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Figure 1. Protein Structure and Gene Expression of xHtrA1(A) SDS-PAGE of supernatant from transfected and 35S-methionine/cysteine-labeled HEK293T cells. The 50 kDa band corresponds to the secreted xHtrA1 protein.(B) Xenopus HtrA1 and related proteins. SP, signal peptide; IB, insulin-like growth factor binding domain; K, kazal-type serine protease inhibitor domain; Trypsin, trypsin-like serine protease domain; PDZ, PDZ domain. The numbers indicate the percentages of amino acid identity with the corresponding domains in xHtrA1. The GenBank accession numbers are Xenopus laevis HtrA1 (EF490997), human HtrA1/L56 (NP 002766), mouse HtrA1 (AAH13516), Drosophila serine protease (NP 650366), and E. coli HtrA/DegP (X12457).(C–J) Whole-mount in situ hybridization of xHtrA1 (C–F) and FGF8 (G–J) at midgastrula (C and G, vegetal view), early neurula (D and H, dorsal view), midneurula (E and I, anterior view), and tail bud stage (F and J, head). The arrowhead indicates the midbrain-hindbrain boundary. anp, anterior neural plate; ba, branchial arch; bpl, blastopore lip; fb, forebrain; nf, neural folds; pm, posterior mesoderm.(K) RT-PCR of animal cap explants at stage 18 after injection of FGF8 (450 pg), FGF4 (3 pg), or BMP4 mRNA (200 pg) at the 4-cell stage. H4, histone H4 for normalization.
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Figure 2. xHtrA1 Blocks Head Formation and Induces Ectopic Tail-like StructuresEmbryos were injected into the animal pole of one blastomere at the 4-cell stage with 80 pg of the indicated mRNAs.(A) Uninjected tail bud embryo (dorsal view, anterior to the left).(B) xHtrA1 mRNA induces a secondary axis (arrowhead) fused with the primary body axis at the anterior end.(C) Uninjected embryo at tadpole stage.(D) xHtrA1 induces loss of head tissue and ectopic tail-like structures.(E) Histological section of xHtrA1-injected tadpole embryo. sc, spinal cord; nc, notochord; so, somites.(F and G) Ectopic N-tubulin expression in xHtrA1-induced tail outgrowth (arrowhead).(H–K) Ectopic expression of Shh and MyoD in early neurulae (dorsal view) adjacent to cells coinjected with xHtrA1 and nuclear lacZ mRNA as lineage tracer (red nuclei).(L and L′) After a single ventral injection of xHtrA1 and GFP mRNA, injected green cells populate the ectopic tail structures (arrowhead).(M and M′) Coinjection of xHtrA1 and GFP mRNA into one dorsal blastomere induces an ectopic tail outgrowth distant to injected cells.(N–Q) While xHtrA1 mRNA causes anencephaly and ectopic tail formation, xHtrA1δTrypsin or xHtrA1 (S307A) mRNA are without effects.(R) Western blot analysis of lysates from mRNA-injected embryos at stage 9 probed for xHtrA1. GAPDH is a loading control.Ectopic axial structures were induced in B, 21/88; D, 61/203; G, 15/18; I, 3/15; K, 6/19; L, 3/12; M, 4/37; O, 110/124 (head reduction), 37/124 (ectopic outgrowth), P, 0/406, and Q, 0/280 embryos.
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Figure 3. Effects of xHtrA1 on Early Pattern FormationWhole-mount in situ hybridization of embryos in lateral (A–D), anterior (E–H), ventral (I–L), and dorsal view (M–T). Embryos were animally injected into one blastomere at the 4-cell stage with nlacZ mRNA as control or xHtrA1 mRNA (each 80 pg).(A–D) In early gastrula embryos, xHtrA1 mRNA blocks expression of Otx2 in the anterior ectoderm and expands Xbra expression into the animal hemisphere.(E–L) At the neurula stage, xHtrA1 mRNA blocks expression of BF1, Rx2a, Krox20, and Nkx2.5. Sizzled expression is reduced anteriorly but significantly expanded at the posterior end of the embryo.(M–T) xHtrA1 mRNA causes expansion of Sox2 and downregulation of Slug and Cytokeratin expression. N-tubulin expression is ectopically induced.Indicated effects on gene expression were observed in B, 45/45; D, 20/23; F, 22/23; H, 16/20 (Rx2a) and 6/20 (Krox20); J, 28/36; L, 31/36; N, 62/67; P, 9/9; R, 19/30; and T, 34/49 embryos.
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Figure 4. Knockdown of xHtrA1 Promotes Anterior Development and Impairs Mesoderm and Neuronal DifferentiationMorpholino oligonucleotides (MOs) were injected at the 2-cell stage (8 pmol per embryo); xHtrA1 or nontargeted xHtrA1* mRNA (see Experimental Procedures) were injected into each blastomere at the 4-cell stage (160 pg total).(A) xHtrA1-MO targets both Xenopus laevis HtrA1 pseudoalleles.(B) xHtrA1-MO, but not control-MO, inhibits translation of injected xHtrA1 mRNA at stage 9. xHtrA1-MO does not affect the protein synthesis of xHtrA1* mRNA.(C) xHtrA1-MO blocks endogenous xHtrA1 protein expression at stage 12.(D–G) xHtrA1-MO leads to enlarged head, decreased eye and shortened tail formation after injection into the animal pole and margin of each blastomere.(H and I) A single marginal injection of xHtrA1-MO blocks Xbra expression.(J–M) Animally injected xHtrA1-MO broadens the expression domains of BF1 and En2, and restricts Rx2a expression.(N and O) A single animal injection of xHtrA1-MO reduces N-tubulin expression.(P–S) Marginally injected xHtrA1-MO depletes the posterior Sizzled expression domain (arrowhead). xHtrA1* mRNA reverts the effect of xHtrA1-MO.Indicated phenotypes were observed in D, 92/92; E, 60/60; F, 18/18; G, 51/51; H, 17/19; I, 22/28; J, 20/20; K, 35/57; L, 10/10; M, 42/43; N, 15/19; O, 16/18; Q, 41/74; R, 19/19; and S, 15/15 embryos.
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Figure 5. Cooperation between xHtrA1 and FGF Signals(A) Ectopic tail-like outgrowth (arrowhead) induced after a single ventral injection of pCS2-FGF4 DNA (4 pg) at the 4-cell stage.(B) Expansion of Xbra expression induced by FGF4 mRNA (6 pg).(C) Ectopic N-tubulin expression induced by FGF8 mRNA (5 pg).(D–G) Animal caps extracted from stage 8 embryos and cultured until stage 25. Caps extend only slightly when injected with xHtrA1 (360 pg) or FGF4 (3.2 pg) but show robust elongation when injected with both mRNAs together.(H) RT-PCR of animal cap explants at stage 11.(I and J) Animal injection of XFD mRNA at the 4-cell stage (110 pg) causes enlargement of head structures.(K and L) XFD restores head development and blocks ectopic tail outgrowth induced by xHtrA1 mRNA (80 pg).(M and N) A single marginal injection of XFD mRNA (220 pg) disrupts Xbra expression.(O and P) XFD inhibits ectopic Xbra expression by xHtrA1 mRNA (80 pg).(Q and R) A single animal injection of DnFGFR4a mRNA (110 pg) suppresses N-tubulin expression. nlacZ mRNA was coinjected as lineage tracer (red nuclei).(S and T) DnFGFR4a blocksN-tubulin expression induced by xHtrA1 mRNA (80 pg) on the injected side.Frequency of embryos with the indicated phenotype was A, 13/66; B, 23/23; C, 118/120; J, 54/68; K, 68/80 (head reduction) and 32/80 (ectopic tail), L, 13/16; N, 38/47; O, 13/13; P, 16/18; R, 18/18; S, 10/12; and T, 10/10.
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Figure 6. xHtrA1 Activates Long-Range FGF Signaling(A) Western blot analysis of stage 14 embryos. Injection of xHtrA1 mRNA (80 pg) induces diphosphorylated ERK (dpERK). xHtrA1-MO (8 pmol), but not control-MO, reduces endogenous dpERK.(B–D) Whole-mount immunohistochemistry of early gastrula embryos probed for diphosphorylated ERK (dpERK). xHtrA1 mRNA (80 pg) induces ectopic dpERK in the animal cap. XFD mRNA (220 pg) blocks endogenous and xHtrA1-induced dpERK expression.(E–J) Whole-mount in situ hybridization of early neurula embryos (lateral view). xHtrA1 mRNA (80 pg) induces robust expression of FGF8 and FGF4. The effects of xHtrA1 on FGF8 and FGF4 are reverted by coinjection of DnFGFR4a (110 pg) and XFD mRNA (220 pg), respectively.(K) Reciprocal stimulation of xHtrA1 and FGF gene activities.(L) Experimental design of animal cap conjugate assay. The doses of mRNAs injected into each blastomere at the 4-cell stage were FGF4 (16 pg) and xHtrA1 (130 pg).(M) Control animal cap conjugates with nlacZ mRNA-injected inducer cap (blue), and uninjected responder cap remain round and do not express Xbra.(N) Slight elongation and narrow Xbra expression (red) in the responder cap at the interface to the inducer cap injected with FGF4 mRNA.(O and P) xHtrA1 mRNA alone has only little effect but, when coinjected with FGF4 into the inducer cap, induces significant elongation and robust Xbra expression in the responder cap.(Q and R) FGF4 in the inducer cap juxtaposed to an xHtrA1-injected responder cap causes significant elongation of the conjugate and Xbra expression far away from the signaling source.Frequency of specimen with the indicated phenotype was B, 11/11; C, 21/21, D, 26/26; E, 10/10; F, 26/27; G, 25/27; H, 16/16; I, 12/12; J, 10/12; M, 16/16; N, 12/12; O, 10/12; P, 15/18; Q, 10/10; and R, 22/25.
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Figure 8. Heparan Sulfate and Dermatan Sulfate Induce Posteriorization, Mesoderm, and Neuronal Differentiation in an FGF-Dependent Manner(A–E) Heparan sulfate (HS, 30 ng), dermatan sulfate (DS, 3000 ng), and the chondroitin sulfates (CSE and CSA, each 3000 ng) were injected into the blastocoel at stage 8. Note the loss or reduction of head structures by HS, DS, and CSE, and the induction of an ectopic tail-like outgrowth by DS (arrowhead).(A′–E′) Injection of XFD mRNA at the 4-cell stage (110 pg/embryo) reverts the posteriorizing effects of HS, DS and CSE.(F–J, F′–J′) HS and DS injected at stage 6.5 significantly expand Xbra expression, which is blocked by XFD mRNA (220 pg).(K–O, K′–O′) HS, DS, CSE, and CSA injected at stage 8 expand N-tubulin expression, which is blocked by DnFGFR4a mRNA (110 pg).(P–S, P′–S′) Blastocoelic injection at stage 6.5 of Heparitinase (16.5 microunits per embryo) or Chondroitinase B (25 microunits) affect axial development and Xbra expression. Heat-inactivated enzymes have no effects.(P′′–S′′) Heparitinase or Chondroitinase B injected at stage 8 influences N-tubulin expression.Frequency of embryos with the indicated phenotype was B, 63/63; C, 98/110 (anencephaly) and 20/110 (ectopic tail); D, 47/47; E, 53/53; B′, 11/11; C′, 7/7; D′, 5/6; E′, 9/13; G, 29/30; H, 48/51; I, 10/20 (slight expansion); J, 9/9; G′, 46/46; H′, 41/41; I′, 50/50; J′, 43/43; L, 63/63; M, 52/59 (slight expansion); N, 35/36 (slight expansion); O, 24/33 (slight expansion); L′, 22/22; M′, 13/13; N′, 35/35; O′, 16/17; P, 45/45; Q, 92/96; R, 48/48; S, 58/62; P′, 19/19; Q′, 27/27; R′, 29/29; S′, 26/31; P′′, 19/19; Q′′, 20/26; R′′, 35/35; and S′′, 27/27.(T) Model for the stimulation of long-range FGF signaling by the secreted serine protease xHtrA1. xHtrA1 cleaves the protein moiety of proteoglycans and releases biologically active FGF bound to glycosaminoglycan (GAG). The FGF-GAG complex activates the FGF receptor on cells distant to its site of origin.
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htra1 (HtrA serine peptidase 1) gene expression in bisected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, blastoporal view, dorsal up.
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htrA1 (HtrA serine peptidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior up.
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htra1 (HtrA serine peptidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
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htra1 (HtrA serine peptidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, head and anterior trunk region only, lateral view, anterior left, dorsal up.
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