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Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in the nervous system in Xenopus embryos. Knock-down of Xisl-1 by specific morpholino leads to severe developmental defects, including eye and heart failure. Staining with the neuronal markers N-tubulin and Xisl-1 itself reveals that the motor neurons and a group of ventral interneurons are lost in the Xisl-1 morphants. Terminal dUTP nick-end labeling (TUNEL) analysis shows that Xisl-1 morpholino injection induces extensive apoptosis in the ventral neural plate, which can be largely inhibited by the apoptosis inhibitor M50054. We also find that over-expression of Xisl-1 is able to promote cell proliferation and induce Xstat3 expression in the injected side, suggesting a potential role for Xisl-1 in the regulation of cell proliferation in co-operation with the Jak-Stat pathway.
Fig. 1.
Differential expression of Xenopus Isl-1 (A–C) and Isl-2 (D–F) at indicated stages revealed by in situ hybridization. (A) A dorsal view (anterior on the left) of a stage 16 embryo showing the expression of Xisl-1 in the motor neurons (white arrowheads) and the dorsal interneurons 3 (black arrowhead) in the neural plate and also in the anterior neural placodal regions (red arrowheads). (B) A lateral view (anterior on the left) of a stage 26 embryo showing the expression of Xisl-1 in the neural tube (NT), pineal gland (PG), heart (He), proctodeum (Pr) region and cranial ganglia. (C) A transverse section through the trunk (indicated by the dashed line in B) showing the expression of Xisl-1 in the dorsal interneurons 3 (black arrowhead) and motor neurons (white arrowheads). (D) A dorsal view (anterior at the top) of a stage 16 embryo showing the expression of Xisl-2 the neural plate region. (E) A lateral view (anterior on the left) of a stage 28 embryo showing the expression of Xisl-2 the axial structures (arrowheads), the eye and the branchial arches regions. (F) A transverse section through the trunk (indicated by the dashed line in E) showing the expression of Xisl-2 in the neural tube (NT), somite (So) and notochord (No). (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)
Fig. 2.
Inhibition of Xisl-1 expression by specific morpholino (islMO) leads to severe developmental defects. (A) Microinjection of 30 ng islMO causes gross developmental defects at tadpole stage including eye defects, edema, and curled and shortened trunks. (B) The effects of islMO can be largely rescued by co-injection of 1 ng Islet1 mRNA, which is not recognized by the MO. (C) Statistics of the embryos shown in (A) and (B), indexed by the development of the eyes in the two groups of embryos. (D,D′) islMO injection leads to eye defects on the injected side (D′, arrowhead), while the eye develops normally on the control side (D, arrowhead). (E) Anterior views of embryos at stage 20 injected on one side with islMO, the cement glands do not form on the injected sides (red arrowheads) while developing normally on the control sides (black arrowheads). (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)
Fig. 3.
Knockdown of Xisl-1 by morpholino induces apoptosis of ventral neurons. (A, B, D, and E), in situ hybrizization using Xisl-1 (A and D) and N-tubulin (B and E) probes on embryos injected on the right sides with the islMO. (C and F) TUNEL analysis showing the apoptotic cells (dark blue color) in embryos injected on the right sides with the islMO. In (D–F), the embryos were treated with the apoptosis inhibitor M50054 before harvest. (A) The medial stripe of Xisl-1 expressing cells is lost on the islMO injected side (white arrowheads), but not on the control side (black arrowheads). (B) The medial and middle stripes of N-tubulin expressing cells are lost on the islMO injected side (white arrowheads and arrows), but not on the control side (black arrowheads and arrows). (C) TUNEL assay indicate that extensive cell apoptosis occurred in the ventral neural plate on the islMO injected side (right) but not on the control side (left). (D–F) Inhibition of apoptosis by M50054 can largely rescue the loss of ventral neurons induced by islMO. All embryos are viewed from dorsal, with anterior end at the top. The injected sides are labeled by expression of co-injected β-gal (stained in weak red, the right sides of all figures). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Fig. 4.
Over-expression Xisl-1 promotes cell proliferation. (A) Embryos injected with Xisl-1 mRNA on one sides bend to the uninjected sides. The injected sides (black arrow heads) are indicated by the expression of the co-injected β-gal (stained in light blue). (B) Embryos injected on one sides with β-gal alone develop normally. The axial structures are slightly thicker on the injected sides (white arrowheads) than on the uninjected sides at this stage, but the differences are small and recover at later stages (not shown). (C and D) Transverse sections of embryos injected on one side with Isl-1 (C) or β-gal alone (D), showing that the neural tube and somite on the Isl-1 injected side are markedly thicker than on the control side, while the differences are small in β-gal injected embryos. (E and F) Whole-mount staining with phosphorylated histone 3 antibody of embryos injected on one side with Xisl-1 mRNA (E) or β-gal alone (F), dorsal views of the trunk regions are shown. More positive cells (dark purple) are detected in the Isl-1 injected side than in the control side (E). No significant difference was observed in the β-gal alone injected embryos (F). (G and H) Xisl-1 promotes Xstat3 expression in Xenopus embryos. The injected embryos were harvested at neurula stages and checked for GFP expression (E). Those with clear GFP expression on the left or right side were selected into two groups for in situ hybridizations with Xstat3 (F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
