Nejigane S et al. (2013), Hippo signaling components, Mst1 and Mst2, act ...

XB-ART-48046

Int J Dev Biol 2013 Jan 01;575:407-14. doi: 10.1387/ijdb.130010st.

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Hippo signaling components, Mst1 and Mst2, act as a switch between self-renewal and differentiation in Xenopus hematopoietic and endothelial progenitors.

Nejigane S , Takahashi S , Haramoto Y , Michiue T , Asashima M .

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Hippo signaling is a conserved pathway that regulates cell proliferation and organ size control. Mst1 and Mst2 were identified as homologs of hippo and as core kinases of the Hippo pathway in mammals. Here, we have characterized the role of Mst1 and Mst2 during Xenopus primitive hematopoiesis. We showed that Mst1 and Mst2 were strongly expressed in the Xenopus ventral blood island, where primitive hematopoiesis is initiated. Loss-of-function analysis of Mst1/2 revealed morphogenetic defects, including short axis, smaller eyes and abnormal epidermis, and decreased phosphorylation of Yap. Mst1/2 morphants did not exhibit any change in the expression of hematopoietic and endothelial progenitor markers in early hematopoietic development. In addition, we have shown that such progenitor markers were continuously expressed through to the late hematopoietic development stage. As a result, the expression of erythroid, myeloid and endothelial differentiation markers were decreased in Mst1/2 morphants. Our results indicate that Mst1/2 act as a differentiation switch in Xenopus hematopoietc and endothelial progenitors.

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Species referenced: Xenopus
Genes referenced: actl6a aplnr cebpa cfd etv2 fli1 gata1 gata2 hba1 hbg1 lmo2 mpo mst1 odc1 runx1 spib stk3 stk4 tal1 tek yap1
???displayArticle.antibodies??? Cytoskeleton Ab1 HA Ab2 O-dianisidine Lectin 1 Yap1 Ab2 Yap1 Ab3
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	Fig. 1. Expression pattern of Mst1 and Mst2 in Xenopus tropicalis development. (A) RT-PCR analysis revealed the temporal expression patterns of Mst1 and Mst2. RNA was extracted from Xenopus tropicalis embryos at the stages indicated above each lane. ODC were used as internal controls. ODC(-), RT-PCR without reverse transcriptase in ODC reaction. e, unfertilized egg. (B-P) Whole-mount in situ hybridization analysis of Mst1 and Mst2. (B-H) Localized expression pattern of Mst1. (B�) The anterior view corresponding to (B). (C�-F�) The ventral view corresponding to C-F. (I) Hemi-sectioning of a stage-18 embryo revealed detailed localization of Mst1 in the aVBI. (I�) High magnification view of the boxed region in (I). (J-M,O,P) Localized expression pattern of Mst2. (J�-M�,O�) The ventral view corresponding (J-M, P,O). (N) Transverse sectioning of a stage-28 embryo revealed detailed localization of Mst2 in the pVBI. (N�) High magnification view of the boxed region in (N). Scale bars, 100 +m. View direction is indicated in the upper right of each panel. Developmental stage is indicated in the lower right. Abbreviations: ba, branchial arch; du, duodenum; in, intestine.
	Fig. 2. Phenotype of Mst1 and Mst2 morphants. (A) Mst1 and Mst2 translational blocking MOs (Mst1t MO and Mst2t MO) targeting Mst1 and Mst2 genes. The target regions of Mst1 and Mst2 are indicated in black, the sequences of the Mst1t MO and Mst2t MO are shown in blue, and the start codons are shown in red. (B) Western blotting analysis showing the translation level of HA-tagged Mst1 or Mst2 protein in Xenopus laevis embryo injected with standard control MO (20 ng), Mst1t MO (20 ng), or Mst2t MO (20 ng). Mst1t and Mst2t MO completely blocked the translations of Mst1-HA and Mst2-HA mRNA (500 pg), but did not block the translations of Mst1mt-HA and Mst2mt-HA mRNA (500 pg), which were mutated in the MO complementary sequences, respectively. Actin was used as an internal control. (C) Genomic structure including exon 1(1) and exon 2 (2) of Mst2. The primer pair designed to amplify in the case of the proper splicing of Mst2 mRNA. The pre-mRNAs of Mst2 generated by splice blocking have a termination codon immediately following exon 1. The primer pair is indicated by arrows. The target region of Mst2 pre-mRNA is indicated in black. The sequence of the Mst2 splicing blocking MO (Mst2s MO) is indicated in blue. The start codon is indicated in red. Exon is shown in upper case, and the intron in lower case. Amino acid sequences are also indicated. Asterisk corresponds to stop codon. (D) RT-PCR analysis of Mst2 mRNA in MO-injected embryos. Embryos were injected with Mst2 MO (MO, 5 ng) or standard control MO (SC, 5 ng), in the animal pole of both blastomeres at the 2-cell stage, and cultured for RT-PCR until various stages as indicated above each lane. In Mst2 MO-injected embryos, the mature mRNA was not detected from stage 14. Splicing of Mst2 pre-mRNA was not affected by standard control MO injection. UN, uninjected embryo. EF1_ was used as an internal control. EF1_(-), RT-PCR without reverse transcriptase in EF1_ reaction. (E) Western blotting analysis of the Mst1/2 knockdown embryos. Embryos were injected with Mst1t MO and Mst2s MO (MO, Mst1t+2s MO 2.5 ng/2.5 ng) or standard control MO (SC, 5 ng), in the animal pole of both blastomeres at the 2-cell stage, and cultured until stage 30. Phospho-Yap levels were described in the bottom of the panel. UN, uninjected embryo. (F) Phenotypes of Mst morphant. Mst1 or Mst2 MO injection induced a morphological abnormality in a concentration-dependent manner. Discriminations of phenotypes were indicated using the following abbreviations. Mst1 morphant; Type I, short axis. Type II, short axis and abnormal epidermis. Mst2 morphant, Type A; smaller eyes and bent axis. Type B; smaller eyes, bent axis and abnormal epidermis. Type C, smaller eyes abnormal episermis and short axis. Mst1/2 morphant; Type 1+2, short axis and smaller eyes. Arrowheads indicate overgrowth of epidermis. (G) Frequency of observed Mst morphant. Injected MOs are described at the left side of the panel. Numbers of injected embryos are indicated on the right side. Distinctions of phenotypes were indicated using the following colors. blue; Normal, lightgreen; Type I, green; Type II, light-pink; Type A, pink; Type B, red, Type C, purple; Type 1+2, yellow, Gastrulation defect.
	Fig. 3. Effects of Mst1/2 MO injection on embryonic erythrocyte formation. (A,A�) Erythrocyte marker, _-globin, was normally expressed in the VBI of embryos injected with standard control MO (Std. MO, 5 ng). (B-D�) Knockdown of Mst1 (Mst1t MO, 5 ng), Mst2 (Mst2s MO, 5 ng) and Mst1/2 (Mst1t+2s MO, 2.5/2.5 ng) decreased _-globin expression in VBI. (E) At the later stage, _-globin was detected in circulating erythrocytes in the vein of standard control MO-injected embryos. (F-H) Knockdown of Mst1, Mst2 and Mst1/2 inhibited the circulation of erythrocytes. (I) O-dianisidine staining also visualized circulating erythrocytes in controls, (K-L) whereas erythrocytes were hardly visualized in Mst1, Mst2 and Mst1/2 morphants. (M) RT-qPCR analysis revealed that knockdown of Mst1/2 decreased `-globin expression. Expression levels were normalized relative to ODC. Values represent means + SD of three independent experiments, P < 0.05, *P < 0.01.
	Fig. 4. Effects of Mst1/2 MO injection on embryonic myeloblast formation. (A) Mpo is detected in myeloblast in standard control MO-injected embryo. (B-D) Knockdown of Mst1, Mst2 and Mst1/2 decreased myeloblast numbers, and (E) Mpo-positive cell number was synergistically decreased by Mst1/2 MO injection. (F) RT-qPCR analysis revealed that the knockdown of Mst1/2 decreased expression of Mpo, a myeloblast marker. Values represent means + SD of three independent experiments, P < 0.05, *P < 0.01.
	Fig. 5. Effects of Mst1/2 MO injection on embryonic vascular plexus formation. WISH analysis of embryonic vascular markers, Ami, Tie2, and Msr. (A,E,I) The embryonic vascular network had started forming at stage 32 in standard control MO-injected embryo, (B-D,F-H,J-L) and this was disrupted by Mst1, Mst2, and Mst1/2 knockdown. Sample numbers are indicated in the upper right of each panel. Developmental stage is indicated in the lower left. Assessed marker is indicated in the lower right all panels.
	Fig. 6. Effects of Mst1/2 MO injection on hematopoietic and endothelial progenitors. (A-H) At the early stage, no differences in the expression of hematopoietic and endothelial progenitor markers, Scl, Runx1, Lmo2, and Gata2, were observed between standard control MO (Std. MO, 5 ng)- and Mst1/2 MO (Mst1t+2s MO, 2.5/2.5 ng)-injected embryos. (I-N) In the Mst1/2 morphant, expressions of early myeloid progenitor marker, C/EBP_ and Spib were maintained in aVBI, but reduction of migrating differentiated myeloid cells was observed. Arrow indicate migrating myeloid cells from aVBI. (O-V) At the later stage, following the differentiation of hematopoietic cells, Scl, Runx1, Lmo2, and Gata2 expression gradually decreased in the VBI of standard control MO-injected embryos, whereas these markers were continuously expressed at similar levels in the Mst1/2 knockdown embryo. (W,X) The pre-erythroblast marker, Gata1, was markedly decreased by Mst1/2 knockdown. (Y-b) The expression patterns of the endothelial progenitor marker, Fli1 and Etv2, were disrupted by depletion of Mst1/2. Sample numbers are indicated in the upper right of each panel, and developmental stage is indicated in the lower left. Assessed marker is indicated in the lower right of each panel.
	Supplemental Fig. S2. Loss of function phenotype was rescued by Mst1 or Mst2 DNA injection. (A-E) After injection of 5 ng of standard control MO (Std. MO) or Mst1/2 MO (Mst1t+2s MO) into the animal pole at the 2-cell stage, embryos were injected with 20 pg of LacZ (pENL), Mst1 (pE-Mst1), or Mst2 (pE-Mst2) DNA into the vegetal pole. Coinjection of Mst1/2 MO and Mst1, or Mst2 DNA partially rescued morphological abnormality as compared to phenotype of Mst1/2 morphant (see also Fig.2). Injected embryos were cultured until stage 38. Injected MO is indicated in the upper right of each panel. Injected DNA is indicated in the lower right. (F) RT-qPCR analysis of b-globin in embryos coinjected with MO and DNA. The coinjection of standard control MO (Std. MO) and LacZ DNA did not alter the expression level of b-globin. In Mst1/2 MO and LacZ DNA-injected embryos, expression of b-globin was decreased (see also Fig. 3). As compared to LacZ DNA, Mst1, or Mst2 DNA slightly rescued decreased b-globin expression of Mst1/2 morphant. Expression levels were normalized relative to ODC. Values represent means + SEM of three independent experiments. (G) The embryonic vascular network was formed in standard control MO and LacZ DNA-coinjected embryo. (H) Impaired vascular formation in the Mst1/2 knockdown embryo could not be rescued by injection of LacZ DNA. (D,E) Coinjection of Mst1/2 MO and Mst1, or Mst2 DNA partially rescued abnormal vascular formation as compared to phenotype of Mst1/2 MO and LacZ DNA-coinjected embryos. Sample numbers are indicated in the upper right of each panel. Developmental stage is indicated in the lower left. Injected MO and DNA are indicated in the lower right all panels.
	stk4 (serine/threonine kinase 4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, lateral view, anterior left, dorsal up.
	stk4 (serine/threonine kinase 4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
	stk3 (serine/threonine kinase 3) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	stk3 (serine/threonine kinase 3) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 45, lateral view, anterior left, dorsal up.