Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration
- Vertebrate Developmental Biology Laboratory, Department of Laboratory Medicine, Lund Stem Cell Center, University of Lund, Sweden
- iPSC Laboratory for CNS Disease Modeling, Department of Experimental Medical Science, Lund Stem Cell Center, Strategic Research Area MultiPark, Lund University, Sweden
Figures
Figure 1 with 1 supplement
SerpinE2 and HtrA1 are expressed in the neural crest.
Xenopus embryos were analyzed by whole-mount in situ hybridization. (A–C) Anterior view of embryos at stage 17. The brackets point to pre-migratory NC cells on each side of the neural plate. The …
Figure 1—figure supplement 1
Twist, SerpinE2, and HtrA1 gene expression in sections of tailbud stage embryos after whole-mount in situ hybridization.
(A) Schematic of embryo at stage 26. Labeled in violet are the Twist-positive NC cells in the mandibular arch (1), hyoid arch (2), anterior branchial arch (3), and posterior branchial arch (4). …
Figure 2 with 1 supplement
Knockdown of SerpinE2 mimics the phenotype of neural excision and inhibits migration of neural crest cells.
(A) Scheme of extirpation. Dorsal view of Xenopus embryo at stage 17, from which NC tissue was removed on both sides. (B, C) Tadpole embryo at stage 40 following NC excision (B) and sibling control …
Figure 2—figure supplement 1
SerpinE2 depletion does not affect the specification of neural crest cells.
(A–H) Anterior view of embryos at stage 18 after whole-mount in situ hybridization. Note that neither control-MO (A, C, E, G) nor SerpinE2-MO (B, D, F, H) affect the expression of the neural crest …
Figure 3
HtrA1 protease inhibits the formation of neural crest (NC)-derived structures and reduces NC migration.
Embryos were injected into all animal blastomeres (A–D) or a single dorsal animal blastomere (E–L) at the eight-cell stage. (A–D) Tadpoles at stage 40. HtrA1 mRNA causes reduction of head tissue, …
Figure 4 with 2 supplements
HtrA1 overexpression and SerpinE2 knockdown decrease cartilaginous elements and craniofacial structures.
Xenopus embryos were injected into four animal blastomeres at the eight-cell stage with a total of 100 pg mRNA and 40 ng morpholino oligonucleotides (MOs). (A–D’) Tadpoles at stages 40/41 after …
Figure 4—figure supplement 1
Overexpression of HtrA1 decreases cartilaginous elements and craniofacial structures.
Embryos were injected into four animal blastomeres at the eight-cell stage with a total of 100 pg mRNA. (A–D) Ventral view of embryos at stage 46 after Alcian Blue staining. Note that HtrA1 mRNA …
Figure 4—figure supplement 2
Knockdown of SerpinE2 reduces craniofacial skeleton formation.
Morpholino oligonucleotides (MOs) were injected into Xenopus embryos at the eight-cell stage. (A–C) Ventral view of tadpoles at stage 46 after Alcian Blue staining. Note that SerpinE2-MO decreases …
Figure 5 with 2 supplements
HtrA1 overexpression and SerpinE2 knockdown inhibit cranial neural crest cell migration and adhesion to fibronectin in vitro.
(A) Scheme of migration experiment. The cranial neural crest was explanted from uninjected or injected embryos at stage 17 and cultured on a fibronectin-covered plastic plate. (B–E’’’) Time-lapse of …
Figure 5—video 1
Collective migration of neural crest (NC) cells in vitro.
Embryos were injected into the animal pole at the four-cell stage with 40 ng standard control morpholino oligonucleotide. The cranial NC was explanted at stage 16/17, plated on fibronectin and …
Figure 5—video 2
Knockdown of SerpinE2 prevents adhesion and migration of neural crest (NC) cells.
Embryos were injected into the animal pole at the four-cell stage with 40 ng SerpinE2 morpholino oligonucleotides. The cranial NC was explanted at stage 16/17, plated on fibronectin and imaged 7 hr …
Figure 6 with 2 supplements
HtrA1 inhibits neural crest (NC) migration as an extracellular protease.
Embryos were injected into a single dorsal animal blastomere at the eight-cell stage. A star labels the injected side.Twist expression demarcates the NC in embryos at stage 20 (B–G; anterior view) …
Figure 6—figure supplement 1
HtrA1 mRNA inhibits neural crest (NC) migration in a concentration-dependent manner.
If not otherwise indicated, embryos were injected with 65 pg HtrA1-derived mRNAs into a single dorsal animal blastomere at the eight-cell stage. Quantification of epithelial-mesenchymal transition …
Figure 6—figure supplement 2
Cytoplasmic HtrA1 causes reduction of αTubulin protein levels in mammalian cells and Xenopus embryos.
(A) Schematic representation of the protein constructs. Indicated are antibody binding sites. RCL, reactive center loop; SP, signal peptide. (B, C) Western blot analysis of cell lysate and culture …
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Figure 6—figure supplement 2—source data 1
Uncropped western blots of HEK293T cell lysates after transfection of Flag-tagged HtrA1, HtrA1ΔSP, SerpinE2, and SerpinE2ΔSP cDNAs.
- https://cdn.elifesciences.org/articles/91864/elife-91864-fig6-figsupp2-data1-v1.pdf
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Figure 6—figure supplement 2—source data 2
Uncropped western blots of HEK293T cell supernatants after transfection of Flag-tagged HtrA1, HtrA1ΔSP, SerpinE2, and SerpinE2ΔSP cDNAs.
- https://cdn.elifesciences.org/articles/91864/elife-91864-fig6-figsupp2-data2-v1.pdf
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Figure 6—figure supplement 2—source data 3
Uncropped gels and western blots of Xenopus embryo lysates after injection of Flag-tagged HtrA1, HtrA1ΔSP, SerpinE2, and SerpinE2ΔSP mRNAs.
- https://cdn.elifesciences.org/articles/91864/elife-91864-fig6-figsupp2-data3-v1.pdf
Figure 7 with 1 supplement
SerpinE2 and HtrA1 interact with Syndecan-4 (Sdc4) in neural crest (NC) cell migration.
mRNAs were injected into one dorsal animal blastomere at the eight-cell stage. Embryos are shown in anterior view (stage 20, injected side labeled with a star, B–G) and lateral view (stage 26, …
Figure 7—figure supplement 1
SerpinE2 and Sdc4 mRNAs partially rescue neural crest (NC) migration defects that are induced by HtrA1 overexpression.
Quantification of epithelial-mesenchymal transition (EMT) and migration of Twist+ NC cells in embryos at stages 20 and 26. Defects were assessed based on a comparison of injected embryos and …
Figure 8 with 1 supplement
SerpinE2 functions in neural crest (NC) cell migration in an HtrA1- and Syndecan-4 (Sdc4)-dependent manner.
mRNAs and morpholino oligonucleotides (MOs, 10 ng) were injected into one dorsal animal blastomere at the eight-cell stage. Embryos are shown in anterior view (stage 20, injected side labeled with a …
Figure 8—figure supplement 1
SerpinE2-MO causes neural crest (NC) migration defects which are rescued by HtrA1-MO and Sdc4 mRNA.
Epithelial-mesenchymal transition (EMT) and NC migration defects were quantified in embryos at stages 22 and 26 after whole-mount in situ hybridization with the NC marker Twist. Defects were …
Figure 9
Model for a proteolytic pathway of SerpinE2 and HtrA1 that regulates collective neural crest migration.
(A) SerpinE2 stimulates collective NC migration by a double-inhibitory mechanism involving the secreted serine protease HtrA1 and its proteolytic substrates Syndecan-4 and fibronectin. (B) Opposing …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293T | TakaraBio | Cat# 632180 | Used for transient transfection and protein production |
| Biological sample (X. laevis, male) | Sperm | Nasco | Cat# LM00715M | Used for in vitro fertilization |
| Biological sample (X. laevis, male) | Sperm | Xenopus 1 | Cat# 5215 | Albino; used for in vitro fertilization |
| Biological sample (X. laevis, female) | Egg | Nasco | Cat# LM00535M | Used for in vitro fertilization |
| Biological sample (X. laevis, female) | Egg | Nasco | Cat# LM00510M | Albino; used for in vitro fertilization |
| Biological sample (X. laevis, female) | Egg | Xenopus 1 | Cat# 4280 | Used for in vitro fertilization |
| Antibody | Anti-flag M2-Peroxidase (HRP) (Mouse monoclonal) | Sigma-Aldrich | Cat# A8592 | WB (1:1000) |
| Antibody | Anti-αTubulin (Mouse monoclonal) | Sigma-Aldrich | Cat# T5168 clone B-5-1-2 | WB (1:1000) |
| Antibody | Anti-βActin (Mouse monoclonal) | Sigma-Aldrich | Cat# A5441 clone AC-15 | WB (1:10,000) |
| Antibody | Anti-HtrA1 (Rabbit polyclonal) | Hou et al., 2007 PMID:17681134 | Immuno-purified GST5057, G22-7 | WB (1:2500) |
| Antibody | Mouse IgG HRP (Goat polyclonal) | R&D Systems | Cat# AF007 | WB (1:2000) |
| Antibody | Rabbit IgG HRP (Goat polyclonal) | R&D Systems | Cat# HAF008 | WB (1:2000) |
| Commercial assay or kit | NucleoBond PC 100, Midi Kit | Macherey-Nagel | Cat# 740573.100 | DNA purification |
| Commercial assay or kit | NucleoSpin Gel and PCR Clean-up, Mini kit | Macherey-Nagel | Cat# 740609.50 | DNA purification |
| Commercial assay or kit | mMessage mMachine SP6 Transcription kit | Invitrogen | Cat# AM1340 | mRNA synthesis |
| Commercial assay or kit | RNeasy Mini Kit | QIAGEN | Cat# 74104 | RNA purification |
| Commercial assay or kit | Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | Cat# 23225 | Protein measurement |
| Chemical compound, drug | Pfu DNA Polymerase | Thermo Fisher Scientific | Cat# EP0572 | DNA synthesis |
| Chemical compound, drug | Lipofectamin 3000 Transfection Reagent | Thermo Fisher Scientific | Cat# L3000001 | Transfection |
| Chemical compound, drug | RIPA Lysis and Extraction Buffer | Thermo Fisher Scientific | Cat# 89901 | Protein purification |
| Chemical compound, drug | Halt Protease and Phosphatase Inhibitor Cocktail | Thermo Fisher Scientific | Cat# 78442 | Protein purification |
| Chemical compound, drug | cOmplete, EDTA-free Protease Inhibitor Cocktail | Roche | Cat# 11873580001 | Protein purification |
| Chemical compound, drug | Bolt Bis-Tris Plus Mini Protein Gels, 4–12% | Thermo Fisher Scientific | Cat# NW04125BOX | Protein electrophoresis |
| Chemical compound, drug | Bolt Sample Reducing Agent (10×) | Thermo Fisher Scientific | Cat# B0009 | Protein electrophoresis |
| Chemical compound, drug | Bolt LDS Sample Buffer (4×) | Thermo Fisher Scientific | Cat# B0007 | Protein electrophoresis |
| Chemical compound, drug | Bolt MES SDS Running Buffer (20×) | Thermo Fisher Scientific | Cat# B0002 | Protein electrophoresis |
| Chemical compound, drug | PageRuler Prestained Protein Ladder, 10–180 kDa | Thermo Fisher Scientific | Cat# 26616 | Protein electrophoresis |
| Chemical compound, drug | Ponceau S Solution | Sigma-Aldrich | Cat# P7170 | Western blotting |
| Chemical compound, drug | Pierce ECL Western Blotting Substrate | Thermo Fisher Scientific | Cat# 32106 | Western blotting |
| Chemical compound, drug | Restore PLUS Western Blot Stripping Buffer | Thermo Fisher Scientific | Cat# 46430 | Western blotting |
| Chemical compound, drug | Human Plasma Fibronectin Purified Protein | Sigma-Aldrich | Cat# FC010 | Neural crest explant culture |
| Chemical compound, drug | Gentamycin Solution | Sigma-Aldrich | Cat# G1272 | Embryo culture |
| Software, algorithm | ImageJ | NIH https://imagej.net/ij/ | RRID:SCR_003070 | Neural crest explant measurement |
| Software, algorithm | AxioVision 4.8 | Zeiss https://www.zeiss.com/ | RRID:SCR_002677 | Neural crest explant imaging |
| Other | 35 mm Dish, No. 0 Coverslip, 10 mm Glass Diameter, uncoated | MatTek | Cat# P35G-0-10C | Neural crest explant culture, time-lapse imaging |
| Other | Vivaspin 2 MWCO 10,000 | Cytiva | Cat# 28932247 | Protein concentration |
| Other | iBlot Transfer Stack, PVDF, mini | Thermo Fisher Scientific | Cat# IB401002 | Western blotting |
