Cao Y et al. (2001), XCL-2 is a novel m-type calpain and disrupts mo...

XB-ART-8331

Dev Growth Differ 2001 Oct 01;435:563-71. doi: 10.1046/j.1440-169x.2001.00592.x.

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XCL-2 is a novel m-type calpain and disrupts morphogenetic movements during embryogenesis in Xenopus laevis.

Cao Y , Zhao H , Grunz H .

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We identified a novel cDNA, XCL-2, encoding an m-type calpain, a calcium-dependent intracellular protease. This protein has all characteristic structures and active sites of canonical calpains. Zygotic transcription of the gene was first detected at stage 10. It is expressed exclusively in the ventral circumblastoporal collar and the mesoderm-free zone at the most anterior tip of neural fold in late gastrulae and neurulae. In later stages, expression is only found in cement gland and proctodeum. It is also expressed in a tissue-specific manner. In adult tissues, various levels of expression were detected in brain, eye, heart, intestine, kidney, lung, stomach and testis, but not in liver, muscle, nerve, ovary, skin and spleen. Overexpression of wild-type XCL-2 suggests that this gene is involved in gastrulation movement and convergent extension during gastrulation and neurulation. Overexpression of a dominant-negative mutant caused a phenotype morphologically similar to, but histologically different from, that caused by overexpression of wild-type XCL-2. The mutant phenotype can be rescued by injection of wild-type XCL-2. These data suggest that XCL-2 plays an important role in convergent extension movements during embryogenesis in Xenopus laevis.

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Species referenced: Xenopus laevis
Genes referenced: capn8.1 chrd fh myod1 ncl odc1 otx2 sox3 tbxt uqcc6 ventx1.2

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	Fig. 1. Nucleotide and deduced amino acid sequence of XCL-2. The four domains are separated with asterisks, the tentative active sites Cys105, His262 and Asn286 in proteolytic domain are in bold and the four calciumbinding EF-hand motifs in the Ca2+ domain are underlined.
	Fig. 2. Schematic structure of XCL-2, nCL-2 and the ubiquitous m-type calpain large subunit (mCL). The four domains are indicated with I, II, III, and IV, respectively; percentages represent the identities between XCL-2, nCL-2 and ubiquitous calpains in human or chicken; and C, H and N are the three active sites in Fig. 1. aa, amino acid.
	Fig. 3. Spatial expression of XCL-2. (A) A posterior view of stage 12.5 embryos. Dorsal is up. Signal appears at a restricted area ventral to blastopore. (B,C) Anterior and posterior views, respectively, of the same stage 15 embryo. Dorsal is up. In (B), expression is detected at the most anterior of neural plate; in (C) it shows the same expression zone as in (A). (D) Ventral view of a late neurula. Anterior is left. It shows the localized expression in cement gland and proctodeum. (E) Ventral view of an early tail-bud. Anterior is left. The expression in cement gland and proctodeum becomes even stronger. (F) In late tail-bud stages, expression is only detected in the cement gland. (G) A sagittal section through a stage 15 embryo, confirming that the expression in (B) and (C) is only localized to the mesoderm-free zone and the ventral circumblastoporal collar. The expression zone is highlighted with boxes. (H,I) Sagittal sections through the head and tail of a stage 23 embryo, respectively, confirming that the expression in (E) is only localized to cement gland and proctodeum. a, anterior view; bl, blastopore; cg, cement gland; mfz, mesoderm-free zone; nf, neural fold; nt, neural tube; p, posterior view; pc, prosencephalon; pr, proctoduem; pv, procencephalic ventricle; vbc, ventral circumblastoporal collar.
	Fig. 4. Expression patterns of XCL-2 detected with RT–PCR. (A) Temporal expression during embryogenesis. Numbers indicate developmental stages. Expression was detected from the onset of gastrulation (stage 10), then its level continuously increased until the middle of tail-bud stage. After approximately stage 30, the expression level decreased gradually. (B) Expression in adult tissues. Various levels of expression of XCL-2 were detected in adult tissues such as brain, eye, heart, intestine, kidney, lung, stomach and testis, but not in liver, muscle, nerve, ovary, skin and spleen. ODC was used as loading control in both cases. br, brain; ey, eye; he, heart; in, intestine; ki, kidney; li, liver; lu, lung; mu, muscle; ne, nerve; ov, ovary; sk, skin; sp., spleen; st, stomach; te, testis; RT–, reverse transcription without reverse transcriptase.
	Fig. 5. Overexpression of XCL-2 disrupts gastrulation movements and causes a significant phenotype. mRNA was injected into two dorsal blastomeres at the 4-cell stage. (A) Control embryos at stage 11 with normal blastopores. (B) Injected embryos showing blastopores of a much larger size than in the controls. (C) Control normal swimming tadpoles at stage 38. (D) Injected embryos at the same stage as in (C). The embryos show significant microcephaly (top) and even no discernible head structure (bottom), and shortening of the anterior–posterior axis. The foreheads shift dorsalwards. The endoderm of the embryos can be clearly seen externally and bifurcate tails are formed. (E,F) Transversal sections through the otic vesicle (E) and the trunk region behind the otic vesicle (F) of an injected embryo at stage 30, showing bifurcation and further shifting of notochord towards one side of the body. cc, central canal; no, notochord; ov, otic vesicle; rc, rhombencephalon; rv, rombencephalic ventricle; sp, spinal cord.
	Fig. 6. Overexpression of XCL-2 affects the localization of mesodermal or neural markers and disrupts convergent extension movements. mRNA was injected into two dorsal blastomeres at the 4-cell stage. (A) Expression of Xbra is localized to a ring surrounding the blastopore of control midgastrulae. In injected embryos the expression is absent at dorsal blastopore lip (D). (B) In control embryos of midgastrulae, Xvent-1 is expressed in ventral and lateral mesoderm, while overexpression of XCL-2 renders the expression shift more ventralwards (E). (C) Xotx2 in control midgastrulae is expressed in the dorsal blastopre lip, but in injected embryos its expression extends laterally (F). (G) Chordin (chd) is expressed at the dorsal blastopore lip of control midgastrulae and (M) at the midline in late gastrulae, but in injected embryos its expression extends posteriorly and laterally (J,P), showing disruption of convergent extension movement. (H) XMyoD is expressed in the paraxial mesoderm in late gastrulae and (N) mid-neurulae, but in injected embryos at equivalent stages (K,Q) it is expressed in a distance away from the midline, showing that paraxial mesoderm does not converge to the midline. (I,O) The neural plate is shown by the neural marker Xsox3 in normal late gastrulae and mid-neurulae, respectively, and also undergoes convergent extension. However, overexpression of XCL-2 causes the posterior expression area of Xsox3 to shift away from the midline, a further indication for the failure of convergence of the posterior neural plate towards the midline (L,R).
	Fig. 7. Dominant-negative mutant C105S also disrupts gastrulation movement and results in a significant phenotype, which can be rescued by co-injection with wild-type mRNA. mRNA of C105S or XCL-2 was injected into two ventral blastomeres at the 4-cell stage. (A) Control embryos at stage 11. (B) Injected embryos at an equivalent stage showing larger-sized blastopores. (C) Control embryos at stage 39. (D) Injected embryos at an equivalent stage showing nearly normal head structure, externally visible endoderm and bifurcate tail (top) or even no head in an extreme case (bottom). (F,G) Transversal sections through the otic vesicle (F) and the trunk region behind the otic vesicle (G) of an embryo injected with C105S at stage 30, showing ventralward movement of otic vesicles, flattening, ventralward bending, bifurcation and further shifting of notochord toward each side of the body (compare Fig. 5E,F). (E) Embryos rescued by co-injecting C105S and XCL-2 mRNA. cc, central canal; no, notochord; ov, otic vesicle; rc, rhombencephalon; rv, rombencephalic ventricle; sp, spinal cord.