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Figure 3. Lithium treatment at the onset of gastrulation prevents formation of the heart. Stage 42 embryos that were either (A) non-treated or (B-D) treated with lithium at stage 10+. (A) Immunostaining of a non-treated stage 42 control embryo for sarcomeric myosin shows the position of the heart (arrow) (B-D) Stage 42 embryos previously treated with lithium at stage 10+. (B, C) Lithium-treated embryo imaged with visible or fluorescent light after immunostaining with the extracellular matrix protein fibrillin-2, which normally marks the endocardial tube of the developing heart (Kolker et al. 2000). The lack of fibrillin-2 in the area where the heart would usually form (arrow) indicates that a cardiac structure has not formed in the treated embryo. (D) The absence of the heart is further demonstrated by the lack of sarcomeric myosin in the normal heart-forming region of the embryo (arrow). (E) Drawing of stage 42 control and lithium-treated embryos, which illustrate the areas used for RT-PCR gene expression analysis. (F) RT-PCR amplification of the housekeeping gene ODC and the cardiac genes Nkx2.5 and cTnI. Note the decrease in both cardiac genes in response to lithium-treatment. (G) Summary of experiments where Xenopus embryos were exposed to 300 mmol/L lithium for 10 min at either stages 9, 10, 11, or 12 (n = 9, 21, 9, 6, respectively). In parallel dishes, embryos were treated at stage 10 with 4 mmol/L SB415286 for 10 min (n = 19), which is the optimized dose of this selective GSK3 inhibitor (Martin et al. 2011). While lithium treatments at stage 9 and 10 abolished formation of a functional heart, neither lithium exposure at stage 12 or SB415286 at stage 10 prevented development of a contractile heart. (# refers to lack of contractility in the scored population of embryos). (H, I) Representative examples of stage 12 lithium and stage 10 SB415286-treated embryos, respectively, with arrow showing the position of a contractile heart. |
