Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart

Abstract

Normal vertebrate heart development depends upon the expression of homeodomain containing proteins related to the Drosophila gene, tinman. In Xenopus laevis, three such genes have been identified in regions that will eventually give rise to the heart, XNkx2-3, XNkx2-5 and XNkx2-10. Although the expression domains of all three overlap in early development, distinctive differences have been noted. By the time the heart tube forms, there is little XNkx2-10 mRNA detected by in situ analysis in the embryonic heart while both XNkx2-3 and XNkx2-5 are clearly present. In addition, unlike XNkx2-3 and XNkx2-5, injection of XNkx2-10 mRNA does not increase the size of the embryonic heart. We have reexamined the expression and potential role of XNkx2-10 in development via oligonucleotide-mediated reduction of XNkx2-10 protein expression. We find that a decrease in XNkx2-10 leads to a broad spectrum of developmental abnormalities including a reduction in heart size. We conclude that XNkx2-10, like XNkx2-3 and XNkx2-5, is necessary for normal Xenopus heart development.

Fig. 1

Analysis of XNkx2-10 expression. (A) A cartoon indicating the differences in XNkx2-3, XNkx2-5 and XNkx2-10 expression patterns at developmental stages 24, 28 and 32. Expression regions were estimated from cardiac XNkx2 in situ hybridization analysis (Cleaver et al., 1996; Newman and Krieg, 1998; Newman et al., 2000; Sparrow et al., 2000). The first column delineates XNkx2-10 expression. Strong XNkx2-10 expression is indicated with royal blue while weak XNkx2-10 expression is indicated with baby blue. The second column represents an overlay compilation of XNkx2-3, XNkx2-5 and XNkx2-10 expression regions. XNkx2-3 expression is indicated in yellow while XNkx2-5 expression is indicated in red. Regions where both XNkx2-3 and XNkx2-5 expression overlap is indicated with the color orange. (B) The RT-PCR products of XNkx2-10 and EF1α. expression from stage 10.5 to 12 (lane 1), stage 22 (lane 2), stage 32 (lane 3), stage 46 (lane 4) and adult heart (lane 5). Lane 6 is a negative control (no reverse transcriptase). PCR products were collected at PCR cycle #45. (C) The RT-PCR products of XNkx2-3, XNkx2-5, XNkx2-10 and EF1α. expression from different Xenopus stage 46 and adult tissues. Lane 1 is the RT-PCR product from RNA harvested from Xenopus stage 46 embryo eyes. Lane 2 is from stage 46 embryo hearts. Lane 3 is from stage 46 embryo tails. Lane 4 is from the adult eye. Lane 5 is from an adult heart. Lane 6 is from an adult liver. Lane 7 is from an adult gut. Lane 8 is from adult skin. Lane 9 is from skeletal muscle. Lane 10 is a negative control that does not contain reverse transcriptase. PCR products were collect at PCR cycle #45. (D) Western blots with protein harvested from adult heart (lane 1) and skeletal muscle (lane 2). Lanes 3 and 4 provide evidence that we have generated an antibody that recognizes XNkx2-10. Lane 3 is XNkx2-10 protein generated in vitro. Primary antibodies were against nucleolin (95 kDa) and XNkx2-10 (black arrow) (29.2 kDa). Lane 4 is S-35-labeled XNkx2-10 generated in vitro.

Fig. 2

Reduction of XNkx2-10 leads to abnormal development. (A–G) Pictures of stage 22 embryos. Scale bar is 3 mm. (A) are non-injected embryos. (B) Embryos injected with 2 ng of XNkx2-10 mRNA (overexpression). (C) Morpholino control injected embryos. (D) Morpholino injected embryos. (E) Deed oligonucleotide injected embryos. (F) Embryos injected with both morpholino and 2 ng of XNkx2-10 mRNA (morpholino rescue). (G) Embryos injected with both Deed oligonucleotide and 2 ng of XNkx2-10 mRNA (Deed rescue), (a1) Stage 42 non-injected embryo. Scale bar is 3 mm. (b1) Stage 42 overexpression embryo, (c1) Stage 42 morpholino control injected embryo, (d1) Morpholino injected embryo. It has an abnormal head shape and delayed eye development (white arrows). (d2) Morpholino injected embryo that is stunted, has an abnormal head shape and delayed eye development, (e1) Deed oligonucleotide injected embryo that is stunted, has an abnormal head shape and delayed eye development. (e2) Deed injected embryo that is stunted, has an abnormal head shape, delayed eye development and delayed gut development, (f1) Stage 42 morpholino rescue embryo, (g1) Stage 42 Deed rescue embryo. (H) Western blot demonstrating that injection of either the morpholino or Deed oligonucleotide leads to reduced XNkx2-10 protein levels in stage 22 embryos (black arrows) and a Western blot demonstrating the translation of rescue mRNA (T7) in stage 22 embryos. (I) Western blot demonstrating XNkx2-10 and nucleolin protein levels at stage 36. (J) Western blot demonstrating XNkx2-10 and nucleolin protein levels at stage 46. Nucleolin serves as a positive control. The expected size of XNkx2-10 is 29.2 kDa while the expected size of nucleolin is 95 kDa. Lane 1 is protein from non-injected embryos. Lane 2 is protein from morpholino control injected embryos. Lane 3 protein from morpholino injected embryos. Lane 4 contains protein from Deed oligonucleotide injected embryos. Lane 5 contains protein from Deed rescue embryos.

Fig. 3

RT-PCR analysis of selected genes expressed in the developing heart. Stage 22 RNA was harvested from non-injected (NI), overexpression (OE), XNkx2-10 morpholino (M), XNkx2-10 Deed oligonucleotide (D), morpholino rescue (MR) and Deed rescue (DR) embryos. RT-PCR products were harvested over eight consecutive PCR cycles. Decreases in XTnIc and Tropomyosin expression were observed for the XNkx2-10 knockdown embryos (black arrows). XNkx2-10 expression was reduced in XNkx2-10 Deed injected embryos (red arrow). The number on top of the column of gels indicates the final PCR cycle that product was collected. Lanes marked with an asterisk were determined to have an amount of PCR product that had approximately doubled when compared to the previous lane’s PCR product. Experiments were performed in duplicate. EF1α. served as a positive control.

Fig. 4

Box plot demonstrating the range of maximum ventricular area values. Sample 1 contains data from non-injected embryos. Sample 2 contains data from morpholino control injected embryos. Sample 3 contains data from XNkx2-10 overexpression embryos. Sample 4 contains data from XNkx2-10 morpholino injected embryos. Sample 5 contains data from XNkx2-10 Deed oligonucleotide injected embryos. Sample 6 contains data from morpholino rescue embryos. Sample 7 contains data from Deed rescue embryos. The morpholino and Deed oligonucleotide significantly reduced the maximum ventricular area of their respective hearts. Alternatively, co-injection of either the morpholino or Deed oligonucleotide with 2 ng of XNkx2-10 mRNA (rescue) restored the size of the maximum ventricular areas. The * symbol indicates a p value of less than 0.00001.

Fig. 5

Confocal microscope generated image comparisons of embryonic stage 46 hearts. Scale bar is 200 μm. The embryos were fixed in Dents fixative and then stained with FITC-phalloidin. Images were captured at 5 nm intervals. Panels (A, E, I, M, Q, U and Y) are a compilation of 20–30 images. Panels (B, F, J, N, R, V and Z) are single images demonstrating the maximum ventricular areas (red outline). Panels (C, G, K, O, S, W and α) are single images demonstrating the spiral valve in the outflow tract (cyan arrows). Panels (D, H, L, P, T, X and β) are single images demonstrating the atrial septum (magenta arrows).