|
Fig. 1. The regulation of DV axis formation downstream of redox and oxygen gradients in the sea urchin embryo. (A-D) Diagrams showing sea urchin DV and skeletal patterning in developing sea urchin embryos in normal conditions (based on Chang et al., 2017; Coffman et al., 2009; Coffman and Davidson, 2001; Coffman et al., 2004, 2014; Czihak, 1963; Duboc et al., 2004; Lapraz et al., 2009; Li et al., 2012). (A) The asymmetric distribution of mitochondria in the egg induces a redox gradient. (B) Regulatory interactions between nodal, lefty and HIF1α at the early blastula stage. (C) Nodal-mediated regulation of BMP signaling in the late blastula stage. (D) In the gastrula stage, Nodal activates the expression of Not1, which represses VEGF expression in the ventral ectoderm. Throughout the figure, the ventral side and Nodal expression domain are highlighted in green; the dorsal side and the domain of BMP activity are marked in purple. Nuclei that show pSMAD1/5/8 are highlighted in pink. VEGF expression is marked in red. VEGFR expression is marked in blue.
|
|
Fig. 2. BMP2/4
control skeletal patterning and VEGF expression. (A) Embryos injected with control MO show two normal spicules at 1 dpf (left, 110/110 of scored embryos show this phenotype) and 2 dpf (right, 56/56). (B,C) BMP2/4 MO-injected embryos show either ectopic spicules indicated by numbers (ES, 89/169 1 dpf, 120/135 2 dpf) or ectopic spicule branching (EB, 39/169 at 1 dpf, 15/135 at 2 dpf). Scale bars: 50 μm in A-C. (D) VEGF expression is localized in two lateral patches in the control embryo (top) and is strongly expanded in embryos injected with BMP2/4 MO at 1 dpf (bottom). (E,F) VEGFR and SM30 expression in control embryo (top) and in BMP2/4 morphants (middle and bottom) at 1 dpf. BMP2/4 MO leads to the expansion of the expression either into ectopic skeletal cell clusters indicated by numbers (ES) or to continuous expansion (EB). LV, lateral view; VV, ventral view. Phenotypes are based on n≥3 independent biological replicates and spatial expression was observed in two independent biological replicates where n≥30 embryos were scored in each condition. (G) Ratio between gene expression in BMP2/4 MO compared with control MO embryos at 1 dpf (left graph) and 2 dpf (right graph). Bars show averages and markers indicate individual measurements of three independent biological replicates. Line indicates a ratio of 1, i.e. the expression of the gene is unaffected by the perturbation. Error bars indicate s.d. Statistical significance was calculated using a one-tailed z-test (**P<0.01).
|
|
Fig. 3. Sea
urchin HIF1α does not affect skeletal patterning and VEGF expression. (A) Control (left) and HIF1α MO-injected embryos (right) show comparable skeletal structure at 2 dpf. (B) Ratio between gene expression in HIF1α MO compared with control MO embryos at 15 hpf (left graph) and 19 hpf (right graph). Bars show averages and markers indicate individual measurements of two independent biological replicates. Line indicates a ratio of 1, i.e. the expression of the gene is unaffected by the perturbation. Error bars indicate s.d. (C) VEGF expression is similar in embryos injected with control MO (left) and HIF1α MO (right) at 15 hpf (top) and 19 hpf (bottom). Spatial expression was observed in two independent biological replicates where n≥30 embryos were scored in each condition.
|
|
Fig. 4. Growth in hypoxic
conditions leads to skeletal defects and perturbs the expression of DV and skeletal patterning genes. (A-C) Representative images of embryos at gastrula stage. (A) Embryo grown in normoxic conditions shows normal development of two spicules (arrowheads). (B,C) Embryos grown in hypoxic conditions show ectopic spicules (arrowheads). (D-F) Representative images of embryos at pluteus stage. (D) Embryo grown in normoxic conditions shows a normal skeleton. (E) Embryo grown in hypoxic condition shows a normal DV axis and ectopic spicule branches. (F) Radialized embryo grown in hypoxic conditions that displays multiple ectopic spicules. LV, lateral view; VV, ventral view. (G) Quantification of skeletogenic phenotypes at gastrula stage and pluteus stage. Color code is indicated in the representative images. Error bars indicates s.d. of three independent biological replicates. (H-J) Spatial expression of nodal, BMP2/4 and chordin genes in normoxic (top) and hypoxic (bottom) embryos at blastula stage. (K-N) Spatial expression of nodal, BMP2, BMP4, VEGF and VEGFR genes in normoxic (top) and hypoxic embryos (bottom) at the gastrula stage. Embryos are presented in ventral view and the axis is labeled ventral (V) to dorsal (D). Throughout H-N, the numbers at the bottom right indicate the number of embryos that show this expression pattern out of all embryos scored, based on three independent biological replicates.
|
|
Fig. 5. BMP activity
is reduced in hypoxic conditions. (A,B) Nuclear pSMAD1/5/8 patterning in normoxic and hypoxic conditions at mesenchyme blastula (MB) stage. In normoxic conditions, pSMAD1/5/8 staining is detected in the dorsal ectoderm (A), while in hypoxic embryos the signal is completely abolished (B). (C-E) pSMAD1/5/8 staining in normoxic versus hypoxic embryos at late gastrula (LG) stage. pSMAD1/5/8 is detected in the nuclei of the dorsal skeletogenic cells of normoxic embryos (C), while in hypoxic conditions the signal is either not detectable (D) or strongly reduced (E). DIC images of the embryos are presented in the upper row of each panel; immunostaining of pSMAD1/5/8 of the embryos are presented in the lower row. All embryos are presented in lateral view (LV). The numbers shown on the bottom right of each figure indicate the number of embryos that show this expression pattern out of all embryos scored, based on three independent biological replicates.
|
|
Fig. 6. Late hypoxia affects skeletal structure but not skeletal patterning. (A-F) Representative images of live embryos: normoxic embryos are presented in the upper row and equivalently staged hypoxic embryos are on the bottom. (A,B) Embryos at early gastrula stage show similar morphology in normoxia and hypoxia. (C,D) A hypoxic embryo at late gastrula stage shows two spicules with ectopic spicule branching (D) that are not observed in the normoxic embryo (C). Inset shows the outlined area at higher magnification. (E,F) Embryos at pluteus stage. Arrowhead in F indicates an abnormal spicule growing in the hypoxic embryo. (G) Quantification of late hypoxia experiment over three biological replicates. Color code is indicated in the representative images. Error bars indicates s.d. of three independent biological repeats. (H-K) WMISH results of nodal, BMP2/4, VEGF and VEGFR at early gastrula stage. A normoxic embryo is presented at the top and a hypoxic embryo is at the bottom of each panel. On the bottom right of each figure, the number of embryos that show this expression pattern out of all embryos scored is provided, based on three independent biological replicates.
|
|
Fig. 7. The interactions between the DV and skeletogenic GRNs, the response to early hypoxia and the similarities to the regulation of vertebrate vascularization. (A,B) Diagrams showing our proposed model for skeletal patterning in normal conditions (A) and hypoxic conditions (B). Color codes are indicated at the bottom of the figure. (A) The regulatory interactions between Nodal, BMP, HIF1α and VEGF signaling during normal development. BMP represses VEGF, VEGFR and SM30 expression in the dorsal side, and HIF1α does not regulate VEGF expression in the sea urchin embryo. (B) The modification of the regulatory states in hypoxic conditions applied at early development revealed in this work. Early hypoxia expands nodal expression and reduces BMP activity and the dorsal ectoderm. The reduction of BMP activity leads to an expansion of VEGF, VEGFR and SM30 expression in the dorsal side and to growth of ectopic skeletal centers. Upward arrows near a gene name indicate enhanced activity; downward arrows indicate reduced activity. Gray regulatory links indicate inactive connections under hypoxic conditions. (C) Diagram showing the relevant regulatory interactions during vertebrate vascularization in normal development and in cancer; see text for explanations.
|