Lhomond G et al. (2024), Spatiotemporal requirements of nuclear β-cateni...

ECB-ART-53384

PLoS Biol 2024 Nov 01;2211:e3002880. doi: 10.1371/journal.pbio.3002880.

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Spatiotemporal requirements of nuclear β-catenin define early sea urchin embryogenesis.

Lhomond G , Schubert M , Croce J .

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Establishment of the 3 primordial germ layers (ectoderm, endoderm, and mesoderm) during early animal development represents an essential prerequisite for the emergence of properly patterned embryos. β-catenin is an ancient protein that is known to play essential roles in this process. However, these roles have chiefly been established through inhibition of β-catenin translation or function at the time of fertilization. Comprehensive analyses reporting the totality of functions played by nuclear β-catenin during early embryogenesis of a given animal, i.e., at different developmental stages and in different germ layers, are thus still lacking. In this study, we used an inducible, conditional knockdown system in the sea urchin to characterize all possible requirements of β-catenin for germ layer establishment and patterning. By blocking β-catenin protein production starting at 7 different time points of early development, between fertilization and 12 h post fertilization, we established a clear correlation between the position of a germ layer along the primary embryonic axis (the animal-vegetal axis) and its dependence on nuclear β-catenin activity. For example, in the vegetal hemisphere, we determined that the 3 germ layers (skeletogenic mesoderm, non-skeletogenic mesoderm, and endoderm) require distinct and highly specific durations of β-catenin production for their respective specification, with the most vegetal germ layer, the skeletogenic mesoderm, requiring the shortest duration. Likewise, for the 2 animal territories (ectoderm and anterior neuroectoderm), we established that their restriction, along the animal-vegetal axis, relies on different durations of β-catenin production and that the longest duration is required for the most animal territory, the anterior neuroectoderm. Moreover, we found that 2 of the vegetal germ layers, the non-skeletogenic mesoderm and the endoderm, further require a prolonged period of nuclear β-catenin activity after their specification to maintain their respective germ layer identities through time. Finally, we determined that restriction of the anterior neuroectoderm territory depends on at least 2 nuclear β-catenin-dependent inputs and a nuclear β-catenin-independent mechanism. Taken together, this work is the first to comprehensively define the spatiotemporal requirements of β-catenin during the early embryogenesis of a single animal, the sea urchin Paracentrotus lividus, thereby providing new experimental evidence for a better understanding of the roles played by this evolutionary conserved protein during animal development.

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	Fig 1. Simplified sea urchin GRN.(A) Schematics illustrating the germ layers of a sea urchin at 6 embryonic stages: 16-cell stage (16-cell), 60-cell stage (60-cell), early blastula stage (e. blas.), late blastula stage (l. blas.), mesenchyme blastula stage (mes. blas.), gastrula stage (gastrula). Embryonic domains are color-coded as follows: anterior neuroectoderm, cyan; ectoderm, intense and light blue; ectoendoderm, green; endoderm, yellow; endomesoderm, orange; non-skeletogenic mesoderm, light brown; skeletogenic mesoderm, red; small micromeres, dark brown; undetermined, white. Abbreviations: ma, macromeres; me, mesomeres; mi, micromeres. (B) Simplified sea urchin GRN based on data from Paracentrotus lividus, Lytechinus variegatus, and Strongylocentrotus purpuratus (e.g., [6,17,18,23–25]). In the GRN, each embryonic domain is highlighted by boxes using the same color-code as in (A), plus gray for the aboral (dorsal) non-skeletogenic mesoderm and pink for the oral (ventral) non-skeletogenic mesoderm. Genes are named based on their corresponding proteins and indicated as lines with bent arrows. Proteins are shown in white boxes. The GRN highlights the germ layer distribution of investigated genes and known regulatory relationships between them whether direct or indirect (arrows pointing to transcriptional activation). The GRN further illustrates reported activation mechanisms leading to β-catenin nuclearization and known nuclear β-catenin target genes. *, #, and ^, respectively indicate genes encoding transcription factors, signaling system components, and differentiation marker genes. Developmental progression is from top to bottom. Abbreviations: ane, anterior neuroectoderm; ecto, ectoderm; ma, macromeres; me, mesomeres; mi, micromeres; SM, skeletogenic mesoderm; X, unknown activation mechanism.
	Fig 2. Vegetal germ layer segregation during Paracentrotus lividus embryogenesis.(A–C) Double fluorescent in situ hybridization assays for: (A) delta (purple) plus gcm (green) mRNAs, (B) delta (blue) plus foxA (red) mRNAs, and (C) gcm (green) plus foxA (red) mRNAs. In (A–C), developmental stages are indicated as hpf and are as follows [37]: 9 hpf, mid-blastula stage; 10 hpf, late blastula stage; 11 hpf, hatched blastula stage; 11.5 hpf (11 h 30 min), swimming blastula stage; 12 hpf, swimming blastula stage; 12.5 hpf (12 h 30 min), swimming blastula stage. In (A–C), maximum intensity projections of confocal z-stacks for embryos in vegetal view. The first and second rows show the expression profiles of each indicated gene individually, and the third row shows a merge of the expression profiles of the 2 indicated genes. Numbers in the bottom right corner indicate the number of embryos with at least a couple of cells co-expressing the 2 analyzed genes, with the total number of embryos analyzed indicated in parentheses. In (A–C), color-coded lines, outlining the expression domain of one of the genes analyzed, are provided to facilitate visualization of co-expression.
	Fig 3. Nuclear localization of β-catenin during Paracentrotus lividus embryogenesis.(A, B) Maximum intensity projections of confocal z-stacks for embryos co-labeled with: (A) β-cateninV proteins (green) plus DNA (blue) and (B) β-cateninV proteins (green) plus foxA mRNAs (red). In (A, B), developmental stages are indicated as hpf and are as follows [37]: 5 hpf, 32-cell stage; 6 hpf, 60-cell stage; 9 hpf, mid-blastula stage; 10 hpf, late blastula stage; 11 hpf, hatched blastula stage; 12 hpf, swimming blastula stage; 12.5 hpf (12 h 30 min), swimming blastula stage. In (A), embryos in the top row are in lateral view with the animal pole up, and embryos in the bottom row are in vegetal view. At 5 hpf and 6 hpf, the red line outlines a macromere, the pink lines a large micromere, the light blue lines a small micromere, the orange line a veg1 cell, and the yellow line a veg2 cell. In (B), embryos are in vegetal view.
	Fig 4. Methodology and validation of an inducible, conditional β-catenin knockdown system.(A) Schematic representation of an inducible, conditional gene knockdown approach developed based on the use of classical morpholino antisense oligonucleotides (MASO) and photocleavable morpholino sense oligonucleotides (P_MSO). The 2 morpholino oligonucleotides are complementary and bind to each other, thereby preventing the association of the MASO with its target mRNA. Upon UV light exposure (365 nm), P_MSO is cleaved, hence releasing the MASO, which in turn binds to its target mRNA, blocks its translation, and induces a knockdown phenotype. (B) In vivo validation of the developed inducible knockdown approach using the production of β-cateninV proteins (green) as a readout. Embryos were obtained from oocytes microinjected with β-cateninV mRNAs alone (control), with β-cateninV mRNAs plus β-catenin MASO (β-cateninM, β-catM), with β-cateninV mRNAs plus β-catenin P_MSO (β-cateninPM, β-catPM), or with β-cateninV mRNAs plus a mix of β-cateninM and β-cateninPM (β-catM+PM). Following microinjection, but before fertilization, oocytes were either exposed (+UV) or not exposed (-UV) to UV light. All embryos were then recorded live at 9 hpf (mid-blastula stage) [37] and are shown in lateral view, with the animal pole up. Numbers in the bottom right corner indicate counts of shown phenotypes with numbers in parentheses indicating the total number of scored embryos from 2 biological replicates.
	Fig 5. Developmental and molecular validation of the inducible β-catenin knockdown approach by blocking β-catenin translation before fertilization.(A) Morphological phenotypes observed at 24 hpf (late gastrula stage) in: uninjected control embryos (uninjected control), embryos microinjected with β-cateninPM (β-catPM) alone, with β-cateninM (β-catM) alone or with a mix of β-cateninM+β-cateninPM (β-catM+PM), which were all either irradiated (+UV) or not (-UV) before fertilization. All embryos are shown in lateral view, with the animal pole up. (B) In situ hybridization assays and qPCR expression matrices for markers of the skeletogenic mesoderm (Ske. M), the endomesoderm (Endomeso), the oral (ventral) non-skeletogenic mesoderm (Or. NSke. M), the aboral (dorsal) non-skeletogenic mesoderm (Ab. NSke. M), the endoderm (Endo), the ectoderm (Ecto), and the anterior neuroectoderm (Ant. N.Ecto). The color-code used for each germ layer is as in Fig 1. In situ hybridization and qPCR assays were carried out at 10 hpf (late blastula stage) and/or 15 hpf (mesenchyme blastula stage) [37], on embryos from the same microinjection and experimental setup as in (A). For the in situ hybridization, all embryos are shown in vegetal view, except for he and foxQ2, which are in lateral view. In (A, B), numbers in the bottom right corner indicate counts of shown phenotypes and in situ hybridization patterns, and the numbers in parentheses outline the total number of scored embryos. In (B), * indicates weak signals in the remaining scored embryos. In (B), the qPCR analyses are shown as tables and were carried out in 2 independent biological replicates (Rep1 and Rep2) on the genes analyzed by in situ hybridization (marked by >) as well as on additional marker genes. In the tables, orange backgrounds indicate down-regulation by more than 2-fold compared to uninjected, irradiated control embryos, while blue backgrounds highlight unaltered expression compared to controls. Related raw data and quantification methods are available in S1 Data and fold change values are further provided in S2 Data.
	Fig 6. Morphological phenotypes associated with blocking β-catenin translation starting at different time points after fertilization.(A) Schematic representation of the experimental setup. Upon microinjection (or not) and fertilization, 6 developmental stages, indicated as minutes post fertilization (minpf) or hpf, were targeted for UV treatments and are as follows: 20 minpf, fertilized egg; 4 hpf, 16-cell stage; 6 hpf, 60-cell stage; 8 hpf, early blastula stage; 10 hpf, late blastula stage; 12 hpf, swimming blastula stage [37]. For all time points, embryo morphology was assessed at 24 hpf (late gastrula stage), as indicated by *. Abbreviations: exp. condition, experimental condition; fert., fertilization; microinj., microinjection; neg. exp. condition, negative experimental condition. (B) Quantification of the morphological phenotypes obtained at 24 hpf for uninjected, irradiated control embryos: uninjected control (+UV); β-cateninM+β-cateninPM-injected and not irradiated embryos: β-catM+PM (-UV); β-cateninM+β-cateninPM-injected and irradiated embryos: β-catM+PM (+UV). The top row illustrates the 4 different observed phenotypes named as indicated and presented with embryos in lateral view, with the animal pole up. The bottom graph highlights the quantification of each of the observed phenotypes, per experimental condition. This quantification is displayed using the same color-code as in the top row and is based on 3 biological replicates. The total number (n:) of embryos scored for each experimental condition is indicated on each histogram. Corresponding raw counts and quantification method are included in S3 Data.
	Fig 7. Time point-specific blockage of β-catenin translation and skeletogenic mesoderm development.(A) Schematic representation of the performed analyses, following the experimental procedure detailed in Fig 6A. For the time points marked by , embryos were collected at 10 hpf (late blastula stage) and/or 15 hpf (mesenchyme blastula stage) [37] to carry out in situ hybridization (In situ) and quantitative RT-PCR (qPCR) assays. (B) In situ hybridization assays for the skeletogenic mesoderm (Ske. M) marker ske-T (tbr) [39] and qPCR expression matrices, from 2 independent biological replicates (Rep1 and Rep2), for ske-T and 4 additional skeletogenic mesoderm marker genes (alx1, dri, foxO, sm50). Experimental conditions included uninjected, irradiated control embryos: uninjected control (+UV); β-cateninM+β-cateninPM and not irradiated embryos: β-catM+PM (-UV); β-cateninM+β-cateninPM and irradiated embryos: β-catM+PM (+UV). The germ layer color-code is as in Fig 1. For the in situ hybridization assays, embryos are in vegetal view. Numbers in the bottom right and left corners indicate phenotypic counts relative to the total number of scored embryos (in parentheses). The 2 control conditions are shown in different. and ° indicate, respectively, weak and no signal in the remaining scored embryos. For the qPCR analyses, results are shown as tables, and > marks ske-T that was also analyzed by in situ hybridization. Blue backgrounds highlight unaltered expression compared to uninjected, irradiated control embryos. Related raw data and quantification methods are available in S1 Data and fold change values are further provided in S2 Data.
	Fig 8. Time point-specific blockage of β-catenin translation and endomesoderm development.(A) Schematic representation of the performed analyses, following the experimental procedure detailed in Fig 6A. For the time points marked by , embryos were collected at 10 hpf (late blastula stage) [37] to carry out in situ hybridization (In situ) and quantitative RT-PCR (qPCR) assays. (B) In situ hybridization assays for the 2 endomesoderm (Endomeso) marker genes gcm and foxA [26,28] and qPCR expression matrices, from 2 independent biological replicates (Rep1 and Rep2), for gcm and foxA, plus 4 additional co-expressed non-skeletogenic mesoderm (pks, wnt16) and endoderm (bra, hox11/13) marker genes. Experimental conditions included uninjected, irradiated control embryos: uninjected control (+UV); β-cateninM+β-cateninPM and not irradiated embryos: β-catM+PM (-UV); β-cateninM+β-cateninPM and irradiated embryos: β-catM+PM (+UV). The germ layer color-code is as in Fig 1. For the in situ hybridization assays, embryos are in vegetal view, and numbers in the bottom right and left corners indicate phenotypic counts relative to the total number of scored embryos (in parentheses). The 2 control conditions are shown in different colors, and indicates weak signal in the remaining scored embryos. For the qPCR analyses, results are shown as tables, and > marks foxA and gcm that were also analyzed by in situ hybridization. Orange backgrounds indicate down-regulation by more than 2-fold compared to uninjected, irradiated control embryos, while blue backgrounds highlight unaltered expression compared to controls. Related raw data and quantification methods are available in S1 Data and fold change values are further provided in S2 Data.
	Fig 9. Time point-specific blockage of β-catenin translation and endoderm and non-skeletogenic mesoderm development.(A) Schematic representation of the performed analyses, following the experimental procedure detailed in Fig 6A. For the time points marked by , embryos were collected at 15 hpf (mesenchyme blastula stage) [37] to carry out in situ hybridization (In situ) and quantitative RT-PCR (qPCR) assays. (B) In situ hybridization assays for the endoderm (Endo) marker gene foxA, the oral (ventral) non-skeletogenic mesoderm (Or. NSke. M) marker gcm, and the aboral (dorsal) non-skeletogenic mesoderm (Ab. NSke. M) marker gataC [26,48] and qPCR expression matrices, from 2 independent biological replicates (Rep1 and Rep2), for foxA, gcm, and gataC as well as 5 additional endoderm (bra, endo16, hox11/13) and oral (pks) and aboral (ese) non-skeletogenic mesoderm marker genes. Experimental conditions included uninjected, irradiated control embryos: uninjected control (+UV); β-cateninM+β-cateninPM and not irradiated embryos: β-catM+PM (-UV); β-cateninM+β-cateninPM and irradiated embryos: β-catM+PM (+UV). The germ layer color-code is as in Fig 1. For the in situ hybridization assays, embryos are in vegetal view. Numbers in the bottom right and left corners indicate phenotypic counts relative to the total number of scored embryos (in parentheses). The 2 control conditions are shown in different colors, and indicates weak signal in the remaining scored embryos. For the qPCR analyses, results are shown as tables, and > marks gcm, gataC, and foxA that were also analyzed by in situ hybridization. Orange backgrounds indicate down-regulation by more than 2-fold compared to uninjected, irradiated control embryos, while blue backgrounds highlight unaltered expression compared to controls. Related raw data and quantification methods are available in S1 Data and fold change values are further provided in S2 Data.
	Fig 10. Time point-specific blockage of β-catenin translation and animal territory restriction.(A) Schematic representation of the performed analyses, following the experimental procedure detailed in Fig 6A. For the time points marked by *, embryos were collected at 10 hpf (late blastula stage) and/or 15 hpf (mesenchyme blastula stage) [37] to carry out in situ hybridization assays (In situ). (B) In situ hybridization assays for the ectoderm (Ecto) marker gene he and the anterior neuroectoderm (Ant. N.Ecto) marker foxQ2 [21,54]. Experimental conditions included uninjected, irradiated control embryos: uninjected control (+UV); β-cateninM+β-cateninPM and not irradiated embryos: β-catM+PM (-UV); β-cateninM+β-cateninPM and irradiated embryos: β-catM+PM (+UV). The germ layer color-code is as in Fig 1, and embryos are in lateral view, with the animal pole up. Numbers in the bottom right and left corners indicate phenotypic counts relative to the total number of scored embryos (in parentheses). The 2 control conditions are shown in different colors. -, +, and # indicate that, relative to controls, the remaining scored embryos display, respectively, reduced, expanded, and unchanged expression domains.
	Fig 11. Schematic summary of the spatiotemporal requirements of nuclear β-catenin during early embryogenesis of the sea urchin Paracentrotus lividus.The top row illustrates the developmental stages and time points, in minpf and hpf, when conditional loss of β-catenin translation was induced by exposure to UV light. The bottom rows show the periods of β-catenin protein production required for the initiation and completion of skeletogenic mesoderm (Ske. M) development, for triggering and maintaining non-skeletogenic mesoderm (NSke. M) and endoderm (Endo) fates, and for restricting ectoderm (Ecto) and anterior neuroectoderm (Ant. N.Ecto) territories along the animal-vegetal axis. The germ layer color-code is as in Fig 1.