Tarsis K et al. (2022), Distinct regulatory states control the elongati...

ECB-ART-50387

Dev Dyn 2022 Aug 01;2518:1322-1339. doi: 10.1002/dvdy.474.

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Distinct regulatory states control the elongation of individual skeletal rods in the sea urchin embryo.

Tarsis K , Gildor T , Morgulis M , Ben-Tabou de-Leon S .

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BACKGROUND: Understanding how gene regulatory networks (GRNs) control developmental progression is a key to the mechanistic understanding of morphogenesis. The sea urchin larval skeletogenesis provides an excellent platform to tackle this question. In the early stages of sea urchin skeletogenesis, skeletogenic genes are uniformly expressed in the skeletogenic lineage. Yet, during skeletal elongation, skeletogenic genes are expressed in distinct spatial sub-domains. The regulation of differential gene expression during late skeletogenesis is not well understood. RESULTS: Here we reveal the dynamic expression of the skeletogenic regulatory genes that define a specific regulatory state for each pair of skeletal rods, in the sea urchin Paracentrotus lividus. The vascular endothelial growth factor (VEGF) signaling, essential for skeleton formation, specifically controls the migration of cells that form the postoral and distal anterolateral skeletogenic rods. VEGF signaling also controls the expression of regulatory genes in cells at the tips of the postoral rods, including the transcription factors Pitx1 and MyoD1. Pitx1 activity is required for normal skeletal elongation and for the expression of some of VEGF target genes. CONCLUSIONS: Our study illuminates the fine-tuning of the regulatory system during the transition from early to late skeletogenesis that gives rise to rod-specific regulatory states.

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	FIGURE 1. Sea urchin larval skeletogenesis and gene expression. (A) Sea urchin embryo at the gastrula stage. AL, anterolateral rods; MV, midâventral rods; BR, body rods; D, dorsal skeletogenic chain; L, longitudinal skeletogenic chain; V, ventral skeletogenic chain. The pseudopodia cable between the skeletogenic cells is marked in black lines. VEGFR expression is marked in red and VEGF is marked in blue, based on. 15 , 16 (B) Sea urchin embryo at the pluteus stage. PO, postoral rods; pâAL, proximal anterolateral rods; dâAL, distal anterolateral rods. (CâH) Differential expression of SM50 and SM30 at 2 dpf and the effect of VEGFR continuous and late inhibition. (CâE) SM50 expression in control (C), late VEGFR inhibition (D), and continuous VEGFR inhibition (E). (FâH) SM50 expression in control (F), late VEGFR inhibition (G), and continuous VEGFR inhibition (H). Numbers at the bottom indicate the number of embryos that show this phenotype (left) out of all embryos scored (right), conducted in three independent biological replicates. (IâN) Embryo morphology and gene expression at the time of the addition of VEGFR inhibitor (25 hpf) in the late inhibition experiments. (I) Embryo morphology, arrowheads point to the triâradiate spicules. Gene names are indicated at the bottom in (JâN)
	FIGURE 2. Differential expression of the skeletogenic regulatory genes at prism stage and the effect of VEGFR continuous and late inhibition. (AâL) Representative images of control embryo (top panels), embryos where VEGFR inhibitor, axitinib, was added at 25 hpf (middle panels), and embryos that were exposed to continuous VEGFR inhibition (bottom panels), at the prism stage (~34 hpf in Paracentrotus lividus). (A) DIC images, scale bar indicates 50âÎ¼m. MV, midâventral; AL, anterolateral; PO, postoral rods. (B) Skeletogenic cell marker, 6a9. (CâL) Gene names are indicated at the top of each panel. Arrowheads point to expression at the tips of the AL rods (orange) and PO rods (cyan). Yellow arcs show the expression at the body rods, lines showing expression along the AL rods (orange) or the MV rods (dark blue). Numbers at the bottom of each image in (CâL) indicate the number of embryos that show this phenotype (left) out of all embryos scored (right), conducted in at least three independent biological replicates. (M, N) Tables summarizing gene expression in control embryos (M) and under late VEGFR inhibition (N) at the different skeletogenic domains (left), illustrated in an embryo diagram (right). Similar color codes are used throughout the figure
	FIGURE 3. Differential expression of the skeletogenic regulatory genes at pluteus stage and the effect of VEGFR continuous and late inhibition. (AâL) Representative images of control embryos (top panels), embryos where VEGFR inhibitor was added at 25 hpf (middle panels), and embryos exposed to continuous VEGFR inhibition (bottom panel). Embryos are at the pluteus stage (~2 dpf). (A) DIC images, scale bar indicates 50âÎ¼m. Rod names are like in FigureÂ 1B and color code is like in FigureÂ 2. (B) Skeletogenic cell marker, 6a9. (CâL) Gene names are indicated at the top of each panel. Numbers at the bottom of each representative image in (CâL) indicate the number of embryos that show this phenotype (left) out of all embryos scored (right). Experiments were conducted in three independent biological replicates for all genes. (M, N) Tables summarizing gene expression in control embryos (M) and under late VEGFR inhibition (N) at the different skeletogenic domains (left), illustrated in an embryo diagram (right)
	FIGURE 4. Effect of VEGFR inhibition on skeletal growth and gene expression in the pluteus stage. (AâD) Relative gene expression levels of treated embryos vs control embryos measured by QPCR at 2 dpf (A,B) and at 3 dpf (C,D). (A, C) Showing results for continuous VEGFR inhibition (>0 hpf) and (B, D) showing results for late VEGFR inhibition (>25 hpf). Bars show averages over three independent biological replicates and markers indicate individual repeats. Ratio of 1 indicates that the expression of the gene is unaffected by VEGFR inhibition. Error bars indicate SD. The significance was calculated by oneâtailed zâtest. One star represents PÂ <â.01 and two stars represent PÂ <â.001. (EâG) Representative embryos at 3 dpf showing skeletogenic phenotypes (E), myoD1 (F), and pitx1 gene expression (G). In each panel, we present control embryo (top), embryo in late VEGFR inhibition (>25 hpf, middle), and embryo where VEGFR was continuously inhibited (>0 hpf, bottom). Embryos are oriented similarly along the dorsalâventral axis where the dorsal side is at the bottom, scale bars indicate 50âÎ¼m. Arrowheads point to expression at the tips of the PO rods. Numbers at the bottom of each representative image in (F, G) indicate the number of embryos that show this phenotype (left) out of all embryos scored (right), conducted in three independent biological replicates
	FIGURE 5. Spatiotemporal expression of myoD1 and pitx1 from gastrula to pluteus stage (24â44 hpf). (AâH) myoD1 spatiotemporal expression. During the gastrula, stage myoD1 is expressed at the skeletogenic ventrolateral clusters (AâD). At prism and pluteus stages, myoD1 is expressed at the cells at the tips of the growing postoral rods (EâH). (IâP) pitx1 spatiotemporal expression. At the early gastrula stage, pitx1 is expressed at the skeletogenic ventrolateral clusters (I). (JâL) During late gastrula stage, pitx1 is expressed within cells of dorsal (D), longitudinal (L), and ventral (V) chains. At prism stage (M, N) shrinks to the cells that form the postoral rods and is expressed at the tips of these rods in pluteus stage (OâP). Embryos at the gastrula stage are presented in lateral view except A and J where they are presented in ventral view (VV). At the prism and pluteus stages embryos are presented in the same orientation along the dorsalâventral axis where the dorsal side is at the bottom of the image
	FIGURE 6. Plâpitx1 MO design and test of splicing MOs. (A) Structure of the Plâpitx1 gene with target sites for the two splicing MOs and PCR primer locations identified. The coordinates correspond to the distance from the beginning of the first exon. The QPCR primers that were used to assess pitx1 level in pitx1 perturbation experiments are located at the fourth exon, a region that is unaffected by the splicing MO. (B, C) Primer design for testing the activity of sMO1 (B) and sMO2 (C) showing the expected PCR transcripts. (D, E) Gels testing sMO1 and sMO2, respectively, showing bands of a long (full) and short (truncated) transcripts. Un, uninjected embryos; raMO, embryos injected with random MO; sMO1, embryos injected with Plâpitx1 splicing MO1; sMO2, embryos injected with splicing MO2; L, long transcript; S, short transcript
	FIGURE 7. The transcription factor, Pitx1, is essential for normal skeletal elongation in the sea urchin embryo. (AâC) pitx1 perturbations. (A) embryo injected with control (random) MO, showing normal skeleton at 3 dpf. (B, C) pitx1 knockdown using two different splicing MOs results in short postoral, body, and distal anterolateral rods. Scale bars are 50âÎ¼m. (D) Measurement of the length of the skeletal rods. Embryos were flattened using the cover slide and the Zeiss software ZEN was used to measure the length of the different rods, as shown in the image. (EâH) Lengths of different rods in control and pitx1 downregulated embryos. Each box plot shows an average, the first and the third quartiles (edges of boxes), and outliers (dots). Experiments were performed in 3 to 5 independent biological replicates and for each condition 99 to 115 embryos were measured. The exact number of studied embryos can be found in TableÂ S2. tâtest; statistical significance is represented by PÂ <â.01, **PÂ <â.0001
	FIGURE 8. Effect of PlâPitx1 downregulation on gene expression at 30 hpf and 2 dpf. (A, B) Representative embryos injected with Random MO (A) and pitx1 sMO2 (B) at 30 hpf. (C) Relative change in gene expression level in pitx1 sMO2 compared to Random MO, measured by QPCR. Bars show averages and markers indicate individual measurements. Ratio of 1 (dashed line) indicates that the expression of the gene is unaffected by pitx1 downregulation. Error bars indicate SD. Oneâtailed zâtest statistical significance is represented by PÂ <â.01; *PÂ <â.001. (DâI) Spatial gene expression in control embryos vs embryos injected with pitx1 sMO2, gene names are indicated at the bottom. (JâR) Similar to (AâI), tested at 2 dpf. QPCR experiments were performed in 3 biological replicates and spatial expression experiments were performed in 2 to 4 biological replicates. Exact numbers of studied embryos are provided in TableÂ S2. (TâS) Pitx putative binding sites in the promoter regions of SpSM30a (S) and SpSM50 (T), that drive expression in the skeletogenic cells. 50 , 51 , 52 Borders of active regions are marked magenta; first exon is marked in blue letters and the start of translation is highlighted in yellow. Pitx1 putative binding sites are highlighted in green, two overlapping sites in SpSM30a are marked in bold
	FIGURE 9. Skeletogenic regulatory states at the prism and pluteus stage and a model of the regulatory interactions in the skeletogenic lineage. (A) Embryo diagrams at the prism and pluteus stages, showing the skeletogenic cells at the body (yellow), PO (cyan), AL (orange), and MV (dark blue). The regulatory genes that are expressed within each territory are listed below using the same color code. VEGFâexpressing ectodermal cells are marked in red. (B) A model of the roles of late VEGF signaling. After spicule initiation at 24 hpf, VEGF signaling is essential for the migration of the skeletogenic cells that form the PO rods and the distal AL rods and for the formation of the spicule cavity in these rods. VEGF signaling is also critical to the expression of the regulatory genes at the tips of the PO rods (cyan box). The transcription factor Pitx1 positively regulates the expression of SM30 and SM50 and negatively regulates its own gene expression

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