Dep1 in arteriolvenous cell fate

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Dep1 in arteriol/venous cell fate

• Carla H, Kelsey H, Lucas H

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introduction.

• The receptor protein-tyrosine phosphatase, Dep1,
  acts in arterial/venous cell fate decisions in
  zebrafish development

• Fiona Rodriguez, Andrei Vacaru, John Overvoorde,
  Jeroen den Hertog.
• 2008.

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PTPs vs. PTKs.

• tyrosine residue (de)phosphorylation
• PTK protein tyrosine kinase
• PTP protein tyrosine phosphatase
• large families
• typically "paired"
• less known about PTPs

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PTPs, subfamilies.

• 2 main classes ? 17 subfamilies
• RPTPs "receptor" PTPs
• R3 RPTPs four members
• single PTP domain
• Dep1 in this subfamily

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Dep1.

• "density-enhanced PTP1"
• vascular endothelial cells of arterial, capillary
  vessels of some organs
• Dep1 cancer
• Dep1 overexpression ? tumor suppression in
  cultures
• mouse Ptprj gene cand. for colon cancer
  susceptibility
• often, PTPRJ gene deleted in human cancer cases

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Dep1 mice.

• gene-targeting of Dep1 ? death before 11.5 days
• in-frame replacement of cytoplasmic sequences
• vascularization failure, disorganized vasc.
  structures
• homozygous mutants ? death (similar timing)
• "subtle effects" e.g. larger vessels
• implicates Dep1, but w/o molecular mech.
• genetic ablation of Ptprj ? viable fertile
• immunoblotting confirms absence of Dep1

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Dep1 C. elegans

• Dep1 ? binary cell fate decisions
• negative regulation of EGFR signaling
• amplifies small changes in EGFR
• complex feedback mechanism
• full activation EGFR/RAS/MAPK in 1o vulval cells
• inactivation in 2o vulval cells

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Dep1 zebrafish.

• Dep1a
• 12 fibronectin-like repeats 4 gt mammalian
  Dep1
• Dep1b
• not fully sequenced yet

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Dep1.
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Dep1 zebrafish.

• morpholino injections
• Dep1a, Dep1b essential for normal blood
  circulation
• Dep1 knockdown ?
• reduced arterial markers
• expanded venous markers
• rescue experiments
• Phosphatidylinositol-3-Kinase
• Grl/hey2
• Notch

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Zebrafish.

• why Zebrafish?
• 12 wk life cycle
• hatch 2 days after fertilization
• transparent embryo
• A/V axis

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Zebrafish.

• VIDEO.

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establishing dep1 expression.
R3 family RPTP interspecies DEP1 homology gt
intraspecies R3 PTP homology
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zebrafish Dep1s, human PTPRJ
comparison of flanking genes.
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whole mount expression of dep1a during early
zebrafish development
whole mount expression of dep1b during early
zebrafish development
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sections of 30 hpf and 48 hpf embryos
DA dorsal aorta NC notochord NT neural
tube PCV posterior cardinal vein
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O-dianisidine staining of hemoglobin of wild-type
(A) and Dep1a-MO1
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does Dep1a knockdown affect vasculogenesis?
No, overall vasculature appears normal.
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functionality of vessels?
WT fluorescence detected through the entire
aorta Dep1a-MO1Rhodamine dextran
staining Only found in aortic bifurcation Dep1a-
MO2 rhodamine dextran Did not reach posterior
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tbx20.

• tbx20 a dorsal aorta marker

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tbx20.
class 1 (normal) wild type expression in dorsal
aorta class 2 (mild) patchy expression in some
cells class 3 (severe) expression completely
absent
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Observed defects were specific
rescue!
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Dep1a, Dep1b synergy.

• cdh5 marker for vascular endothelial cells
• not affected
• myoD somite marker
• somitogenesis not affected by Dep1 knockdown

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arterial fate.
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Position of dorsal aorta indicated by the arrow.
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venous fate.
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signaling pathway.
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discussion.

• comparisons with mice experiments
• Zebrafish knockdown ?? Mouse GFP replacement
• Dep1 mutants had reduced arterial differentiation
• accd by ectopia of venous markers
• Dep1a/Dep1b-MOs vs. grl-MOs ?
• Dep1a/Dep1b mutants phenocopies of grl knockdown
• coinjection of these MOs was additive
• Dep1-MOs abolished grl expression in aorta
• upstream activation?

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discussion.

• VEGF also involved in sim. processes
• Dep1 ? VEGFR2?
• they do interact in tissue culture in vitro?
• parallels between Dep1 function amongst homologs
• Zebrafish arteriol/venous cell fate
• C. elegans vulval cell fate
• Mouse vascular cell fate
• TAKE-HOME MESSAGE
• Dep1 definite role in cell fate determination

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critique.

• Dep1s role in cancer?
• mentioned, not explored
• not fully explaining reasons for results in some
  cases
• e.g. knockdown did not cause the same phenotype
  mechanism?
• overall well done
• experiments results - thorough, clear, logical
• intro discussion glossed over some issues
• some topics in discussion data not provided?

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future experiments.

• role of Dep1 in cancer?
• overexpression ? tumor suppression
• many cases, deletion ? cancer
• poor vascular development ? tumor?
• exact mechanism of action?
• confirmation of the hypothesized signaling
  pathway
• possibly acts on VEGFR2/RAF/MEK/ERK pathway?
• whats with the mouse thing!?

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references.

• Haffter P, Granato M, Brand M, Mullins MC,
  Hammerschmidt M, Kane DA, Odenthal J, van Eeden
  FJ, Jiang YJ, Heisenberg CP, Kelsh RN,
  Furutani-Seiki M, Vogelsang E, Beuchle D, Schach
  U, Fabian C, Nüsslein-Volhard C. The
  identification of genes with unique and essential
  functions in the development of the zebrafish,
  Danio rerio. Development 123 1-36.
• Rodriguez F, Vacaru A, Overvoorde J, den Hertog
  J. 2008. The receptor protein-tyrosine
  phosphatase, Dep1, acts in arterial/venous cell
  fate decisions in zebrafish development.
  Developmental Biology 324, 122130.
• Takahashi T, Takahashi K, St John PL, Fleming PA,
  Tomemori T, Watanabe T, Abrahamson DR, Drake CJ,
  Shirasawa T, Daniel TO. 2003. A mutant receptor
  tyrosine phosphatase, CD148, causes defects in
  vascular development. Mol Cell Biol. 23,
  18171831.
• Trapasso F, Drusco A, Costinean S, Alder H,
  Aqeilan RI, Iuliano R, Gaudio E, Raso C, Zanesi
  N, Croce CM, Fusco A. 2006. Genetic ablation of
  Ptprj, a mouse cancer susceptibility gene,
  results in normal growth and development and does
  not predispose to spontaneous tumorigenesis. DNA
  Cell Biol. 25, 376382.
• all images presented under Fair Use.