Gessert S et al. (2011), The spatio-temporal expression of ProSAP/shank ...

XB-ART-42998

Dev Dyn 2011 Jun 01;2406:1528-36. doi: 10.1002/dvdy.22613.

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The spatio-temporal expression of ProSAP/shank family members and their interaction partner LAPSER1 during Xenopus laevis development.

Gessert S , Schmeisser MJ , Tao S , Boeckers TM , Kühl M .

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Members of the ProSAP/Shank family are important scaffolding proteins of the postsynaptic density (PSD). We investigated for the first time the expression of the three family members named Shank1, ProSAP1/Shank2, and ProSAP2/Shank3 during Xenopus laevis development. Shank1 is expressed in the neural tube, the retina, and the cranial ganglions. In contrast, ProSAP1/Shank2 transcripts could be visualized in the otic vesicle, the pronephros, the liver, the neural tube, and the retina. ProSAP2/Shank3 could be detected in the cardiovascular network, the neural tube, the pronephros, and the retina. Furthermore, we showed that LAPSER1 interacts with all three ProSAP/Shank family members in Xenopus embryos and co-localizes with ProSAP/Shank in a cell-based assay. In Xenopus, LAPSER1 is expressed in somites, brain, proctodeum, pronephros, and in some cranial ganglions. Thus, we suggest that members of the ProSAP/Shank family and LAPSER1 not only play a role in PSD formation and plasticity, but also during embryonic development.

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Species referenced: Xenopus laevis
Genes referenced: ank1 gad1.2 lzts2 myc psd shank1 shank2 shank3 tbx2

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	Figure 1. The spatio-temporal expression of Shank1 in Xenopus development. A–D: Lateral views of Xenopus embryos. A: At stage 28, Shank1 is expressed in the neural tube (green arrowhead). Dotted lines indicate the level of sections shown in E–H. B: Shank1 transcripts are detected in the neural tube (green arrowhead) and the trigeminal ganglion (gV) at stage 32. Dotted lines indicate the level of sections shown in I–K. C: At stage 35, Shank1 can be visualized in the neural tube (green arrowhead), the trigeminal ganglion (gV), as well as the anteriordorsal lateral line ganglion (gAD). Dotted line indicates the level of section in L. D: Shank1 is expressed in the posterior neural tube (green arrowhead), fore- and midbrain (blue and yellow arrowhead). In addition, Shank1 is detected in the trigeminal ganglion (gV) as well as the anteriordorsal lateral line ganglion (gAD) to this time point of development. E–H: Transverse sections through an embryo at stage 28. E: Shank1 mRNA molecules are visualized in the trigeminal ganglion (gV) and very weakly in the ventral neural tube (green arrowhead). F: Shank1 is expressed in the ventral neural tube (green arrowhead) and the anteriordorsal lateral line ganglion (gAD). Dotted lines surround the neural tube (nt) and the otic vesicle (ov). G,H: Shank1 transcripts are detected in the posterior ventral neural tube (green arrowhead). I–K: Transverse sections through an embryo at stage 32. I: Shank1 is expressed in the anteriordorsal lateral line ganglion (gAD) and the ventral neural tube (green arrowhead). J: Shank1 transcripts can be visualized in the proximal pronephros (red arrow). K: Shank1 mRNA molecules are located in the distal pronephric tubule (red arrowhead). L: Transverse section at stage 35. Shank1 is expressed in the ventral portion of the midbrain. In addition, Shank1 transcripts are detected intensely in the ganglion cell layer (GCL) and more weakly in the inner nuclear layer of the retina. e, eye; gAD, anteriordorsal lateral line ganglion; GCL, ganglion cell layer; gV, trigeminal ganglion; INL, inner nuclear layer; nt, neural tube; ONL, outer nuclear layer; ov, otic vesicle.Download figure to PowerPoint
	Figure 2. Spatial expression pattern of ProSAP1/Shank2 during Xenopus embryogenesis. A: Lateral view of an embryo at stage 23. ProSAP1/Shank2 is expressed in the otic vesicle (white arrowhead). B: Lateral view of an embryo at stage 25. ProSAP1/Shank2 transcripts are detected in the otic vesicle (white arrowhead), pronephric anlage (black arrow), forebrain (red arrowhead), isthmus (green arrowhead), and hindbrain (white arrow). C: Anterior view of the embryo shown in C. ProSAP1/Shank2 can be visualized in the forebrain (red arrowheads) and the isthmus (green arrowhead). D–G: Lateral views of Xenopus embryos at stages indicated. ProSAP1/Shank2 expression is demonstrated in the otic vesicle (white arrowhead), developing pronephros (black arrow and arrowhead), forebrain (red arrowhead), isthmus (green arrowhead), and hindbrain (white arrow). In addition, ProSAP1/Shank2 transcription in the liver begins (red arrow). Dotted lines indicate the level of transverse sections shown in H-O. H: ProSAP1/Shank2 is expressed in the proximal and intermediate tubule (arrow). I: ProSAP1/Shank2 transcripts are detected in the connecting tubule anlage (arrowhead). J: ProSAP1/Shank2 expression is clearly seen in the isthmus (green arrowhead). K: The transverse sections through an embryo at stage 36 display ProSAP1/Shank2 expression in the ventral interneurons of the hindbrain (white arrow). L: ProSAP1/Shank2 is expressed in the otic vesicle (white arrowhead) as well as the hindbrain (white arrow) at stage 36. M: ProSAP1/Shank2 transcripts can be visualized in the proximal and intermediate pronephric tubules (black arrow). N: At stage 40, ProSAP1/Shank2 is expressed in the retina of the eye (blue arrowhead). O: The transverse section shows that ProSAP1/Shank2 transcripts are strongly detectable in the liver (red arrow).Download figure to PowerPoint
	Figure 3. ProSAP2/Shank3 expression during Xenopus embryonic development. A: Lateral view of an embryo at stage 27. ProSAP2/Shank3 is expressed in the proximal tubule (pt) anlage of the pronephros. B: At stage 32, ProSAP2/Shank3 is predominantly expressed in the cardiovascular system. C: Ventral view. ProSAP2/Shank3 transcripts can be visualized in the ventral aorta (va) and the endocardium (ec). D, E: Lateral views of Xenopus embryos at stage 34 and 38. ProSAP3/Shank3 mRNA is localized in the cardiovascular system as indicated. F: Ventral view of an embryo at stage 38. ProSAP2/Shank3 is expressed in the ventral aorta (va) and the vitelline veins (vv). G–O: Transverse sections through Xenopus embryos at different stages as indicated. G: ProSAP2/Shank3 mRNA molecules are stained in the ventral interneurons of the neural tube (green arrowhead) as well as the developing head arteries (ha). H: ProSAP2/Shank3 is expressed in the endocardium (ec) of the heart. I: At stage 32, ProSAP2/Shank3transcripts can be visualized in the vitelline veins (vv). J: ProSAP3/Shank3 is expressed in the ventral interneurons of the neural tube (green arrowhead) and in the proximal tubule (pt) of the pronephros. K: At stage 32, ProSAP2/Shank3 transcripts are detected in the posterior cardinal veins (pcv) and in the media-lateral portion of the neural tube, the marginal and subventricular zones (green arrowhead). L: ProSAP2/Shank3 is expressed in the isthmus (yellow arrowhead) and the ventral-lateral neural tube. M: ProSAP3/Shank3 transcripts are detectable in the ventral interneurons of the midbrain (green arrowhead), the retina of the eye (white arrowhead), and the ventral aorta (va). N,O: ProSAP2/Shank3 is expressed in the medio-lateral neural tube (green arrowhead), the proximal tubule (N; pt) and connecting tubule (O; ct) of the pronephros, the vascular vitelline network (black arrowheads; vvn), as well as the hypochord (red arrowhead). The notochord is surrounded by a dotted line. aa, aortic arch arteries; acv, anterior cardinal vein; cg, cement gland; ct, collecting tubule of the pronephros; dc, Duct of Cuvier; e, eye; ec, endocardium; ha, head arteries; ia, intersomitic arteries; pcv, posterior cardinal vein; pt, proximal tubule of the pronephros; va, ventral aorta; vv, vitelline veins; vvn, vascular vitelline veins.Download figure to PowerPoint
	Figure 4. The interaction of LAPSER1 and all members of the ProSAP/Shank family is conserved among species. A: GFP-LAPSER1 and Myc-Shank1A co-localize within clustered and filamentous structures in COS-7 cells. B: Punctate, vesicular structures mark sites where GFP-LAPSER1 and Myc-ProSAP1/Shank2 co-localize with each other in COS-7 cells. C: GFP-LAPSER1 and Myc-ProSAP2/Shank3 are physically attached to each other within prominent clusters in COS-7 cells. D: LAPSER1 can be detected in whole Xenopus laevis embryos (St. 33) using a rat anti-LAPSER1 antibody. LAPSER1 protein is enriched after immunoprecipitation with the rat anti-LAPSER1 antibody from a whole embryo lysate. Immunoprecipitation without an antibody (beads only) served as negative control. Whole embryo lane reflects 1/10 of the IP input. E: Endogenous ProSAP1/Shank2, ProSAP2/Shank3, and Shank1 are precipitated from Xenopus whole embryo lysate (St. 31–35) and LAPSER1 is detected in all precipitates by Western blot analysis. Immunoprecipitation using an unrelated IgG antibody against GFP (IgG) or without any antibody (beads only) serve as negative controls.Download figure to PowerPoint
	Figure 5. Spatio-temporal expression of LAPSER1 during X. laevis development. A: Ventral view of an embryo at stage 11. LAPSER1 is expressed in the migrating meso-ectoderm (white arrow). B: Dorsal view of an embryo at stage 13. Posterior is to the top. A faint LAPSER1 expression can be seen in the dorsal part of the embryo. C: Dorsal view of an embryo at stage 17, anterior orientated to the right. The blue arrow depicts LAPSER1 expression in the segmenting somites. D: Ventral view of a Xenopus tail. LAPSER1 transcripts can be detected in the proctodeum (green arrow). E: Anterior view of an embryo at stage 23. LAPSER1 can be visualized in the forebrain (red arrowheads). F–J: Lateral views of Xenopus embryo at indicated stages. F: LAPSER1 expression is shown in the somites that undergo segmentation (blue arrow). G: LAPSER1 is expressed in the isthmus (green arrowhead). Dotted lines indicate level of sections shown in L–Q. H: At stage 32, LAPSER1 is expressed in the posterior somites that undergo segmentation (blue arrow), the segmented somites (blue arrowhead), the pronephros (black arrow and arrowhead), and the trigeminal ganglion (gV). I: LAPSER1 is clearly expressed in some cranial ganglions as indicated. In addition, LAPSER1 transcripts are found in the proximal part of the pronephros (arrow) and slightly in the collecting tubule (arowhead). Dotted lines indicate level of sections shown in R and S. J: At stage 40, LAPSER1 expression is detected in the head mesenchyme (white arrowhead). K–M, R,S: Horizontal sections. N–Q: Transverse sections. K: LAPSER1 is strongly expressed in the somites undergoing segmentation. L: LAPSER1 transcripts can be detected in the segmenting and rotating somites (blue arrows). M: LAPSER1 is slightly expressed in the somites (blue arrow) and the trigeminal ganglion (gV). N: LAPSER1 can be visualized in the trigeminal ganglion (gV). O: LAPSER1 is expressed in the somites (blue arrowhead), the hypochord (yellow arrowhead), and the proximal pronephros anlage (black arrow). P: LAPSER1 is detectable in the forming collecting tubule of the pronephros. Q: LAPSER1 transcripts in the somites (arrow). R: LAPSER1 is expressed in the proximal tubule (arrow). S: LAPSER1 mRNA can be localized in diverse cranial ganglions as indicated. e, eye; egVII, facial epibranchial ganglion; egIX, glossopharyngeal epibranchial ganglion; epXI, first vagal epibranchial ganglion; gV, trigeminal ganglion; gVPL, cells that contribute to the vagal and posterior lateral line ganglion.Download figure to PowerPoint

	shank1 ( SH3 and multiple ankyrin repeat domains 1 ) gene expression in Xenopus laevis embryos, NF stage 35, as assayed by in situ hybridization. Lateral view: anterior right, dorsal up.
	shank2( SH3 and multiple ankyrin repeat domains 2 ) gene expression in Xenopus laevis embryos, NF stage 31, as assayed by in situ hybridization. Lateral view: anterior right, dorsal up. Copyright Wiley-Liss , 2011 Image published in: Gessert S et al. (2011)
	shank2( SH3 and multiple ankyrin repeat domains 2 ) gene expression in Xenopus laevis embryos, NF stage 33, as assayed by in situ hybridization. Lateral view: anterior right, dorsal up. Copyright Wiley-Liss , 2011 Image published in: Gessert S et al. (2011)
	shank2( SH3 and multiple ankyrin repeat domains 2 ) gene expression in Xenopus laevis embryos, NF stage 36, as assayed by in situ hybridization. Lateral view: anterior right, dorsal up. Copyright Wiley-Liss , 2011 Image published in: Gessert S et al. (2011)
	shank3( SH3 and multiple ankyrin repeat domains 3 ) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization. Lateral view: anterior right, dorsal up. note although expression is attributed to tropicalis, this is a laevis embryo