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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.
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Figure 4. Histology of the Xenopus embryo. (A) Drawing of stage 42 control and lithium-treated embryos displaying the transverse sectional planes shown in the corresponding panels. (B) Cross sectional slice of non-treated embryo from the pericardial cavity through the head region. (C) Transverse section of a lithium-treated embryo at the plane analogous to that shown for the control embryo in panel B. (D, E) Transverse anterior sections of control and lithium-treated embryos, respectively, immunostained for sarcomeric myosin. (F, G) Cross sectional slice through the tail of control and lithium-treated embryos, respectively. da, dorsal aorta; df, dorsal fin; ht, heart; oc, otic capsule; nc, notochord; nt, neural tube; phx, pharynx, ppc, pericardial cavity; som, somites; spc, spinal cord; vf, ventral fin.
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Figure 5. Immunofluorescent analysis of cell proliferation and apoptosis. Transverse frozen sections of stage 40 embryos that were (A, B) non-treated, or exposed to (C, E, F) lithium or (D) SB415286 at stage 10. (A) Non-treated sectioned embryo stained with the mitotic marker phospho-histone H3 (phosH3). Note the presence of multiple phosH3-positive cells, which indicate cells undergoing mitosis. (B) High magnification view of sectioned non-treated frog embryo, which was stained for phosH3 and co-stained with the nuclear label 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI), demonstrating the nuclear localization of phosH3 antibody staining. (C) Sectioned tissue from lithium-treated embryo, showed an absence of phosH3-staining. (D) Embryos treated with the selective GSK3 inhibitor SB415286 exhibited positive phosH3 staining. (E, F) Brightfield and immunostained sectioned tissue from a lithium-treated embryo that was immunostained for the apoptotic marker capsase-3 and co-stained with DAPI. Exposure to lithium produced embryos that displayed high numbers of apoptotic cells, as indicated by positive staining for caspase-3. Scale bars = 50 μm.
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Figure 6. Cardiac tissue differentiation of DMZ explants cultured in the presence of lithium. (A) Outline of experimental protocol involving the dissection of DMZ tissue from stage 10+ embryos, which were exposed to either lithium or media alone, and then cultured for 3 days. Subsequently, explants were either (B-F) fixed and immunostained for sarcomeric myosin, or (G) harvested for RNA for reverse transcription-polymerase chain reaction (RT-PCR) amplification. (B) Summary of the immunohistochemical data, with explants displaying a large aggregate of sarcomeric myosin-positive cells within the interior of the tissue being scored for a cardiac phenotype. Examples of sarcomeric myosin-positive (C, D) non-treated control and (E, F) lithium-treated explants, shown in brightfield and fluorescent images. The asterisks overlaid on the brightfield images show position of the sarcomeric myosin positive aggregates revealed in the adjacent panels displaying immunofluorescent staining. (G) RT-PCR analysis of the cultures examined their expression of the housekeeping gene ODC and cardiac genes Nkx2.5 and cTnI. While lithium treatment decreased expression of the late cardiac gene cTnI, levels of Nx2.5 were only moderately reduced in response to lithium exposure.
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Figure 7. Removal of DMZ from lithium-treated embryos rescued the ability to form contractile cardiac tissue. (A) For these experiments, stage 10+ embryos were either mock or lithium-treated, prior to the removal and culture of DMZ tissue. (B) Following 6 days of culture, explants were scored for contractility, as summarized in this graph. Although explants from lithium-treated embryos showed decreased formation of a contractile phenotype as compared to controls, >50% of the lithium-exposed tissue produced beating tissue. This is in contrast to embryos left intact following lithium exposure, which never developed a contractile heart (Figs 2�4). (C, D) Brightfield and fluorescent view of a representative explant taken from a lithium-treated embryo that was subsequently immunostained for sarcomeric myosin. The arrow in both panels shows position of the sarcomeric myosin-positive beating tissue within the explant. (E) Image of a live explant harvested from a lithium-treated embryo, which displays a well-formed heart tube (arrow) that was contractile (see Fig. S1). (F) Cardiac gene expression in DMZ tissue, as expressed as a ratio of relative mRNA levels displayed in tissue obtained from lithium-treated embryos as compared to non-treated embryos. DMZ tissue was cultured for 2, 3, and 5 days prior to RNA isolation and PCR analysis of cardiac transcription factors Nkx2.5, GATA6, Tbx5, Tbx20 and the muscle genes cTnI, Actc, and cMHCα.
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Figure 8. Cardiac tissue differentiation of precardiac tissue removed from embryos 10 h after lithium exposure. (A) For these experiments, stage 10+ embryos were either mock or lithium-treated and incubated for 10 additional hours to stage 18, prior to the removal and culture of the heart forming region (HFR). Explants were cultured for 5 days and scored for beating. (B) Summary of the results, which showed a significant decrease but not complete abolition of contractile tissue formation in response to lithium treatment. The asterisk in the right hand column denotes the nine explants from each group that were subsequently immunostained for sarcomeric myosin. All these explants were scored positive for cardiac tissue, with both control and lithium-exposed tissue exhibiting three-dimensional sarcomeric myosin-positive structure within the explant interior. (C) Reverse transcription�polymerase chain reaction (RT�PCR) amplification of the housekeeping gene ODC and cardiac genes Nkx2.5 and cTnI. The expression of the two heart genes by explants obtained from lithium embryos confirms the cardiac phenotype of the differentiated tissue. Individual examples of sarcomeric myosin-positive explants from (D, E) non-treated control and (F, G) lithium-treated embryos, as imaged in brightfield and fluorescence. Arrows in panels E and G identify cardiac tissue within the explants that stained positive for sarcomeric myosin.
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