Current Biology Supplemental Information Org 1 Dependent …€¦ · · 2015-04-07Org-1-Dependent...
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Current Biology Supplemental Information Org-1-Dependent Lineage Reprogramming Generates the Ventral Longitudinal Musculature of the Drosophila Heart Christoph Schaub, Johannes März, Ingolf Reim, and Manfred Frasch
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Transcript of Current Biology Supplemental Information Org 1 Dependent …€¦ · · 2015-04-07Org-1-Dependent...
Current Biology
Supplemental Information
Org-1-Dependent Lineage Reprogramming
Generates the Ventral Longitudinal
Musculature of the Drosophila Heart
Christoph Schaub, Johannes März, Ingolf Reim, and Manfred Frasch
Supplemental data
Figure S1. Lack of larval alary muscle phenotypes upon org-‐1-‐driven knock-‐
down of Mef2, tup, EcRDN, and HtlDN and the role of cardioblast-‐intrinsic EcR
signals. Related to Figures 3 and 4.
(A-‐D) Control stainings of late 3rd instar larvae carrying org-‐1-‐RFP showing that
org-‐1-‐GAL4-‐driven expression of dsRNA against Mef2 (A), tup (B), and dominant-‐
negative versions of EcR (C) and Htl (D) do not elicit any effects on alary muscle
morphologies prior to pupariation. Shown are dorsal-‐anterior views of live
larvae. (E) Phalloidin-‐staining of dorsal vessel from adult with tinCΔ4-‐GAL4-‐
driven EcRDN in cardioblasts showing only mild effects on VLM formation. Scale
bars of (A)–(E) represent 100 µm.
Supplemental Material & Methods
Analysis of pupal and adult phenotypes
Individuals were kept and GAL4/UAS-‐induced overexpression was carried out at
22°C, except for the experiments using TARGET [S1], which involved
temperature shifts to 28oC during embryonic stages. Control experiments with
the TARGET system without this temperature shift did not show any phenotype
or G-‐TRACE expression, thus ruling out that the observed effects are due to leaky
expression during later development.
Pupal stages until P4 were dissected with microsurgery scissors in toto. For
pupae from P4 and later as well as for pharate adults the pupal cases were
removed prior to dissection.
Drosophila strains
In this study the following strains were used: org-‐1-‐HN39-‐GFP [S2], hand-‐nGFP
[S3, S4], duf-‐rP298-‐lacZ [S5] and duf-‐rP298-‐GAL4 [S6], tupF4-‐nGFP [S7], tin346
[S8], tinEC40 [S9], tin-‐ABD [S10], and tinCΔ4-‐Gal4 [S11]. UAS-‐Abd-‐A was a gift from
Juan Botas (Baylor, Houston). UAS-‐lifeact-‐GFP was a gift from Frank Schnorrer
(MPI, Martinsried). UAS-‐dsRNA-‐org-‐1 (Transformant ID 104393), UAS-‐dsRNA-‐tup
(Transformant IDs 103585 and 45859) and UAS-‐dsRNA-‐Mef2 (Transformant IDs
15549 and 15550) were obtained from the VDRC (Vienna) [S12]. The two
different RNAi lines for tup and Mef2, respectively, showed essentially the same
effects. tubP-‐GAL80ts20;TM2/TM6B and UAS-‐FLP-‐Exel3,Ubi-‐p63E(FRT.STOP)
Stinger [S13] as well as UAS-‐EcR-‐B1DN [S14] and UAS-‐heartlessDN [S15] were
obtained from the Bloomington stock center (Indiana).
Construction of org-‐1-‐HN18-‐RFP, org-‐1-‐HN39-‐GAL4 and tup-‐ADME-‐GFP
reporters
For the creation of org-‐1-‐HN18-‐RFP, the genomic region chrX: 8442048...
8444555 (R6.01) was amplified using yw genomic DNA as template and cloned
into BglII/NaeI of pRed H-‐Pelican [S16].
For org-‐1-‐HN39-‐GAL4 the HN39 fragment from org-‐1-‐HN39-‐pH-‐Stinger-‐AttB [S2]
was cloned into EcoRI/BamHI of p221-‐GAL4 (a gift from C. Klämbt).
For tup-‐ADME-‐GFP the genomic region chr2L: 18899122…18900688 (R6.01)
(tup-‐ADME) [S17] was amplified using yw genomic DNA as template and cloned
into KpnI/XhoI of pH-‐Stinger-‐AttB [S18]. For the analogous creation of tup-‐
ADMEorgI-‐IIImut-‐GFP reporter the Org-‐1 binding sites within the tup-‐ADME
sequence [S17] were mutated via site directed mutagenesis as follows: tup-‐
ADME-‐orgI TAACACAT -‐> tup-‐ADME-‐orgImut TAAGCTTT, tup-‐ADME-‐orgII
GGGTGCCA -‐> tup-‐ADME-‐orgIImut GGCTCGAG and tup-‐ADME-‐orgIII TGGTGGGA -‐
> tup-‐ADME-‐orgIIImut TGTCTAGA.
All constructs were transformed into yw using standard transgenesis techniques.
Immunofluorescence
For antibody stainings the dissected animals were fixed in 3,7 % formaldehyde
for 1 hour, washed several times in PBT, and incubated in the primary antibody
for two days at 4 °C. Subsequent staining procedures were performed as
described [S19].
The following primary antibodies were used: rat anti-‐Org-‐1 (1:100, [S2]), mouse
anti-‐Isl1/Tup 40.3A4 (1:25, DSHB) guinea-‐pig anti-‐Ubx (1:400, a gift from I.
Lohmann, Heidelberg), rabbit anti-‐RFP (1:300, Millipore) and mouse anti-‐GFP
(1:100, Molecular Probes). Filamentous actin was visualized using Phalloidin-‐
Atto 647N (1:1500, Sigma-‐Aldrich). Confocal pictures were taken with a Leica
SP5 II (20x/1.3 PL APO Glycerol). Projections were done with Leica Application
Suite Advanced Fluorescence (LAS AF).
In vivo time-‐lapse imaging
Pupae were aligned on a strip of double-‐faced adhesive tape connected to a slide,
covered with a drop of halocarbone oil and a coverslip. Time-‐lapse imaging was
essentially performed as described in [S20, S21]. Movies were generated using
LAS AF.
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