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First published online 5 January 2005
doi: 10.1242/dev.01599

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XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFß proteins in Xenopus development

¹ Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
² Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97201, USA
³ Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
⁴ Wellcome Trust/Cancer Research Gurdon UK Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (91K): [in a new window]   Fig. 1. Alignment of Xenopus PACE4 (XPACE4) protein sequence with human PACE4 splice isoform AII (hPACE4AII) and rat PACE4 (rPACE4). The clustal alignment algorithm of MacVector software is used to align the translated protein sequences and BOXSHADE is used for display. The table shows the percent identity and similarity of these proteins. XPACE4 is cloned as described in the Materials and methods procedures. Accession Numbers: hPACE4AII, NP 612192; rPACE4, NP 037131; XPACE4, AY836768.

View larger version (49K): [in a new window]   Fig. 2. Spatiotemporal expression pattern of XPACE4. (A) Real-time RT-PCR analysis of the wild-type embryos at the indicated stages shows that XPACE4 mRNA is abundant maternally and its level increases pre-MBT (stage 6 and 8) and gradually declines after gastrulation (stage 10 and 12.5). (B) The comparison of oligo dT (dT) versus random hexamer (R6) primed cDNA shows the increase in XPACE4 levels pre-MBT is due to polyadenylation of the maternal mRNA. (C) Whole-mount in situ hybridization shows that XPACE4 is localized during early oogenesis. Inset shows XPACE4 mRNA localization in the mitochondrial cloud of stage 1 oocytes. No signal is detected with the sense probe (S). (D) XPACE4 is localized to the vegetal hemisphere of full-grown oocytes. (E,F) Hemisected stage 10 (E) and stage 11 (F) embryos show XPACE4 mRNA remains localized to the endodermal precursors during gastrulation (dorsal side on the right). (G) At stage 30, XPACE4 is detected in a group of cells in the endoderm after the embryos are bleached and cleared. (H) At stage 35/36, XPACE4 is detected in the olfactory bulb, the brain and the notochord. The lower embryo is the sense control. Inset shows the notochord in transverse sections. S, sense control.

View larger version (29K): [in a new window]   Fig. 3. Depletion of XPACE4 using an antisense oligo approach. (A) Real-time RT-PCR analysis of oocytes (oo) injected with 5-10 ng unmodified antisense oligos shows depletion of maternal XPACE4 mRNA to variable degrees. (B) Phosphorothioate modified AS-5 oligo (AS-5MP) depletes XPACE4 mRNA without affecting the mRNA levels of a closely related maternal convertase Furin (XFurA) as analyzed by real-time RT-PCR. (C) XPACE4 mRNA levels in control and XPACE4-depleted embryos: XPACE4-depleted embryos (AS-5MP+) are generated by the host transfer technique and are compared with sibling uninjected control embryos at blastula (7 and 8) and gastrula (10, 11, 12) stages. Real-time RT-PCR shows that XPACE4 is depleted down to 5% and it does not reach the control levels (AS-5MP-). (D) Antisense oligo depletion of XPACE4 reduces signaling activity of Xnr1 in oocytes. A schematic presentation of the paracrine assay is shown at the top. Animal caps co-cultured with uninjected control oocytes have very low or no detectable levels of organizer genes chordin (Chd), goosecoid (Gsc), mesodermal gene Xbra or endodermal gene XSox17. All of these genes are induced when caps are co-cultured with control oocytes injected with Xnr1 mRNA. Animal caps co-cultured with XPACE4-depleted oocytes injected with Xnr1 mRNA show a reduction in the expression levels of these mRNAs. Sibling whole embryo (WE) is used for the dilution series and the quantification in real-time RT-PCR.

View larger version (66K): [in a new window]   Fig. 4. Depletion of XPACE4 disrupts gastrulation, mesoderm induction and normal development. (A) XPACE4-depleted embryos [P(-)] compared with controls (Un) show delay in gastrulation and rescue by XPACE4 mRNA [P(-)+mRNA]. (B) Gastrulation delay of XPACE4 depletion is rescued by the reintroduction of XPACE4 mRNA. XPACE4 mRNA injected into control embryos (Un+mRNA) speeds up gastrulation and rescues the gastrulation delay of XPACE4-depleted embryos [P(-)+mRNA]. (C) XPACE4-depleted embryos develop patterning defects and anterior abnormalities. Depletion of XPACE4 [P(-)] results in a variety of late phenotypes ranging from ventralization to small heads. These phenotypes can be partially rescued by the reintroduction of XPACE4 mRNA [P(-)+mRNA]. (D) Equatorial explants (Eq explants) of XPACE4-depleted embryos fail to elongate. Explants dissected from control embryos (Un) show normal convergent extension movements and elongation. XPACE4-depleted embryos [P(-)] show severe reduction in elongation; XPACE4 mRNA rescues the elongation defect [P(-)+mRNA]. (E) Analysis of gene expression of equatorial explants from XPACE4-depleted embryos. Explants (Eq explants) dissected from control (AS-5MP-) and XPACE4-depleted (AS-5MP+) embryos at mid-blastula stage are cultured and analyzed together with stage 28 whole embryos (WE). The expression of general mesodermal marker MyoD, dorsal mesoderm marker cardiac actin and neural marker NCAM are all reduced in XPACE4-depleted whole embryos and explants. XPACE4 mRNA rescues the expression of markers. (F) Analysis of gene expression of XPACE4-depleted gastrulae. XPACE4-depleted whole embryos are harvested together with sibling control embryos. The expression of Xbra, chordin (Chd), Xnr1 and Xnr3 is analyzed using real-time RT-PCR. At stage 10.5, XPACE4-depleted embryos (AS-5MP+) show a reduction in the levels of these markers and the reduction is partially rescued by XPACE4 mRNA. The mRNA alone increases marker levels. (For rescue experiments 75-100 pg of XPACE4 mRNA is injected vegetally into both cells at the two-cell stage.)

View larger version (29K): [in a new window]   Fig. 5. Induction of mesoderm is disrupted by XPACE4 depletion. (A) A schematic presentation of the Nieuwkoop Assay details the Materials and methods. The marker expression of animal cap explants (ac) co-cultured with control (Un vm) and XPACE4-depleted vegetal masses [P(-) vm] is analyzed by real-time RT-PCR. Animal caps incubated alone (ac alone) do not express significant levels of mes-endodermal genes. The induction of organizer genes chordin, goosecoid, mesodermal genes Fgf8 and Xbra, and endodermal gene XSox17 are all reduced in caps cultured with XPACE4-depleted vegetal masses [ac P(-) vm] compared with controls (ac Un vm). Uninjected whole embryo (Uninj WE) is used for the dilution series and the quantification. (B) XPACE4-depleted embryos [P(-)+ARE] have reduced ARE-luciferase reporter activity compared with controls (Un+ARE). (C) Western blot analysis of control (UN) and XPACE4-depleted [P(-)] gastrulae show phospho-Smad2 levels are reduced by XPACE4 depletion. Total Smad2 levels are shown as loading control.

View larger version (67K): [in a new window]   Fig. 6. XPACE4 depletion affects the processing of a specific subset of TGFß proteins. (A) Maturation of Xnr2 is reduced in XPACE4-depleted embryos. Western blots from control (Un) and XPACE4-depleted [P(-)] whole embryos and blastocoel fluids overexpressing Xnr2-HA mRNA (200 pg) show the mature form of Xnr2-HA (26 kDa) is reduced in whole embryo homogenates of XPACE4-depleted embryos. In the blastocoel fluid of XPACE4-depleted embryos, a significant reduction in mature Xnr2 level and accumulation of the unprocessed (57 kDa) and an intermediate form (33 kDa) are detected. (B) Maturation defect of Xnr2 in XPACE4-depleted embryos is rescued by injection of XPACE4 mRNA (150 pg). In whole embryos, the level of mature Xnr2 is rescued by XPACE4 mRNA injection. In the blastocoel fluid the unprocessed and intermediate forms are rescued by XPACE4 mRNA injection. (C) Maturation of Xnr1 is reduced in XPACE4-depleted embryos expressing 200 pg of Xnr1-HA mRNA. The mature form of Xnr1-HA (24 kDa) is reduced in whole embryo homogenates of XPACE4-depleted embryos. In the blastocoel fluid, a significant reduction in mature Xnr1 level and accumulation of the unprocessed form (57 kDa) are detected. (D) Maturation of Xnr3 is reduced in XPACE4-depleted embryos expressing 300 pg of Xnr3-HA mRNA. The mature form of Xnr3-HA (22 kDa) is reduced in whole embryo homogenates of XPACE4-depleted embryos. In the blastocoel fluid, a significant reduction in mature Xnr3 level is detected. (E) Maturation of Xnr5 is not affected in XPACE4-depleted embryo lysates or blastocoel fluids. Xnr5-HA mRNA (100 pg) is injected. (F) Maturation of ActivinB is not affected in XPACE4-depleted embryos. A very low level of mature ActivinB-HA (15 kDa) is more concentrated in the blastocoel fluid both in controls and XPACE4-depleted whole embryos. However, there is no significant change in levels of mature (15 kDa) or unprocessed forms (54 kDa) of ActivinB-HA. ActivinB-HA (400 pg) mRNA is injected. (G) Maturation of Derrière is not affected in XPACE4-depleted embryos. There is no significant change in levels of mature (21 kDa) or unprocessed (50 kDa) forms of Derrière-HA, either in whole embryo homogenates or in the blastocoel fluid of XPACE4-depleted embryos. Derrière-HA mRNA (400 pg) is injected. (H) Maturation of Vg1 is reduced in XPACE4-depleted embryos. The unprocessed Vg1 as a doublet (46 and 44 kDa), and intermediate form (35 kDa) are easily detected in whole embryo lysates and the blastocoel fluid. The mature form (18 kDa) is reduced in the blastocoel fluid of XPACE4-depleted embryos. Vg1-HA mRNA (600 pg) is injected. (-tubulin is used as a loading control.)