Fig 1.
The Tv4-enhancer faithfully reports FMRFa expression exclusively in Tv4 neurons and contains conserved putative binding sites for Ap and Smads.
(A) Tv neurons in the embryonic/larval VNC; Tv1 neurons (green), Tv2/3 neurons (blue) and Tv4 neurons (red). Tv1 neurons express the neuropeptide Nplp1 and Tv4 neurons express FMRFa. Segment number indicated on the left side of the VNC. (B) Transcription factors postulated to regulate FMRFa in Tv4 neurons. (C) Genome coordinates (Release 5) and scale image of FMRFa gene locus (exons denoted by thick blue lines, introns denoted by thin blue line, promoter denoted by arrow) and the 445 bp Tv4-enhancer (red box). Below is a conservation histogram through the Tv4-enhancer across 12 Drosophila species (high peaks = best conserved) from UCSC Browser. Below that, we show the relative location of putative homeodomain (green box), Mad (red and magenta boxes) and Medea sites (yellow boxes). (D) Nuclear-localized EYFP reporter expression driven from the wildtype 445 bp Tv4-enhancer (TvWT-nEYFP). TvWT-nEYFP is only expressed in Tv4 neurons (side panels; anti-FMRFa upper panel, TvWT-nEYFP lower panel). Scale bar is 30 μm. (E) Sequence of Drosophila melanogaster Tv4-enhancer showing putative Homeodomain (green box), Mad (red or magenta box) and Medea (yellow box) binding sites. Conservation of nucleotide identity was identified using the Relaxed EvoPrint (EvoprinterHD), and is shown here using two layers of conservation. The first layer is shown by bolded capital letters to denote nucleotides conserved in 11 of 12 sequenced Drosophila species (see S4 Fig). The second layer is shown as small bolded letters that are conserved in 9 of 12 Drosophila species.
Fig 2.
The Tv4-enhancer responds appropriately to known FMRFa transcriptional regulators.
(A-C) Representative images of TvWT-nEYFP (green) and FMRFa immunoreactivity (magenta) in controls (ctrl), and in ap and wit mutants (side panels; FMRFa upper panel, TvWT-nEYFP lower panel), showing partial and full loss of reporter and FMRFa peptide expression, respectively. (D) Graph showing % fluorescence intensity for FMRFa immunoreactivity and TvWT-nEYFP reporter expression in mutant backgrounds for known regulators of FMRFa, relative to the mean of the control. Numbers below columns represent the number of Tv4 neurons that express detectable FMRFa or TvWT-nEYFP. (E) In sqz mutants, T1 segment Tv4 neurons are often not generated, so we show intensity and FMRFa expression for T2 and T3 segments only. (D,E) All data represented as mean±SEM. Data compared using one-way ANOVA with Tukey HSD post-hoc test. * = p<0.05 compared to controls. n = 10–20 animals per genotype. (F-H) OK6-GAL4 drove combinations of UAS-ap with either UAS-dac or UAS-dimm. Representative images of TvWT-nEYFP and FMRFa expression in whole VNCs are shown; these were imaged through the entirety of their z-axis. (I) The number of neurons expressing ectopic nEYFP was counted when we overexpressed ap, dac or dimm in the combinations shown from OK6-GAL4. Only UAS-dac or UAS-ap,UAS-dac together induced ectopic TvWT-nEYFP-positive cells. Data is represented as mean number of ectopic nEYFP cells ± SEM. n = 10–20 VNCs per genotype. Data compared using one-way ANOVA with Tukey HSD post-hoc test. * = p<0.001 compared to controls. Scale bars are 30 μm in all images. Genotypes in A-E: ctrl (TvWT-nEYFP). wit (TvWT-nEYFP,witA12/ TvWT-nEYFP,witB11). ap, (apGAL4/ apP44; TvWT-nEYFP). sqz (TvWT-nEYFP,sqzie/ TvWT-nEYFP,sqzie). dac (Df(2L)Exel7066/ dac3; TvWT-nEYFP). eya (eyaCli-IID/eyaD1; TvWT-nEYFP). grh, (grhIM/grhDf; TvWT-nEYFP). dimm (dimmrev4/dimmP1; TvWT-nEYFP).
Fig 3.
Mutant sequence analysis of the Tv4-enhancer.
(A) Conservation track from UCSC Browser showing conservation islands between 12 sequenced Drosophila species. (B1-B9) Graphic representation of the position of putative HD (green), Mad (red and magenta) and Med motifs (yellow) as the coloured vertical bars within the Tv4-enhancer. These overlay the depicted deletion series of the Tv4-enhancer B1-B9. We generated a series of deletion transgenes (B1-B9; dotted region deleted). B1 is the full length wild-type Tv4-enhancer, denoted by the complete horizontal solid black line. B2-B9 show the region present in the reporter transgene (horizontal black bar) after removal of certain sequences (dotted regions). B4,5,7 show a deletion between two regions; the points of fusion are shown by vertical bars, between which the intervening sequence is removed. B8,9 show the type of sequence conversion performed within the region shown. (C) Reporter expression driven from control or deletion mutants B1-B9, or the empty reporter vector, expressed in the bar graph as the % nEYFP fluorescence intensity relative to mean of the B1 control. The right-most panel provides the number of Tv4 neurons (out of six) expressing significant nEYFP above the 99% confidence interval of the empty vector control. Removal of the HD-A motif (green, B3,B4) or the Mad-D motif (magenta, B3,B5) severely reduced expression. The enhancer fragment spanning the HD-A to Mad-D motifs (B6) expressed at moderate levels, but alteration of the intervening sequence severely reduced reporter expression, whether by sequence elimination (B7), conversion to complementary sequence (B8) or non-canonical sequence conversion (B9). (D) Point substitution mutants of Mad, HD and Med motifs, showing (bar graph) expression levels as % nEYFP fluorescence intensity relative to mean of the control, or (bottom-most panel) the number of Tv4 neurons (out of six) expressing significant nEYFP above the 99% confidence interval of the empty vector control. Only substitution mutants that alter the HD-A or Mad-D motifs essentially eliminate reporter expression. n = 10–20 animals per genotype. All data represented as mean±SEM. Data compared using one-way ANOVA with Tukey HSD post-hoc test. * = p<0.05 compared to controls.
Fig 4.
The 6xHD-A and 4xMad-D cis-elements encode sufficient information for Tv4-specific expression.
(A) Relative position of putative HD, Mad and Medea motifs. We concatemerized fragments from the Tv4-enhancer (shown in A). The number of concatemeric direct sequence repeats is shown, as is the number of nucleotides within each direct repeat. (B-F) Reporter expression for each concatemer, shown above, in the early L1 VNC. Two regions generate reporter expression in Tv4 neurons, the 25 bp HD-A-containing conserved region (B) and the 39 bp Mad-D containing conserved region (D). All other regions generate weak widespread neuronal expression (C), ectopic expression (E), or fail to express (F). Scale bars are 30 μm in all images.
Fig 5.
Genetic and biochemical analysis of the HD-RE and BMP-RE concatemers.
(A,B) HD-RE (6xHD-A-nEYFP) and (C,D) BMP-RE (4xMad-D-nEYFP) reporter expression in the genotypes shown. We show the relative intensity of reporter expression as the % of the mean of the genetic control. (A) Expression of HD-RE is reduced in ap mutants and eliminated in eya mutants, but not altered in wit or sqz mutants. Expression of UAS-GluedDN (retrograde traffic blocker) using apGAL4 had no effect on HD-RE. (B) UAS-dac overexpression (by OK6-GAL4) strongly increased HD-RE expression in Tv4 neurons. (C) Expression of BMP-RE is reduced in ap, wit and eya mutants. BMP-RE expression was also eliminated by overexpression of UAS-Mad1 (DNA-binding defective Mad) or UAS-GluedDN (retrograde trafficking blocker) using apGAL4. (D) UAS-dac overexpression increased BMP-RE expression in Tv4 neurons. (E-G) EMSA studies show sequence-specific binding of Apterous to the HD-A motif of HD-RE and of Mad to the Mad-D motif of BMP-RE. (E) HD-RE labeled probes (sequences shown below as WT HD-RE) are shifted in the presence of recombinant GST-CtermAp (C-terminal half of Apterous containing the Homeodomain) and efficiently out-competed by wildtype unlabeled probe (WT comp). The binding is not outcompeted by unlabeled HD-RE with a mutated HD-A motif (MutHD HD-RE). The number under each lane indicates the stoichiometric ratio between unlabeled and labeled probe. (F) The BMP-RE is not shifted in the presence of GST-CtermAp, indicating a lack of Ap binding. (G) The BMP-RE is shifted when presented with GST-MH1-Mad (that contains the DNA-binding domain). The band shift is out-competed by increasing amounts of unlabeled wild-type probe (WT comp). Competition is not observed when unlabelled probe with a mutated putative Mad-binding site (MutMad comp) is added. Data in A-C represented as mean±SEM. n = 10–20 animals per genotype and compared using one-way ANOVA with Tukey HSD post-hoc test. * = p<0.05 compared to control. Genotypes. [cis-element] in the following refers to either 6xHD-A-nEYFP or 4xMad-D-nEYFP. (A,C) ctrl (Tv[cis-element]/+). ap, (apGAL4/apP44; Tv[cis-element]/+). wit (Tv[cis-element],witA12/ witB11). sqz (Tv[cis-element],sqzie/sqzie). eya (eyaCli-IID/eyaD1; Tv[cis-element]/+). UAS-Mad1 (apGAL4/UAS-Mad1; Tv[cis-element]//UAS-Mad1). UAS-GluedDN (apGAL4/UAS-GluedDN; Tv[cis-element]/+) (B,D) Ctrl (OK6-GAL4/+; Tv[cis-element]/+). UAS-dac (OK6-GAL4/+; Tv[cis-element]/UAS-dac).
Fig 6.
Svp represses FMRF via the HD-RE and BMP-RE prior to target contact.
(A-D) The HD-RE (6xHD-A-nEYFP) and BMP-RE (4xMad-D-nEYFP) reporters are not expressed at late Stg. 16 (during Svp expression) but become expressed by late Stg. 17 (after mouth hook appearance). (E-H) Precocious, robust activation of BMP (shown by pMad immunoreactivity, red) is seen by Stg. 16 in Tv neurons (green), using apGAL4 to drive UAS-tkvAct,UAS-saxAct and UAS-myc::mad transgenes (G). However, even when BMP is precociously activated, FMRFa expression (blue) is not detected until late Stg. 17, its normal initiation time (F,H). Tv4 neurons are indicated by dotted circles. (I,J) Maintaining UAS-svp expression using apGAL4 results in total loss of FMRFa immunoreactivity in all Tv4 neurons, here shown at early L1. Also, pMad accumulation is unaffected in Tv4 neurons in these animals; numbers under insets represent fraction of Tv clusters with pMad or FMRFa expressing cells (n = 30 and 48 for controls and UAS-svp, respectively.) (K-N) Expression of HD-RE and BMP-RE reporters is strongly reduced by early L1 larvae when UAS-svp is overexpressed. Numbers in lower right corner indicate the mean number of nEYFP cells per animal ± SEM (n = 10–16 animals per group, p<10−3 two-tailed t-test between experimental and control for each reporter). (O,P) Svp expression is not detectable in early L1 larvae in control or wit mutant animals. Thus, BMP signaling is not required to downregulate Svp expression. The HD-RE reporter is used to identify the Tv4 neuron in these wit mutants. Tv4 neurons are indicated by dotted circles. Svp immunoreactivity can be detected in Tv2 and Tv3 neurons. Genotypes: (A-D) Concatemerized cis-elements [cis-element] (6xHD-A-nEYFP or 4xMad-D-nEYFP) were analyzed as homozygotes. (E-F) BMP gain of function (apGAL4/+;+/+ vs. apGAL4/UAS-tkvAct,UAS-saxAct;+/UAS-myc::mad). (I-N) Svp gain of function (apGAL4/+;+/+ vs. apGAL4/+;+/UAS-svp) (apGAL4/+; Tv[cis-element]/+ vs. apGAL4/+; Tv[cis-element]/UAS-svp). (O,P) BMP loss of function (aplacZ/+;witA12/ Tv6xHD-A-nEYFP + vs. aplacZ/+;witA12/ witA12,Tv6xHD-A-nEYFP).
Table 1.
Reduction in svp gene dosage results in premature 6xHD-A-nEYFP reporter expression.