January 1, 2013;
Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.
Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3
overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3
function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3
plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3
loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3
-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3
on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3
is required for cephalic neural crest migration in vivo during embryonic development.
[+] show captions
References [+] :
Figure 1. Spatial expression profile of xlPTP4A3 during embryogenesis.
A-K) Whole-mount in situ hybridization showing the spatial expression of xlPTP4A3 during early Xenopus development. A'-K') Corresponding negative control using a sense probe of xlPTP4A3 mRNA. L) Whole-mount in situ hybridization of xlPTP4A3 in NC explants dissected at stage 17. L') Corresponding negative control using a sense probe of xlPTP4A3 mRNA. M) Whole-mount in situ hybridization of the NC marker xlSNAI2 on stage 17-dissected NC explants. s1-s3) Transverse sections through a stage 25 embryo stained by whole-mount in situ hybridization for xlPTP4A3 mRNA. Sense) Corresponding negative control using an xlPTP4A3 mRNA sense probe. sc: spinal cord, nc: notochord, ov: otic vesicle, ac: archenteron.
Figure 2. Depletion of xlPTP4A3 reduces NCC territory.
Control 5MM-MO or xlPTP4A3-MO were injected at the four-cell stage in the animal pole region and embryos were cultured until stage 22. A) Morpholino sequence targeting xlPTP4A3. B) xlPTP4A3-MO specifically knocked down the translation of xlPTP4A3 in vitro using the TNT® Coupled Reticulocyte Lysate System. C) xlPTP4A3 depleted embryos showed blockade of NCC territory (a-a') and can be rescued by co-injection of mouse mPTP4A3 (c-c'), as shown by whole-mount in situ hybridization on xlTWIST. The injected side was monitored using the co-injection of nuclear-targeted β-galactosidase (red dotted signal). Injection of the control 5MM-MO does not affect the NCCs territory, as in the control noninjected embryos (b-b'). D) Quantitative results of relative phenotype (**p<0,01). E) The phenotype quantification was determined as the length of the NC migratory streams showing that the migratory streams are shorter in the injected side than in the uninjected side (n=15) (***p<0,001). F) Immunodetection of cleaved caspase 3 on xlPTP4A3-MO injected embryos. Two phenotypes are observed: one of weaker apoptosis in the injected side compared to the uninjected side (a-a') and one of no differential apoptosis between the two sides (b-b').
Figure 3. xlPTP4A3 gain of function induces strengthening of the anterior NC migration streams in vivo.
xlPTP4A3, mmPTP4A3 or the mutant form mmPTP4A3(C104S) were injected at the four-cell stage in the animal pole region, and embryos were cultured until stage 22. A) Overexpression of xlPTP4A3 induces a strengthening (b, d vs. d') and/or lengthening (a, c) of the anterior NC migration streams as showed by whole-mount in situ hybridization on xlTWIST. B) xlPTP4A3 corresponding phenotype quantification was determined as the area of the NC migratory streams showing that the migratory streams surface is greater in the injected side than in the uninjected side (n=15) (**p<0,01). C) Overexpression of mmPTP4A3 causes a strengthening and/or lengthening (e vs.e', f) of the anterior NC migration streams while overexpression the mutant form mmPTP4A3(C104S) does not seem to significantly affect the migration of the NC (g vs.g', h). D) mmPTP4A3 or mmPTP4A3(C104S) corresponding phenotype quantification was determined as the area of the NC migratory streams showing that mmPTP4A3 injected embryos display a greater anterior NC area in the injected side than in the uninjected side while no difference in the anterior NC area could be observed in the mmPTP4A3(C104S) injected embryos between the two sides (n=15) (**p<0,01). E) Immunodetection of phosphoHistone 3 (Ser10) on xlPTP4A3 gain of function injected embryos. Injection of xlPTP4A3 does not seem to affect cell proliferation in the injected side relative to that of the uninjected side (a vs.b).
Figure 4. Depletion of xlPTP4A3 inhibits both migration of NCC and uveal melanoma cells.
A) Isotopic and isochronic grafts of xlPTP4A3-depleted NC explants into host embryos showed a delayed NCC migration (b-b') when compared to the control histone 2b-GFP NC explants (a-a'). B) Timelapse videomicroscopy of stage 17-derived NCC treated with a pharmacological inhibitor of PTP4A3, a rhodanine derivative (PRL-3 inhibitor I), shows that inhibition of PTP4A3 increases the average of pausing NC cells and strongly decreases the migration velocity of the treated cells (relative to that of vehicle treated cells). The result is representative of three independent experiments and the average of counted cells is 60 cells per condition (***p<0,001). C) Timelapse videomicroscopy of uveal melanoma OCM1 cells stably expressing EGFP-PTP4A3, EGFP-PTP4A3(C104S) or EGFP on collagen I shows that inhibition of PTP4A3, using PRL-3 Inhibitor I, decreases specifically the migration velocity of the PTP4A3 expressing cells (relative to the velocity of untreated and control cells). The result is representative of three independent experiments and the average of counted cells is 50 cells per condition (***p<0,001).
ptp4a3 (protein tyrosine phosphatase type IVA, member 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, animal view (left) and vegetal (right).
ptp4a3 (protein tyrosine phosphatase type IVA, member 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
ptp4a3 (protein tyrosine phosphatase type IVA, member 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
Aceto, Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop. 2012, Pubmed