Urry LA et al. (1998), Thrombospondins in early Xenopus embryos: dynam...

XB-ART-15100

Dev Dyn 1998 Apr 01;2114:390-407. doi: 10.1002/(SICI)1097-0177(199804)211:4<390::AID-AJA10>3.0.CO;2-8.

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Thrombospondins in early Xenopus embryos: dynamic patterns of expression suggest diverse roles in nervous system, notochord, and muscle development.

Urry LA , Whittaker CA , Duquette M , Lawler J , DeSimone DW .

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The thrombospondins (TSPs) are a family of extracellular matrix (ECM) glycoproteins that modulate many cell behaviors including adhesion, migration, and proliferation. Here we report the molecular cloning of the Xenopus homologs of TSP-1 and TSP-3, and the developmental patterns of expression of Xenopus TSP-1, TSP-3, and TSP-4 mRNAs. Xenopus TSP-1 and TSP-3 protein sequences each share approximately 80% amino acid identity with their mammalian counterparts. TSP-1 mRNAs are detectable at low levels in fertilized eggs indicating that this TSP is a maternally deposited transcript. Zygotic expression of TSP-1, TSP-3, and TSP-4 begins at the end of gastrulation and transcripts encoding each protein accumulate through the tadpole stages of development. Whole mount in situ hybridizations reveal that each TSP mRNA is localized in the embryo with distinct, developmentally regulated patterns of expression. TSP-1 mRNAs are detected in a wide range of tissues including the floor plate of the neural tube, epidermis, somites, notochord and, most notably, alternating rhombomeres. Transcripts encoding TSP-3 are expressed in the notochord, floor plate, sensorial layer of the epidermis and sensory epithelia. TSP-4 mRNAs are restricted to somitic mesoderm and skeletal muscle. These data suggest that the TSPs represent a functionally diverse family of ECM proteins with tissue-specific functions during embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: thbs1 thbs3 thbs4

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	Fig. 6.Spatial pattern of expression of TSP-1 mRNA. Whole mount in situ hybridization, using digoxygenin-labeled antisense TSP-1 probes, was performed on albino Xenopus embryos. Experiments with TSP-1 probe in the sense orientation were regularly performed to control for specificity and these embryos lacked detectable staining (data not shown). Each embryo is oriented with anterior to the right. Embryos were staged according to Nieuwkoop and Faber (1994). Stage 19 neurula, (A) dorsal view (B) side view. Arrows indicate single line of cells along the dorsal midline. Arrowheads indicate bilaterally symmetrical staining in the hindbrain. C: Stage 26 tailbud, side view. Arrow indicates floor of forebrain. Arrowhead indicates endoderm ventrally apposed to the notohord. D: Stage 28 tailbud, side view. Arrowhead indicates epidermal staining surrounding the cement gland. E: Stage 30 tailbud, side view. Arrowhead indicates hindbrain staining in alternating rhombomeres. White arrow points to expression in the caudal somites. Black arrow indicates notochord staining. F: Stage 32 tailbud, side view. Note the increased staining within caudal somites and rhombomeres in comparison to (E). G: Stage 34 tailbud, side view. Arrow indicates intense expression in the pre-chordal plate. Arrowhead points to expression in anterior epidermis. H: Stage 40 tadpole, side view. Arrow indicates staining in the heart. Note the lack of somitic expression.
	Fig. 7. The spatial pattern of TSP-1 mRNA expression in tailbud stage embryos. Whole mount in situ hybridization was performed on albino Xenopus embryos fixed at 2 hour intervals from stage 24 through stage 34. Pictured embryos are stage 34 and oriented with anterior to the right and dorsal to the top unless otherwise noted. (C) are views of different optical sections of the embryo in (A), (F) are views of the embryo in (B). (C) and (F) are views from the dorsal side (horizontal) and (D, E, G, H) are different focal planes when viewed from the front (transverse). White arrows indicate staining concentrated in the anterior and posterior limits of r2 and white arrowheads point to the notochord. Black arrowheads indicate expression in the anterior region of r1. Large arrows in (C) and (G) point to nonspecific background staining in the brain ventricle and gut respectively. (D) and (G) are optical transverse sections at the level of the anterior region of r1. (E) is an optical transverse section at the level of r2 and (H) at the level of r4. I: Stage 33 tailbud, frontal view with dorsal oriented to the top of the page. Arrowhead indicates central region of facial epidermis that is devoid of TSP-1 staining. J: Side view of a stage 34 tailbud, arrow indicates intense expression in the pre-chordal plate. Note the strong staining in the region of the brain in contact with the pre-chordal plate. Arrowhead indicates facial epidermal staining. K: Stage 34 tailbud with the focal plane at the level of r4. Arrowhead indicates intense expression within the otic vesicle. L: Stage 33 tailbud, side view in trunk region. Arrowhead indicates TSP-1 mRNA expressing cells at the location of a major blood vessel.
	Fig. 8. Spatial patterns of expression of TSP-3 mRNA. Whole mount in situ hybridization was performed on albino Xenopus embryos with digoxygenin-UTP labelled antisense TSP-3 probes. All embryos are oriented so that anterior is to the right except for F and G which are transverse sections with dorsal oriented to the top. A: Side view of a stage 13 neurula, arrowhead indicates staining along the dorsal midline corre- sponding to the notochord. B: Similar stage embryo to that in (A) viewed from the top. Arrowhead indicates the notochord. C: Stage 19 neurula, side view. Arrow points at the notochord and the arrowhead indicates the floor-plate of the neural tube. D: Stage 26 tailbud, side view. Stained notochord and floor plate are visible along the length of the embryo. Arrowhead indicates sensorial layer of the ectoderm. E: Side view of a stage 33 tailbud. F: Optical transverse section in the trunk of a stage 30 tailbud. Arrowhead indicates sensorial layer of ectoderm, arrow points to the floor-plate of the neural tube. G: Transverse paraffin section of a whole-mount stained, stage 33 tailbud, through the hindbrain. Arrowhead points to expression in the roof plate, which was cracked during process- ing. H: Stage 35 tailbud, side view. White arrowhead indicates the intensely stained floor plate of the brain. I: Top view of the head of a stage 40 tadpole. Arrowhead indicates auditory epithelium. Black arrow points to olfactory epithelium, slightly out of the plane of focus and white arrow indicates staining in the epidermis.
	Fig. 9. Spatial pattern of expression of TSP-4 mRNA. Whole mount in situ hybridization was performed on albino Xenopus embryos using digoxygenin-UTP labelled antisense TSP-4 probes. All embryos are oriented with anterior to the right. A: Dorsal view of a stage 14 neurula, showing staining in the paraxial mesoderm, which flanks the unstained notochord (arrowhead). B: Stage 19 neurula, dorsal view. C: Side view of a stage 26 tailbud, arrowhead indicates segmented somite. D: Top view of a tailbud of similar stage to embryo in (C). E: Stage 33 tailbud, side view. Arrow indicates progressive loss of staining from the somites. The faint blue staining in the gut is non-specific background. F: Stage 35 tadpole, side view. Arrowhead points to intense staining in the dorsal region of the somite and the arrow indicates peripheral skeletal muscle. G: Stage 39 tadpole, side view. Arrow indicates skeletal muscle staining. H: High magnification side view of the head of the embryo in (G). Arrow indicates stained muscles surrounding the eye.
	thbs1 (thrombospondin 1) gene expression in Xenopus laevis embryos, NF stage 19, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	thbs1 (thrombospondin 1) gene expression in Xenopus laevis embryos, NF stage 26, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	thbs1 (thrombospondin 1) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	thbs1 (thrombospondin 1) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	thbs1 (thrombospondin 1) gene expression in Xenopus laevis embryos, NF stage 34, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	thbs1 (thrombospondin 1) gene expression in Xenopus laevis embryo, NF stage 40, assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	thbs3 (thrombospondin 3) gene expression in Xenopus laevis bisected embryo, assayed via in situ hybridization, NF stage 13, lateral view, anterior right, dorsal up.
	thbs3 (thrombospondin 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, lateral view, anterior right, dorsal up.
	thbs3 (thrombospondin 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior right, dorsal up.

	thbs3 (thrombospondin 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, dorsal view, head region, anterior left, dorsal up.