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The tetraspanin family of four-pass transmembrane proteins has been implicated in fundamental biological processes, including cell adhesion, migration, and proliferation. Tetraspanins interact with various transmembrane proteins, establishing a network of large multimolecular complexes that allows specific lateral secondary interactions. Here we report the identification and functional characterization of Xenopus Tetraspanin-1 (xTspan-1). At gastrula and neurula, xTspan-1 is expressed in the dorsal ectoderm and neural plate, respectively, and in the hatching gland, cement gland, and posterior neural tube at tailbud stages. The expression of xTspan-1 in the early embryo is negatively regulated by bone morphogenetic protein (BMP) and stimulated by Notch signals. Microinjection of xTspan-1 mRNA interfered with gastrulation movements and reduced ectodermal cell adhesion in a cadherin-dependent manner. Morpholino knock-down of endogenous xTspan-1 protein revealed a requirement of xTspan-1 for gastrulation movements and primary neurogenesis. Our data suggest that xTspan-1 could act as a molecular link between BMP signalling and the regulation of cellular interactions that are required for gastrulation movements and neural differentiation in the early Xenopus embryo.
Fig. 1 Expression of xTspan-1 during early Xenopus development.
(A) Maternal transcripts were detected throughout the embryo until late blastula. (B) At gastrula, xTspan-1 transcripts were detected in a dorsoventral gradient. (B0) Paraffin sections of the embryo shown in B. The expression was restricted to the dorsal surface ectoderm and not detectable in the involuting mesendoderm (insert). (C) At neurula stages, xTspan-1 was expressed in the closing neural plate
and the dorsal blastopore slit. (D) Paraffin section of the embryo shown in (C), demonstrating the specific expression of xTspan-1 in the superficialneuroectoderm. (E) Reverse transcriptase polymerase chain reaction analysis of dorsal embryonic explants of neurula stage embryos. The superficialneuroectoderm (E) had been separated from the mesoderm (M). E-cadherin is specifically expressed in the ectoderm, esr-6e in superficialneuroectoderm, a-actin and myoD were used as mesodermal marker genes and odc served as a
loading control. (F) At tailbud stages, the expression levels decreases and xTspan-1 transcripts were found in the hatching gland, cement gland, the posterior neural tube. (G) Paraffin sections revealed an additional expression domain in the posterior perianal region.
Fig. 2 The expression of xTspan-1 is regulated by bone morphogenetic protein (BMP) and Notch signals. (A) Expression of xTspan-1 in a dorsoventral gradient at gastrula stages. (B) Treatment of embryos with 120 mM LiCl at the 32-cell stage induced uniform expression of xTspan-1 throughout the marginal zone. (C) Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of ventral
marginal zone (VMZ; lane 3) and dorsal marginal zone (DMZ; lanes 2, 4) explants at stage 12. Microinjection of DMZ with BMP7/OP-1 mRNA (lane 4) reduced the expression of xTspan-1 to the levels detected in VMZ (lane 3). The ventral markers sizzled and vent-1 and the neural marker sox-2 were used as control for BMP signalling activity. (D) RT-PCR analysis of animal cap explants at stage 12 microinjected with a dominant-negative BMP-receptor (tBR). The expression of xTspan-1 was increased in tBR-injected explants, similar to the neural marker genes otx-2 and sox-2, whereas the ventral marker msx-1 was reduced. The panmesodermal marker xbra was used as a control for mesoderm contamination, odc served as a loading control. (E) Whole-mount in situ analysis of xTspan-1 expression at late gastrula in embryos microinjected with 100 pg Notch-ICD mRNA (NICD) together with 100 pg LacZ mRNA. (E0) Paraffin section of the embryo shown in (E).
Fig. 3 Microinjection of xTspan-1 mRNA interferes with gastrulation movements and ectodermal cell adhesion. (A) Embryos microinjected with 25 or 100 pg xTspan-1 mRNA developed with a shortened body axis and anterior truncations. (B) At gastrula, blastopore closure was delayed in embryos microinjected with
100 pg xTspan-1 mRNA. (C–E) Whole-mount in situ analysis for chordin (C), myoD (D) and rx2a, krox-20 and shh (E) of uninjected control embryos (left) and embryos microinjected with 100 pg xTspan-1 mRNA (right) at late gastrula (C, D) and neurula (E) stages. (F) Microinjection of 1ng xTspan-1 mRNA into the animal
region of four-cell embryos induced lesions in the ectoderm at gastrula (right). This effect could be rescued by co-injection of 1 ng Ccadherin mRNA (G). (H) Reverse transcriptase polymerase chain reaction analysis of the animal cap explants at gastrula. The expression of E-cadherin was not reduced and expression of mesodermal (xbra), endodermal (mixer) marker genes and the
expression of the direct Wnt target gene xnr-3 were not induced by ectopic xTspan-1 protein. (I–K) Dissociation-reaggregation assays using cells from control animal cap explants and animal cap explants microinjected with mRNAs coding for green fluorescent protein (GFP) (I), GFP and xTspan-1 (J) or GFP, xTspan-1, and C-cadherin (K). In two independent experiments at least three aggregates were analyzed and displayed similar distributions of the microinjected cells in the aggregates.
Fig. 4 Xenopus Tspan-1 is required for gastrulation movements. (A) In vitro transcription translation of xTspan-1 cDNA that included (lanes 1–3) or lacked (lanes 4, 5) the morpholino target sequence in the 50-untranslated region (50
-UTR), in the presence of the xTspan1-specific antisense morpholino oligomer (Tspan-MO) or control morpholino oligomer. (B) The translation of a xTspan–GFP fusion protein (xTspan–GFP) was inhibited by co-injection of the TspanMO, dependent on the presence of the morpholino target sequence (150-UTR). An unrelated control morpholino had no effect. (C) Embryos microinjected with Tspan-MO at the four-cell stage displayed a delay in epiboly and developed with a shortened and bended body axis. (D) Whole-mount in situ analysis of neurula
stage embryos microinjected with control morpholino (co-MO), Tspan-MO or Tspan-MO together with 5 pg of xTspan-1 mRNA lacking the 50-UTR sequence. (E) Elongation of ectodermal explants after treatment with 2 ng/ml recombinant activin was inhibited by Tspan-MO and could be rescued by co-injection of 5 pg xTspan-1 mRNA. The induction of dorsal mesodermal or ectodermal marker genes by activin (lane 3) was not affected by the Tspan-MO (lane 4) or the co-injection of Tspan-MO and xTspan-1 mRNA (lane 5). Lane 1 shows the results obtained from whole embryos and lane 2 the results from untreated control explants.
Fig. 5 xTspan-1 is required for primary neurogenesis and modulates N-tubulin and xdelta-1 expression. (A) Whole-mount in situ hybridization of stage 13 embryos for sox-2 and xdelta-1. (B) Embryos injected radially with the Tspan-1 morpholino display a significant reduction of xdelta-1 staining, whereas sox-2 expression was unaffected. (C–H) Whole-mount in situ hybridization of stage 13 embryos for N-tubulin (C–E) and xdelta-1 (F–H) microinjected into dorsal animal blastomeres at the eight- to 16-cell stage with mRNA coding for LacZ together with xTspan-1 morpholino (C, F), xTspan-1 morpholino together with 5 pg xTspan-1 mRNA
lacking the morpholino target sequence (D, G) and 40 pg mRNA coding for xTspan-1 (E, H). The injected cells visualized by b-galactosidase can be seen on the right side of the embryos. (A–E) Two embryos for each staining and (F–H) a dorsal and an anterior view of the same embryo is shown.
