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The GTPase activating proteins (GAPs) specific for Rho family GTPases serve as either negative regulators or downstream effectors of Rho-GTPases through their ability to interact with specific Rho proteins and additional cytosolic factors. Here we report cloning and expression of a novel Xenopus Rho-GTPase activating protein cDNA designated as XrGAP. It encodes a protein of 1902 amino acids that contains a PDZ domain, a PH motif and a Rho-GAP domain. Sequence analysis shows that XrGAP protein shares 60% overall amino acids identity with a human Rho-GTPase activating protein (ARHGAP10) that is highly expressed in the brain. Northern blot and RT-PCR analysis show that a single XrGAP transcript is maternally expressed and gradually decreases afterwards. Whole-mount in situ hybridization shows that maternal XrGAP transcripts are specifically expressed in the animal hemisphere of the eggs and blastula stage embryos. During gastrulation, XrGAP is detected in the prospective neuroectoderm and the mesoderm. At neurula stages, the expression is in the neural regions including the neural folds, eye analgen and neural crest cells, and subsequently restricted to notochord, developing brain, spinal cord, eye, branchial arches, and otic vesicle at tailbud stages.
Fig. 3. In situ hybridization of XrGAP. (A) Laternal view of a blastula (stage 8) embryo showing enhanced signal in the animal hemisphere. (B) Vegetal view of a stage 10 embryo. (C) Lateral view of a sagitally sectioned stage 10.5 embryo. XrGAP transcripts are detected in the prospective neuroectoderm and the mesoderm with more enrichment at the dorsal marginal zone. An arrowhead indicates dorsal lip of blastopore. XrGAP expression is also detected in the dorsal involuting cells (arrow). Anterior view (D) and dorsal view (E) of mid-neurula (stage 16) embryo. XrGAP is expressed at the neural folds from anterior to caudal end, but absent from the dorsal midline. Note the expression in eye anlange (two arrowheads) and neural folds (two arrows). (F) Anterior view of late neurula (stage 20) embryo. XrGAP expression remains restricted to eye anlagen (two arrowheads) and antero-ventral portions of neural regions as a crescent-shape. (G) Lateral view of a sagittally sectioned stage 23 embryo. Expression is observed in notochord but absent from other mesodermal tissues. (H) Anterior view of a stage 25 embryo. XrGAP expression remains restricted in the developing brain and eye vesicle (two arrowheads). (I) Lateral view of a tailbud (stage 30) embryo. XrGAP expression persists in the developing brain, spinal cord, eye, branchial arches, and otic vesicle. ba; branchial arches, cg, cement gland; nc, notochord; ey, eye; ov, otic vesicle.
Fig. 2. The temporal expression pattern of XrGAP during early Xenopus embryogenesis. (A) RT-PCR analysis shows that XrGAP transcripts are present maternally and gradually reduced after the onset of gastrulation. Ornithine decarboxylase (ODC) was used as a loading control. The -RT lane is the control on stage 26 whole embryo RNA in the absence of reverse transcriptase. (B) Northern blot analysis reveals a single XrGAP transcript of the predicted size (about 7 kb). Positions of transcripts are indicated by an arrow. Ethidium bromide-stained 28S and 18S ribosomal RNAs are shown in the lower panel as a loading control.
arhgap21 (Rho GTPase activating protein 21) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8.
arhgap21 (Rho GTPase activating protein 21 ) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10.5, dorsal right, anterior up.
arhgap21 (Rho GTPase activating protein 21 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
arhgap21 (Rho GTPase activating protein 21 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
arhgap21 (Rho GTPase activating protein 21 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, sagittally sectioned at NF stage 23, lateral view, anteriorleft, dorsal up.
arhgap21(Rho GTPase activating protein 21 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25. anterio-lateral view, dorsal up.
arhgap21 (Rho GTPase activating protein 21 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
