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Fig. 1. Design and characterization of the caspase reporter constructs. (A) Schematic representation of the caspase reporter constructs. The GAL4VP16
transcription factor is anchored in the membrane by the extracellular and transmembrane domain of IL2R. Activated caspases cleave their recognition site, freeing
the chimeric GAL4VP16 transcription factor, which can then translocate to the nucleus, where it initiates the transcription of the eGFP reporter gene.
A general lay-out of the three designed reporter systems with their distinct DNA and AA sequences is shown. (B) Anti-Myc tag western blot analysis of reporter
cleavage products in 293t HEK cells cotransfected with mouse casp3. The DEVD reporter, but not the DEVA control reporter, was found to be sensitive to the
exogenous casp3 (arrowhead). The DNA coding for the distinct reporter constructs (200 ng) was combined with a DNA gradient (50 ng, 100 ng, 150 ng,
250 ng) coding for casp3. (C) The LEHD reporter system can be proteolytically cleaved by exogenous mouse casp9 in a TNT assay. The three reporter systems
were in vitro transcribed and translated and subsequently exposed to recombinant mouse casp9 proteins. Raf-1 protein was used as a positive control for
cleavage with casp9 (black arrowheads). Both the LEHD and the DEVD constructs are cleaved by casp9 resulting in a 30 kDa fragment (open arrowhead).
(D) The addition of the casp3 inhibitor DEVD-cmk (10 μM) prevented the cleavage of the DEVD construct, but not the LEHD construct (open arrowhead),
suggesting that the observed cleavage of DEVD by casp9 in C is a result of the casp9-mediated activation of casp3 present in the lysate as an inactive
procaspase. The DEVA reporter is insensitive to recombinant casp9. Arrows indicate the uncleaved fragments (â¼70 kDa). hEcad, human CDH1 promoter; EF1α,
Xenopus EF1α promoter; IL2R, transmembrane region of the IL2R gene; GAL4 DBD, GAL4 DNA-binding domain; Vp16 TA, transactivation domain of the
Herpes simplex VP16 gene.
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Fig. 2. Transgenic embryos and larvae carrying the
DEVD reporter show dynamic spatio-temporal GFP
expression patterns during their development.
(A) Dorsal and (B) anterior view of a stage 18â19
X. tropicalis embryo at neural fold stage. GFP expression
emerges in defined stripes on each side of the dorsal
midline, which are most intense in the future brain region
as well as in the anterior patches corresponding to the eye
vesicle. (C) Similar patterns are visible at stage 24 of a
X. tropicalis tailbud embryo. (DâJ) A dynamic and
diversified GFP expression signal is observed in reporter
embryos as development proceeds. Particularly high
expression levels were observed in the developing neural
tissues. (F) Stage 46 X. laevis tadpoles show GFP
positivity in kidney cells (closer view in H), the brain, nasal
pits (closer view in G) and muscle fibers (I). (J) The lens
was still GFP positive. (KâL) Around metamorphosis
(X. laevis, stages 60â64), GFP is expressed in the growth
zones of the long bones. (M) The teeth and some cranial
structures, including the skull and the jawbone, were also
GFP positive. b, brain; bo, bone; e, eye; ev, eye vesicle;
he, heart; k, kidney cells; mf, muscle fibers; np, nasal pits;
nt, neural tube; sc, spinal cord; t, teeth.
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Fig. 3. Highly dynamic caspase activity during eye development. (AâF) Cryosections of the eyes in DEVD reporter X. laevis tadpoles at (A-C) stage 36 and
(DâF) stage 45+. Nuclei are counterstained with DAPI. (AâC) A DEVD transgenic embryo at stage 36 showed consistent GFP signals in all retinal layers
including the ganglion cell layer, the inner nuclear layer and the outer nuclear layer. At this stage, nuclei are still present in the differentiating lens fiber cells.
(B) Higher magnification view of the GFP-positive photoreceptors in the box labeled âbâ in A. (C) Higher magnification view of the GFP-positive neuronal network
between the different retinal layers in the box labeled âcâ in A. (D,E) GFP is expressed in the enucleated lens fiber cells of stage 45+ embryos (a higher
magnification view of the box labeled âeâ is shown in E). A few GFP-positive cells were still observed in the fully developed retina (i.e. photoreceptor layer) (a higher
magnification view of the box labeled âfâ is shown in F.
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Fig. 4. DEVD reporter activity colocalizes with TUNEL and active casp3 staining. Confocal images of cryosectioned eyes isolated at stages (A) 36 and
(B) 32, when massive cell death is occurring in the developing retina (X. tropicalis). Arrowheads indicate GFP-positive retinal cells colocalizing with TUNEL-positive
cells (A) or active casp3 (B) during retinogenesis. Arrows indicate the eye and the brain on the sections.
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Fig. 5. Activation of the LEHD reporter in the VBI. (A,B) Comparison between the transgenic DEVD and LEHD reporter embryos (X. tropicalis) revealed
overlapping GFP-positive regions, such as the brain and eye. However, only the LEHD embryos had GFP expression in the VBI, where the primitive blood is
formed (arrowheads). (CâD) As development continued, the GFP signal in the VBI intensified and localized in the typical outline of the VBI (ventral view in D).
(E) At stage 38â40, GFP-positive cells entered the blood circulation via the vitteline veins. Circulating GFP-expressing blood cells could be detected throughout
the embryo (arrowhead). (F) Individual GFP-expressing erythrocytes collected and imaged at stage 42.
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Fig. 6. Casp9 is ubiquitously expressed in early tadpole
stages and is slightly enriched in the VBI. (AâD) Expression
of casp9 mRNA at Nieuwkoop stages 22, 26 and 32, as revealed
by WISH. Expression is ubiquitous but enriched in some tissues,
including the VBI (arrowhead). D shows the same embryo as
in C, but in a tilted ventro-lateral view to better expose the VBI.
(E) Casp9 mRNA expression in a vibratome section of the eye
vesicle. (F) Control using a sense casp9 probe.
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Fig. 7. Knockdown of casp9 depletes the expression of early blood marker genes. (A) X. tropicalis embryos were injected in two CD4 blastomeres at 16-cell
stage with 2.5 ng C9MOatg1, 4 ng CoMO or a combination of 2 ng C3MOatg and 2 ng C7MOatg in each blastomere. β-Gal RNA (10 pg) was coinjected as lineage
tracer. WISH analysis of T3 globin and gata1 expression in the posterior part of the VBI is shown. Both T3 globin and gata1 expression were affected upon depletion of casp9, but not upon the combined depletion of casp3 and casp7. (B) Graphs representing the percentage of embryos injected in CD4 blastomeres at 16-cell
stage, exposing the defects in marker expression. The embryos were scored using the phenotype in A as a reference (based on six independent experiments).
(C) Depletion of T3 globin expression could be rescued by X. laevis casp9 RNA. X. tropicalis embryos were injected in two CD4 blastomeres at 16-cell stage with
2.5 ng CoMO, 2.5 ng C9MOatg1 or 2.5 ng C9MOatg1, combined with 100 pg X. laevis casp9 RNA in each blastomere. β-Gal RNA was coinjected as a lineage tracer. WISH analysis shows T3 globin expression in the posterior part of the VBI. The overexpression of Xlcasp9 could rescue the defect in T3 globin expression induced by casp9 depletion. (D) The percentages of embryos injected in CD4 blastomeres at 16-cell stage showing the defect in T3 globin marker expression (based on four
independent experiments). (E) For quantitative RT-PCR analysis, X. tropicalis embryos were injected in two CD4 blastomeres at 16-cell stage with 4 ng CoMO,
2.5 ng C9MOatg1 with or without 100 pg X. laevis casp9 RNA, or a combination of 2 ng C3MOatg and 2 ng C7MOatg in each blastomere. RT-PCR analysis of T3
globin expression shows significant reduction upon injection of C9MOatg1, which is rescued upon coinjection of casp9 RNA. T3 globin expression is not significantly affected in C3/C7 double morphants. The graph shows the combination of three biological replicates. ns, not significant; *P<0.05; **P<0.005 (Student’s t-test).
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Figure S1. Embryos transgenic for the DEVA reporter do not display eGFP expression. The F0
transgenic X. tropicalis embryos can be easily selected by their DsRed expression. However, the
transgenic DEVA reporter embryos failed to generate a GFP signal despite strong DsRed expression.
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Figure S2. Whole mount TUNEL-staining did not show apoptotic cells in the ventral blood island
(VBI). (A-B) Stage 32-34 embryo revealed a great number of apoptotic cells at the otic vesicle (ov) and eye (ey). No TUNEL-positivity was seen at the VBI. (C-E) Stage 32-34 embryo showed also apoptotic cells in the brain area (b) (clearly visible on the dorsal view in (E)). Again no apoptotic cells were present at the VBI (see ventral view in (D)).
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Figure S3. Caspase-3 and caspase-7 are ubiquitously expressed in early tadpole stages. Expression of caspase-3 (panels A-E) and caspase-7 (panels F,G) mRNA as revealed by whole mount in situ hybridization. Expression is shown for stage 32 (panels A,F) or stage 36 (panels B-E and G). Panels CE and G show mRNA expression in vibratome sections. (C and G) Expression in the eye is detectable in the lens (Le) and the outer nuclear layer (ONL), the inner nuclear layer (INL) and the ganglion cell layer (GCL) of the retina. (D) Expression of caspase-3 mRNA in the hindbrain. (E) Expression of caspase-3 mRNA in the trunk region including the epidermis (Ep), the neural tube (NT), the notochord (No) and the somites (So).
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Figure S4. Similarities and differences in GFP patterns in transgenic DEVD- and LEHD-reporter tadpoles. (A) DEVD- and LEHD-reporter activity is present in the lens and the muscle, consistent with possible non-apoptotic caspase activity. Both the DEVD- (top) and the LEHD-reporter (bottom) reveal GFP expression in the cells forming the lens around stage 32-34 (left pictures, yellow arrowhead) and in individual myocytes in the late tadpole tail (middle pictures, with the boxed area enlarged in the right pictures). (B) Circulating GFP-positive blood cells are only present in LEHD reporter tadpoles. Tadpoles of DEVD-reporter (left) or LEHD-reporter (right) are shown around Nieuwkoop stage 39. GFP signal in the vasculature is due to circulating GFP-positive blood cells. No such activity is present in the DEVD reporter despite high transgene expression.
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Figure S5. Validation of Morpholino injections. (A) C9MOatg1 does not target Xenopus laevis casp9 RNA. Western blot analysis of exogenous casp9 levels after depletion with the translation blocking morpholino C9MOatg1. Embryos were injected at 2-cell stage with 10 ng C9MOatg1 and 10 ng CoMO alone or combined with 250 pg RNA coding for Xenopus laevis casp9 (Myc-tagged) or Xenopus tropicalis casp9 (Myc-tagged). The functionality of C9MOatg1 was proven by the clear reduction in Xtcasp9 protein levels upon depletion while the four mismatches in the morpholino target sequence of the Xlcasp9 mRNA seem to be sufficient for blocking the binding with the morpholino and make the Xlcasp9 transcripts suited for rescue experiments. (B) Splice blocking morpholino C9MOE2I2
retained intron I in the X. tropicalis casp9 transcripts. RT-PCR analysis of Xtcasp9 levels after depletion with the splice blocking morpholino (C9MOE2I2). Embryos were injected at 2-cell stage with 20 ng C9MOE2I2 or 20 ng CoMO and lysed at stage 20. Blockage of the exon2 and intron2 boundary by the morpholino resulted in a intron1 retention, which was noticed by the 579 bp fragment. The intron retention causes a shift in the open reading frame of Xtcasp9 and results in an incorrectly translated protein and a premature stop. (NI, non injected)
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Figure S6. Combined depletion of casp3 and casp7 results in reduced DEVDase and DEVD reporter
activity. (A) X. tropicalis F1 DEVD reporter embryos were injected at the 1 cell stage with the
translational blocking morpholinos directed against casp3 and/or casp7 (C3 and C7 MO), lysed at
stages 20-21 and subjected to a DEVD-linked coumarin-based fluorescent compound (AMC). Only the
combined injection of the two morpholinos was able to decrease the DEVDase activity with 50%,
confirming the functional redundancy between these two executioner caspases. For each injection 6
groups of 15 embryos were lysed and individually measured. Error bars are SEMs (standard errors of
the mean) whereby n represents the 6 groups of embryos/sample and an unpaired student t-test
indicated a P value of 0,0054 (p< 0,05 **). Abbreviation : NI, non-injected wild type embryos. (B)
Transgenic F1 DEVD-reporter embryos (X. tropicalis) were unilaterally injected at the two cell stage
with the combination of 3 ng C3MO and 3 ng C7MO or 6 ng control MO (CoMO). 2 ng of the CoMO
conjugated with a lissamin tracer (red) was included in every injection mix to identify the injected
side. The combined depletion of casp3 and casp7 resulted in a clear reduction of the GFP expression
at the injection side (arrowhead). This was not the case when the control MO was injected.
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Figure S8. Confirmation of specific depletion of the primitive blood markers with additional casp9 morpholinos (C9MOatg2 and C9MOE2I2). Embryos were injected in the two CD4 blastomeres at 16-cell stage with 3.5 ng C9MOatg2, 4 ng C9MOE2I2 or 4 ng CoMO in each blastomere. RNA of cytoplasmic β-Gal (10 pg) was coinjected as lineage tracer. Whole mount in situ hybridization analysis of T3 globin expression in the posterior part of the VBI (arrowheads). T3-globin expression was affected upon depletion of casp9 with these additional morpholinos. Graphs represents the percentage of embryos exposing the defect in the observed marker expression.
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