away. In germband-elongated embryos the two tran-
scripts are expressed in complementary patterns. The probes used
for hybridization reside within 2 kb of one another within the iab-4
transcription unit and are described in Figure 1. (F) Summary of the
strand-exclusion model. A large transcript derived from the Abd-B/
iab-8 domain might preclude transcription from the iab-4 promoter
on the opposite strand.
miRiab-4 represses Ultrabithorax
N. 2005. microRNA target predictions across seven Drosophila spe-
cies and comparison to mammalian targets. PLoS Comput. Biol. 1:
Karch, F., Bender, W., and Weiffenbach, B. 1990. abdA expression in
Drosophila embryos. Genes & Dev. 4: 15731587.
Karch, F., Galloni, M., Sipos, L., Gausz, J., Gyurkovics, H., and Schedl, P.
1994. Mcp and Fab-7: Molecular analysis of putative boundaries of
cis-regulatory domains in the bithorax complex of Drosophila mela-
nogaster. Nucleic Acids Res. 22: 31383146.
Kosman, D., Mizutani, C.M., Lemons, D., Cox, W.G., McGinnis, W., and
Bier, E. 2004. Multiplex detection of RNA expression in Drosophila
embryos. Science 305: 846.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. 2001.
Identification of novel genes coding for small expressed RNAs. Sci-
ence 294: 853858.
Lai, E.C. 2002. Micro RNAs are complementary to 3 UTR sequence
motifs that mediate negative post-transcriptional regulation. Nat.
Genet. 30: 363364.
———. 2003. microRNAs: Runts of the genome assert themselves. Curr.
Biol. 13: R925R936.
Lai, E.C., Tam, B., and Rubin, G.M. 2005. Pervasive regulation of Dro-
sophila Notch target genes by GY-box-, Brd-box-, and K-box-class
microRNAs. Genes & Dev. 19: 10671080.
Lewis, E.B. 1978. A gene complex controlling segmentation in Dro-
sophila. Nature 276: 565570.
Lewis, B.P., Burge, C.B., and Bartel, D.P. 2005. Conserved seed pairing,
often flanked by adenosines, indicates that thousands of human
genes are microRNA targets. Cell 120: 1520.
Lipshitz, H.D., Peattie, D.A., and Hogness, D.S. 1987. Novel transcripts
from the ultrabithorax domain of the bithorax complex. Genes &
Dev. 1: 307322.
Ludwig, M.Z., Patel, N.H., and Kreitman, M. 1998. Functional analysis of
eve stripe 2 enhancer evolution in Drosophila: Rules governing con-
servation and change. Development 125: 949958.
Mann, R.S. and Morata, G. 2000. The developmental and molecular bi-
ology of genes that subdivide the body of Drosophila. Ann. Rev. Cell
Dev. Biol. 16: 243271.
Mansfield, J.H., Harfe, B.D., Nissen, R., Obenauer, J., Srineel, J.,
Chaudhuri, A., Farzan-Kashani, R., Zuker, M., Pasquinelli, A.E.,
Ruvkun, G., et al. 2004. MicroRNA-responsive sensor transgenes
uncover Hox-like and other developmentally regulated patterns of
vertebrate microRNA expression. Nat. Genet. 36: 10331034.
Martin, C.H., Mayeda, C.A., Davis, C.A., Ericsson, C.L., Knafels, J.D.,
Mathog, D.R., Celniker, S.E., Lewis, E.B., and Palazzolo, M.J. 1995.
Complete sequence of the bithorax complex of Drosophila. Proc.
Natl. Acad. Sci. 92: 83988402.
Pirrotta, V. 1997. Chromatin-silencing mechanisms in Drosophila main-
tain patterns of gene expression. Trends Genet. 13, 314318.
Roch, F. and Akam, M. 2000. Ultrabithorax and the control of cell mor-
phology in Drosophila halteres. Development 127: 97107.
Rougeulle, C. and Avner, P. 2004. The role of antisense transcription in
the regulation of X-inactivation. Curr. Top. Dev. Biol. 63: 6189.
Sanchez-Herrero, E., Vernos, I., Marco, R., and Morata, G. 1985. Genetic
organization of Drosophila bithorax complex. Nature 313: 108113.
Shermoen, A.W. and OFarrell, P.H. 1991. Progression of the cell cycle
through mitosis leads to abortion of nascent transcripts. Cell 67:
Stark, A., Brennecke, J., Russell, R.B., and Cohen, S.M. 2003. Identifica-
tion of Drosophila MicroRNA targets. PLoS Biol. 1: E60.
Tanzer, A., Amemiya, C.T., Kim, C.B., and Stadler, P.F. 2005. Evolution
of microRNAs located within Hox gene clusters. J. Exp. Zoolog. B
Mol. Dev. Evol. 304: 7585.
Weatherbee, S.D., Halder, G., Kim, J., Hudson, A., and Carroll, S. 1998.
Ultrabithorax regulates genes at several levels of the wing-patterning
hierarchy to shape the development of the Drosophila haltere. Genes
& Dev. 12: 14741482.
White, R.A. and Wilcox, M. 1984. Protein products of the bithorax com-
plex in Drosophila. Cell 39: 163171.
Xie, X., Lu, J., Kulbokas, E.J., Golub, T.R., Mootha, V., Lindblad-Toh, K.,
Lander, E.S., and Kellis, M. 2005. Systematic discovery of regulatory
motifs in human promoters and 3 UTRs by comparison of several
mammals. Nature 434: 338345.
Yekta, S., Shih, I.H., and Bartel, D.P. 2004. MicroRNA-directed cleavage
of HOXB8 mRNA. Science 304: 594596.
Zhou, J., Barolo, S., Szymanski, P., and Levine, M. 1996. The Fab-7 ele-
ment of the bithorax complex attenuates enhancerpromoter inter-
actions in the Drosophila embryo. Genes & Dev. 10: 31953201.
Ronshaugen et al.
    • "Although canalization is most likely a systemic property, few specific molecular mechanisms have been proposed to support canalization [reviewed in (Salathia and Queitsch, 2007)]. These include buffering of genetic variability by the Hsp90 chaperone (Rutherford and Lindquist, 1998; Queitsch et al., 2002; Milton et al., 2006; Samakovli et al., 2007; Sgro et al., 2010), tissue-specific maintenance of active and repressed state of developmental genes by the Polycomb (and by analogy also the trithorax) system (Lee et al., 2005; Sawarkar and Paro, 2010; Stern et al., 2012), stabilizing negative feedback by microRNAs (Ronshaugen et al., 2005; Hornstein and Shomron, 2006; Li et al., 2009; Wu et al., 2009), and piwi-mediated silencing of transposon activity (Specchia et al., 2010; Gangaraju et al., 2011) or of existing variation (Gangaraju et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: The microbiome is known to have a profound effect on the development, physiology and health of its host. Whether and how it also contributes to evolutionary diversification of the host is, however, unclear. Here we hypothesize that disruption of the microbiome by new stressful environments interferes with host-microbe co-adaptation, contributes to host destabilization, and can drive irreversible changes in the host prior to its genetic adaptation. This hypothesis is based on three presumptions: (1) the microbiome consists of heritable partners which contribute to the stability (canalization) of host development and physiology in frequently encountered environments, (2) upon encountering a stressful new environment, the microbiome adapts much faster than the host, and (3) this differential response disrupts cooperation, contributes to host destabilization and promotes reciprocal changes in the host and its microbiome. This dynamic imbalance relaxes as the host and its microbiome establish a new equilibrium state in which they are adapted to one another and to the altered environment. Over long time in this new environment, the changes in the microbiome contribute to the canalization of the altered state. This scenario supports stability of the adapted patterns, while promoting variability which may be beneficial in new stressful conditions, thus allowing the organism to balance stability and flexibility based on contextual demand. Additionally, interaction between heritable microbial and epigenetic/physiological changes can promote new outcomes which persist over a wide range of timescales. A sufficiently persistent stress can further induce irreversible changes in the microbiome which may permanently alter the organism prior to genetic changes in the host. Epigenetic and microbial changes therefore provide a potential infrastructure for causal links between immediate responses to new environments and longer-term establishment of evolutionary adaptations.
    Full-text · Article · Jun 2014
    • "addition, Hox-embedded microRNAs (miRNAs), such as miR-iab-4, and its antisense miRNA, miR-iab-8 (also known as miR-iab-4as), have been shown to suppress Ultrabithorax (Ubx) expression, whereas only miR-iab-8 appears to inhibit D. melanogaster abd-A expression (Ronshaugen et al., 2005; Stark et al., 2008; Tyler et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Patterning and phenotypic variations of appendages in insects provide important clues on developmental genetics. In the silkworm Bombyx mori, morphological variations associated with the E complex, an analogue of the Drosophila melanogaster bithorax complex, mainly determine the shape and number of prolegs on abdominal segments. Here, we report the identification and characterization of the allele responsible for the supernumerary crescents and legs-like (ECs-l) mutant, a model derived from spontaneous mutation of the E complex, with supernumerary legs and extra crescents. Fine mapping with 1605 individuals revealed a ∼68 kb sequence in the upstream intergenic region of B. mori abdominal-A (Bmabd-A) clustered with the ECs-l locus. Quantitative real-time PCR (qRT-PCR) and Western blotting analyses disclosed a marked increase in Bmabd-A expression in the ECs-l mutant at both the transcriptional and translational levels, compared to wild-type Dazao. Furthermore, we observed ectopic expression of the Bmabd-A protein in the second abdominal segment (A2) of the ECs-l mutant. Our results collectively suggest that the 68 kb region contains important regulatory elements of the Bmabd-A gene, and provide evidence that the gene is required for limb development in abdominal segments in the silkworm.
    Full-text · Article · Oct 2013
    • "Misexpression of some of these miRNAs result in phenotypes similar to that of loss of Notch function. Misexpression of miR-iab-5p can repress Ubx and induce a homeotic phenotype63. However, it remains to be determined whether the corresponding miRNAs mutants will impact Notch signaling or Ubx function in vivo. "
    [Show abstract] [Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small 22-25 nucleotides long non-coding RNAs, that are conserved during evolution, and control gene expression in metazoan animals, plants, viruses, and bacteria primarily at post-transcriptional and transcriptional levels. MiRNAs ultimately regulate target gene expression by degrading the corresponding mRNA and/or inhibiting their translation. Currently, the critical functions of miRNAs have been established in regulating immune system, cell proliferation, differentiation and development, cancer and cell cycle by as yet unknown control mechanism. MiRNAs play an essential role in malignancy, and as tumour suppressors and oncogenes. Thus, discovery of new miRNAs will probably change the landscape of cancer genetics. Significantly different miRNA profiles can be assigned to various types of tumours, which could serve as phenotypic signatures for different cancers for their exploitation in cancer diagnostics, prognostics and therapeutics. If miRNA profiles can accurately predict malignancies, this technology could be exploited as a tool to surmount the diagnostic challenges. This review provides comprehensive and systematic information on miRNA biogenesis and their implications in human health.
    Full-text · Article · Apr 2013
Show more