XB-ART-53727J Cell Sci July 15, 2017; 130 (14): 2371-2381.
Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation.
Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation.
PubMed ID: 28576973
Article link: J Cell Sci
Species referenced: Xenopus laevis
Genes referenced: casp7 casp9 cd4 cdh1 gata1 lgals4.2 myc
Antibodies: Myc Ab11
Morpholinos: casp9 MO1 casp9 MO2 casp9 MO3
Article Images: [+] show captions
|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 (∼70 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.|
|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 18–19 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. (D–J) 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. (K–L) Around metamorphosis (X. laevis, stages 60–64), 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.|
|Fig. 3. Highly dynamic caspase activity during eye development. (A–F) Cryosections of the eyes in DEVD reporter X. laevis tadpoles at (A-C) stage 36 and (D–F) stage 45+. Nuclei are counterstained with DAPI. (A–C) 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 ‘b’ in A. (C) Higher magnification view of the GFP-positive neuronal network between the different retinal layers in the box labeled ‘c’ 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 ‘e’ 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 ‘f’ is shown in F.|
|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.|
|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). (C–D) 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 38–40, 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.|
|Fig. 6. Casp9 is ubiquitously expressed in early tadpole stages and is slightly enriched in the VBI. (A–D) 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.|
|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).|
|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.|
|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)).|
|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).|
|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.|
|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)|
|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.|
|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.|