The Secreted Serine Protease xHtrA1 Stimulates LongRange FGF Signaling in the Early Xenopus Embryo

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The Secreted Serine Protease xHtrA1 Stimulates
Long-Range FGF Signaling in the Early Xenopus
Embryo

• Shirui Hou,1,2 Marco Maccarana,3 Tan H. Min,2 Ina
  Strate,1,2 and Edgar M. Pera1,2, 1 Department of
  Developmental Biochemistry, Institute of
  Biochemistry and Cell Biology, Georg August
  University Go ttingen, 37077 Go ttingen,
  Germany 2 Laboratory of Vertebrate Developmental
  Biology, Lund Strategic Research Center for Stem
  Cell Biology and Cell Therapy 3 Department of
  Experimental Medical Science Lund University,
  22184 Lund, Sweden Correspondence
  edgar.pera_at_med.lu.se DOI 10.1016/j.devcel.2007.07.
  001

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xHtrA1 encodes a Protease

• SP signal peptide
• Trypsin serine protease domain
• PDZ protein interaction zone
• IB insulin-like growth factor binding domain
• K kazal-type serine protease inhibitor domain

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Serine Protease

• Serine residue in active site
• Trypsin cleaves peptide bonds following positive
  amino acid residues

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PDZ domain

• oligomeric proteins involved in heat-shock
  response, chaperone function, and apoptosis.
• Regulates protease activity

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xHtrA1 expression resembles FGF8

• unbiased screen for proteins in the early xenopus
  embryos a cDNA clone of xHtrA1 was isolated
• In Situ hybridization for xHtrA1 revealed similar
  pattern to FGF8 expression

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Microinjection of xHtrA1
Stage 12

• xHtrA1 mRNA (made by RT-PCR from cDNA) was
  injected into stage 12 embryos and observed at
  later stages like stage 42, the swimming tadpole
  stage

mRNA
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Injection of xHtrA1 induces secondary tail
structures

• When xHtrA1 mRNA was injected into xenopus early
  stage embryos secondary tail structures developed

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Tail in the Tummy? GFP as a lineage marker

• Co-injection of xHtrA1 mRNA with GFP on the
  dorsal side of the embryo, secondary tail-like
  structures developed on the belly
• Tail structures were devoid of GFP and
  histologically complete

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Because the tail structures were devoid of GFP,
what can be said about xHtrA1?

• xHtrA1 is not a posteriorizing agent
• xHtrA1 is only important for head development
• xHtrA1 has inductive effects at a considerable
  distance
• A and B
• None of the above

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Trypsin mutants reveal the role of xHtrA1s
proteolytic domain
xHtrA1 (S307A) mutantcatalytic serine residue in
position 307 replaced with alanine
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Trypsin mutants have normal phenotypes

• wild-type xHtrA1 anencephaly and secondary tail
  formation
• xHtrA1?trypsin normal development
• xHtrA1 (S307A) normal development
• xHtrA1 ability to induce ectopic tails is
  dependent upon a functional proteolytic domain

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The loss of the phenotype when mutant xHtrA1 was
injected can be attributed to

• Heparan sulfate could not be cleaved from cell
  surface proteoglycans
• FGF could not bind the mutant xHtrA1 protein
• The RTKs are not cleaved to induce long-term
  signaling
• Mutant xHtrA1 cannot travel through the ECM
• The proteoglycan core proteins could not be
  cleaved

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Model
Cleavage of core proteins releases Heparan-FGF
complexes into ECM
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Review

• Injection with xHtrA1 causes loss of head
  structure and ectopic tail growth

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xHtrA1-MO injection enlarged heads, reduced eye
size and shortened tails

• Injection of xHtrA1-MO had an opposite pheontype
  from xHtrA1 injection

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The xHtrA1-MO revealed xHtrA1s roles as

• Restrictive function in head development
• Supportive role in tail outgrowth
• Head development enhancer
• All of the Above
• A and B

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Cooperation between FGF signals and xHtrA1
FGF4 xHtrA1
Uninjected
FGF4
xHtrA1

• FGF4 xHtrA1 dramatic elongation
• FGF4 alone mild mesoderm elongation
• xHtrA1 alone mild mesoderm elongation

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(No Transcript)
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ERK

• Must be diphosphorilated to be activated
• Mek phosphorilates both ser/thr and tyr on ERK

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Analyzing Activation of ERK
Hepes and protease inhibitor cocktail
Embryos were lysed and proteins were
extracted Separated on SDS Page gel Isolation of
ERK by immunoprecipitation Immunoblotting using
anti-dpERK
Stage 12 Embryo
SDS Page
Co-Ip
Western Blot using anti-dpERK
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xHtrA1 stimulates FGF signal intermediate

• Microinjection of xHtrA1 accumulation of
  activated ERK
• Microinjection of xHtrA1-MO blocked activation of
  ERK

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Creation of Inducer Cap
Animal caps were injected with FGF4, xHtrA1, and
nlacZ
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Animal Caps can be Fused
Fusion of two animal caps
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nlacZ

• Stains nucleus
• Appears blue
• Used to show distinction between inducer and
  responder cap

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xbra

• A.k.a. brachyury (T)
• Functions as switch between cell migration and
  convergent extension
• In the experiment, only used as a mesoderm marker

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Fusion of Inducer and Responder Caps

• Inducer fused with regular animal caps (responder
  caps)
• The caps were analyzed by in situ hybridization
  for Xbra, a mesoderm marker

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xHtrA1 promotes long-term FGF signaling

• FGF4 weak effect
• xHtrA1 weak effect
• FGF4 xHtrA1 robust growth of responder cap
  and greatly elevated Xbra levels far away from
  sites of FGF4 secretion

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Proteoglycan side chains must be removed

• Occurs when proteoglycans are collected from
  embryo
• Side chains must be removed to get definite bands

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Use of Hek293 Cells

• Human Embryonic Kidney Cells transformed with
  sheared adenoveirus 5 DNA
• Harvested from an aborted fetus in late 70s
• Extreme transfectability
• In this experiment
• Transfected cDNA of each type of proeteoglycan
  using calcium phosphate method
• Cells were lysed and proteoglycans were isolated
  by SDS Page
• Result proteoglycans without heparan sulfate
  chains

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xHtrA1 causes proteolytic degradation

• Western blot of xHtrA1 and flag tagged xbiglycan
  and heparan sulfate proteoglycans, xsyndecan and
  xglypican
• Revealed that xHtrA1 degrades all three
  proteoglycans

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Conclusion
xHtrA1 effectively cleaves core proteins from
three types of proteoglycans syndecan, glypican,
and biglycan