XB-ART-54948Stem Cells September 1, 2018; 36 (9): 1368-1379.
Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.
Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.
PubMed ID: 29781215
Article link: Stem Cells
Genes referenced: crabp1 cyp26a1 egr2 en2 gata4 gata6 hoxa10 hoxb1 hoxb9 hoxd4 igf2 klf9 lif mef2c myod1 nes npat otx2 pax3 pax6 pou5f3.1 rarb sox17a sox17b.1 sox17b.2 sp1 stra6 stra8 strap twist1 vim vim.2
GO keywords: stem cell differentiation
Morpholinos: strap MO1
Article Images: [+] show captions
|Figure 1. Strap depletion has no effect on the morphology or viability of ESCs. A. Schematic representation of the strategy used to generate Strap heterozygous mice. The Strap locus (from exons 1 to 10) is shown. Exons are shown as filled boxes and cutting positions of the two zinc finger nucleases (ZFNs) are indicated. Details of targeted se‐ quences are shown in boxes. B. Left panel, phase contrast image showing the morphology of isolated E3.5 blastocysts from Strap+/‐ intercrossings. Right panel, representative images of blastocyst‐derived Strap WT and KO ESCs cultured on MEFs. Image magnification x 40; scale bar = 50 um. C. PCR genotyping of ESCs established from various blasto‐ cysts. D. Fluorescence‐activated cell sorting (FACS) analysis of WT and KO ESC lines. The percentages of cell popula‐ tions at each cell cycle phase are displayed. E. Growth curve of two independent WT and KO ESC lines. A total of 1X104 ESCs were seeded in 12‐well plates and cell numbers were counted every other day. F. Alkaline phosphatase staining of WT and KO ESC cells. G. Western blots of pluripotent markers and STRAP in the indicated ESCs. β‐Actin was used as a loading control.|
|Figure 2. Strap‐deficient EBs are unable to differentiate into primary germ layers. A. Phase‐contrast images of EB morphology during EB formation from Strap WT and KO ESCs at the indicated time points. Scale bar = 600 um. B. The cell cycle distribution of D10 EBs from WT and KO ESC lines was analyzed by FACS. The percentages of cell populations at each cell cycle phase are labeled. C‐E. qPCR analyses of lineage‐specific gene’ expression at Day 0, Day 4, Day 8 and Day 12 during EB formation of two independent WT and KO ESC lines. Error bars represent mean ±s.d. Each experi‐ ment was replicated at least three times.|
|Figure 6. STRAP knockdown influences anterior‐posterior neural patterning and impairs the body axis and eye de‐ velopment in early Xenopus embryogenesis. A. Strap transcript was detected in the entire blastopore (a), early neu‐ rulation (b), late neurula stage (c), and the tailbud and the tadpole stages (d and e, respectively). B. 50 ng Strap‐MO was injected into the animal regions of two‐cell‐stage embryos and resulted in defects in the tadpole stage. C. Strap RNA (0.20 ng) was injected alone or with 25 ng Strap‐MO unilaterally into two‐cell‐ stage embryos. The embryos were stained with the β‐Gal substrate Red‐Gal. D. 50 ng Strap‐MO was injected unilaterally into two‐cell‐ stage embryos. The embryos were examined at neurula stages 17‐18 by ISH for neural markers. E. 50 ng Strap‐MO was injected into the animal regions of two‐cell‐stage embryos. Animal caps were dissected at blastula stage 9 and cultured until neu‐ rula stages 19‐20. RNA was then extracted for RT‐qPCR analysis of marker expression. Bars represent mean ±s.d., n=3. * P<0.05, **P<0.01, when compared with the control.|
|strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view.|
|strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior left.|
|strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.|
|strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
|strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.|