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Dev Dyn
2019 Jul 01;2487:603-612. doi: 10.1002/dvdy.47.
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PDGF-B: The missing piece in the mosaic of PDGF family role in craniofacial development.
Corsinovi D
,
Giannetti K
,
Cericola A
,
Naef V
,
Ori M
.
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BACKGROUND: The platelet-derived growth factor (PDGF) family consists of four ligands (PDGF-A, PDGF-B, PDGF-C, PDGF-D) and two tyrosine kinase receptors (PDGFR-α and PDGFR-β). In vertebrates, PDGF signaling influences cell proliferation, migration, and matrix deposition, and its up-regulation is implicated in cancer progression. Despite this evidence, the role of each family member during embryogenesis is still incomplete and partially controversial. In particular, study of the role of pdgf signaling during craniofacial development has been focused on pdgf-a, while the role of pdgf-b is almost unknown due to the lethal phenotypes of pdgf-b-null mice.
RESULTS: By using a pdgf-b splice-blocking morpholino approach, we highlighted impairment of neural crest cell (NCC) migration in Xenopus laevis morphants, leading to alteration of NCC derivatives formation, such as cranial nerves and cartilages. We also uncovered a possible link between pdgf-b and the expression of cadherin superfamily members cdh6 and cdh11, which mediate cell-cell adhesion promoting NCC migration.
CONCLUSIONS: Our results suggested that pdgf-b signaling is involved in cranial NCC migration and it is required for proper formation of craniofacial NCC derivatives. Taken together, these data unveiled a new role for pdgf-b during vertebrate development, contributing to complete the picture of pdgf signaling role in craniofacial development.
Figure 1
Spliceâblocking morpholino microinjection. A: Schematic representation of the microinjection procedure. Embryos were injected with morpholino oligos and GFP mRNA at 4âcell stage in one dorsal blastomere in order to specifically target the central nervous system. The green dashed line highlights the neural plate on the injected side of a neurulaâstage embryo in brightfield. The same embryo shows GFP fluorescence in the injected side. B: Graphic representation of pdgfâb spliceâblocking morpholino (pdgfâb MO) action on RNA maturation. C: RTâPCR on Co MOâ and pdgfâb MOâinjected embryos. Co MOâinjected embryos show the band corresponding to the full pdgfâb coding sequence, while pdgfâb MOâinjected embryos present both the wildâtype band and a lower one, corresponding to the PCR product obtained after exon 2 excision. GFP, green fluorescent protein; MO, morpholino; PCR, polymerase chain reaction; RTâPCR, reverse transcriptionâpolymerase chain reaction
Figure 2
pdgfâb downâregulation affects NCC migration. A: WISH on pdgfâb MOâ and Co MOâinjected embryos showing expression of twist. Black dashed line shows the middle line of the embryo. B: Graph reporting the percentage of embryos with altered migration of NCC expressing twist (stage 20: Co MO 12%, pdgfâb MO 84%; stage 25: Co MO 5%, pdgfâb MO 66%). C: WISH on pdgfâb MOâ and Co MOâinjected embryos showing expression of sox10. D: Graph reporting the percentage of embryos with impaired migration of NCC expressing sox10 (stage 20: Co MO 10%, pdgfâb MO 83%; stage 25: Co MO 5%, pdgfâb MO 60%). E: Rescue experiment on embryos coâinjected with pdgfâb MO and PDGFâB mRNA showing expression of twist and sox10. F: Graph indicating the mean percentage of stageâ20 embryos with NCC migration defects (twist: pdgfâb MO 84%, pdgfâb MOâ+âPDGFâB mRNA 52%; sox10: pdgfâb MO 83%, pdgfâb MOâ+âPDGFâB mRNA 56%). The injected side is marked with an asterisk (*). Arrowheads indicate impaired NCC migration. BCS, branchial crest stream (branchial arches IIIâIV); HCS, hyoid crest stream (branchial arch II); MCS, mandibular crest stream (branchial arch I); MO, morpholino; N, number of independent experiments; n, number of embryos; NCC, neural crest cell; WISH, wholeâmount in situ hybridization
Figure 3
pdgfâb depletion affects cranial nerves development. A: Wholeâmount immunostaining on stageâ45 tadpoles labeled with the neurofilamentâspecific 3A10 antibody revealing cranial nerves. Co MOâinjected tadpoles display a normal development of the same nerves on both sides, whereas pdgfâbâmorphant tadpoles lack the most anterior portion of the VII cranial nerve on the injected side, detectable in dorsal, ventral, and lateral views (red arrowhead). V cranial nerve (blue arrowhead) appears thinner on the injected side than on the control side and its branching pattern is severely disrupted, as we can observe in dorsal and lateral views. B: Statistical analysis of data shown in A; graph reporting the percentage of tadpoles with altered cranial nerves (Co MO 4%, pdgfâb MO 49%). The injected side is marked with an asterisk (*). Black dashed lines show the middle line of the tadpoles. MO, morpholino; N, number of independent experiments; n, number of tadpoles; V, trigeminal nerve; VII, facial nerve
Figure 4
pdgfâb depletion alters craniofacial cartilages development. A: Dorsal and ventral views of pdgfâb MOâ and Co MOâinjected tadpoles after dissection. The ethmoidal plate and the subocular cartilage are mainly affected on the injected side of pdgfâb morphants (black arrowhead), but a slight reduction of the ceratohyal and the branchial cartilages (gills) size is detectable in ventral view. Skeletal elements of the Co MOâinjected tadpoles are well developed and bilaterally symmetric. B: Statistical analysis of data shown in A; graph reporting the percentage of tadpoles with altered craniofacial cartilages (Co MO 4%, pdgfâb MO 49%). The injected side is marked with an asterisk (*). Black dashed lines show the middle line of the tadpoles. C, ceratohyal; Et, ethmoidal plate; G, gills; M, Meckel's cartilage; MO, morpholino; N, number of independent experiments; n, number of tadpoles. Q, quadrate; So, subocular arc
Figure 5
pdgfâb knockdown causes a reduction of cdh6 and cdh11 expression levels. A: qRTâPCR analysis on stageâ20 embryos revealing no effect of pdgfâb downâregulation on Ecad and Ncad expression levels and a reduction of cdh6 and cdh11 expression levels in pdgfâb morphants compared to Co MOâinjected embryos. B: WISH on pdgfâb MOâ and Co MOâinjected embryos showing cdh6 expression. Black dashed line shows the middle line of the embryo. Arrowheads indicate a strong reduction of cdh6 expression on the injected side of pdgfâb morphants. C: Statistical analysis of data shown in B; graph reporting the percentage of embryos with reduced cdh6 expression (stage 20: Co MO 11%, pdgfâb MO 88%; stage 25: Co MO 5%, pdgfâb MO 80%). D: WISH on pdgfâb MOâ and Co MOâinjected embryos showing cdh11 expression. Arrowheads indicate reduced cdh11 expression. E: Statistical analysis of data shown in D; graph reporting the percentage of embryos with reduced cdh11 expression (stage 20: Co MO 11%, pdgfâb MO 82%; stage 25: Co MO 4%, pdgfâb MO 75%). The injected side is marked with an asterisk (*). MO, morpholino; N, number of independent experiments; n, number of embryos qRTâPCR, realâtime RTâPCR; WISH, wholeâmount in situ hybridization
Figure 6
Control and functional rescue experiments to verify pdgfâb MO specificity. A: Rescue experiment on embryos coâinjected with pdgfâb MO and PDGFâB mRNA showing expression of cdh6 and cdh11 at neurula and tail bud stages. Black dashed line shows the middle line of the embryo. Pdgfâb MO and mRNA coâinjection restored cadherins expression. B: Graph indicating the mean percentage of stageâ20 embryos with reduced cadherins expression level (cdh6: pdgfâb MO 88%, pdgfâb MOâ+âPDGFâB mRNA 45%; cdh11: pdgfâb MO 82%, pdgfâb MOâ+âPDGFâB mRNA 47%). Rescue percentage is indicated. C: TUNEL assay performed on pdgfâb morphants at neurula (n = 61, N = 3) and tail bud (n = 65, N = 3) stages. The injected side is marked with an asterisk (*). N, number of independent experiments; n, total number of analyzed embryos
