XB-ART-56783Dev Dyn July 1, 2020; 249 (7): 847-866.
BACKGROUND: Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS: We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells'' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS: Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.
PubMed ID: 32141178
Article link: Dev Dyn
Genes referenced: bak1 barhl2 bax bcl2l10 bcl2l11 bcl2l2 bid casp3.1 casp3.2 casp7 casp9 chrd.1 mcl1 myc sox3 xiap
GO keywords: programmed cell death
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|Figure 1: Endogenous apoptosis contributes to axial organizer formation independently of Caspase3 activity (A) TUNEL-staining reveals apoptosis in the axial midline. Representative TUNEL-stained wild type (wt) stage (st.) 12 Xenopus embryos. (B) Activated Caspase-3 (Act-Casp3) is not detected before stage 14 in Xenopus embryos. Representative immuno-staining against Act-Casp3 performed on st.10 to st.14 embryos. Act-Casp3 positive nuclei are shown with black arrows; st.10 to 12 ventral view dorsal up; st. 13 and 14 dorsal view anterior up. (C-F) Endogenous apoptosis affects establishment of the posterior neural plate via its activity on axial organizer signaling independently of Casp3. (C) Xenopus embryos were injected into one dorsal blastomere at the four-cell stage with mRNA encoding (a) X. laevis bcl-xl or (b) MOcasp3 together with a tracer (red) and analyzed by ISH with sox3. Representative st.14 embryos are shown. (c) Quantification graph of (a, b). (D) Xenopus embryos were injected into one dorsal blastomere at the four- (a), sixteen- (b, c) or thirty-two- (d) cells stage with mRNA encoding X. laevis bcl-xl or MOcasp3 together with a tracer (red) and analyzed by ISH with sox3. The site of injection (red dot), and the corresponding st.14 targeted territory (red area) are represented. The graph indicates the % of embryos exhibiting the (a-c) posterior, or (d) anterior, neural plate enlargement phenotype (blue). ML: medio-lateral; L: lateral; A: anterior neural plate. (E, F) Bcl-XL acts on the axial organizer cells. Embryos were injected either in the axial organizer (notochord and floor plate) or in lateral ectoderm with (E) bcl-xl or (F) MOcasp3. Representative embryos are shown dorsal view, anterior up, except (Fc) shown dorsal-anterior view. Representative transverse sections (50μm) are shown at the antero-posterior axis positions indicated with a white-dashed arrow (Eb,d; Fb,d). The black arrow indicates the limits of the injected territory. PNE: posterior neuroepithelium. Inj: injected side. White dashed lines indicate the midline. Relative normalized expression is indicated as means ± standard error (s.e.) *** p<0.001.|
|Figure 2: mcl1 expression pattern suggests a role for this protein in axial organizer development. (A) RT-qPCR analysis of apoptotic cascade mRNA in Xenopus early neurula. Analysis performed on RNA extracted from dissected neural plates, and ventral ectoderms (N=15) of X. laevis embryos at stage (st.) 14 for pro-apoptotic factors – bak, bad, bid, bim, including caspases (casp) - casp3, casp7, and casp9 and anti-apoptotic factors – bcl2l10, mcl1, bcl-xl, and bcl2l2. Expression is normalized relative to ef1α. Relative normalized expression is indicated as means ± s.e. (B) mcl1 expression pattern. ISH using a probe for mcl1 from stage 11 to stage 20 embryos; (a) ventral view, dorsal up, (b-e) dorsal view anterior up, (f) anterior view dorsal up. (g) Transverse section of (c) in the posterior part of the embryo. The scale bar stands for 200μm. The white arrow indicates the border between the ectoderm and the neuroectoderm. The black arrow indicates the notochord. (C) bcl-xl expression pattern in X. laevis gastrula and neurula. ISH using a probe for the anti-apoptotic gene bcl-xl from stage 12 to stage 20. Embryos are shown dorsal side up.|
|Figure 4: Mcl1 depletion specifically affects the survival of notochord cells. (A) Depletion of Mcl1 affects notochord development. ISH of stage (st.) 13.5 embryos using probes against chordin (a,b). Embryos injected (inj) with (a) MOmcl1-ct (N=24); or (b) MOmcl1 (N=19); (c) Quantification graph showing notochord width comparison in embryos injected with MOmcl1-ct or MOmcl1. (d-f) Histological vibratome sections of respectively (d) MOmcl1-ct and (e,f) MOmcl1. Blue arrow indicates the notochord. Scale bar stands for 200μm. (B) Quantification of midline apoptosis induced by Mcl1 depletion. TUNEL staining was performed at st.13.5. (a) Schematic representation of TUNEL quantification: red dots represent injected cells; black dots represent TUNEL positive cells. All apoptotic cells were counted in (A) the black rectangle considered the total injected area, and (B) the red rectangle considered the midline area. (b, c) Representative TUNEL staining on stage 13.5 embryos injected with (b) MOmcl1-ct; or (c) MOmcl1 (N=39). (d) Quantification of B. (C, D) The decrease in neural plate width upon depletion of Mcl1 is mirrored by the increase in ectoderm territory. ISH against epidermal keratin (EpKer). Representative stage 13.5 embryo injected with (C) MOmcl1-1; (D) flag-mcl1; (a) dorsal view anterior up; (b) View of the non-injected side: lateral view, anterior up, dorsal side right; (c) View of the injected side: lateral view, anterior up, dorsal side|