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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
Splice‐blocking morpholino microinjection. A: Schematic representation of the microinjection procedure. Embryos were injected with morpholino oligos and GFP mRNA at 4‐cell 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 neurula‐stage embryo in brightfield. The same embryo shows GFP fluorescence in the injected side. B: Graphic representation of pdgf‐b splice‐blocking morpholino (pdgf‐b MO) action on RNA maturation. C: RT‐PCR on Co MO‐ and pdgf‐b MO‐injected embryos. Co MO‐injected embryos show the band corresponding to the full pdgf‐b coding sequence, while pdgf‐b MO–injected embryos present both the wild‐type 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; RT‐PCR, reverse transcription‐polymerase chain reaction
Figure 2
pdgf‐b down‐regulation affects NCC migration. A: WISH on pdgf‐b MO‐ and Co MO–injected 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%, pdgf‐b MO 84%; stage 25: Co MO 5%, pdgf‐b MO 66%). C: WISH on pdgf‐b MO‐ and Co MO‐injected embryos showing expression of sox10. D: Graph reporting the percentage of embryos with impaired migration of NCC expressing sox10 (stage 20: Co MO 10%, pdgf‐b MO 83%; stage 25: Co MO 5%, pdgf‐b MO 60%). E: Rescue experiment on embryos co‐injected with pdgf‐b MO and PDGF‐B mRNA showing expression of twist and sox10. F: Graph indicating the mean percentage of stage‐20 embryos with NCC migration defects (twist: pdgf‐b MO 84%, pdgf‐b MO + PDGF‐B mRNA 52%; sox10: pdgf‐b MO 83%, pdgf‐b MO + PDGF‐B mRNA 56%). The injected side is marked with an asterisk (*). Arrowheads indicate impaired NCC migration. BCS, branchial crest stream (branchial arches III‐IV); 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, whole‐mount in situ hybridization
Figure 3
pdgf‐b depletion affects cranial nerves development. A: Whole‐mount immunostaining on stage‐45 tadpoles labeled with the neurofilament‐specific 3A10 antibody revealing cranial nerves. Co MO‐injected tadpoles display a normal development of the same nerves on both sides, whereas pdgf‐b‐morphant 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%, pdgf‐b 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
pdgf‐b depletion alters craniofacial cartilages development. A: Dorsal and ventral views of pdgf‐b MO‐ and Co MO‐injected tadpoles after dissection. The ethmoidal plate and the subocular cartilage are mainly affected on the injected side of pdgf‐b 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 MO‐injected 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%, pdgf‐b 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
pdgf‐b knockdown causes a reduction of cdh6 and cdh11 expression levels. A: qRT‐PCR analysis on stage‐20 embryos revealing no effect of pdgf‐b down‐regulation on Ecad and Ncad expression levels and a reduction of cdh6 and cdh11 expression levels in pdgf‐b morphants compared to Co MO‐injected embryos. B: WISH on pdgf‐b MO‐ and Co MO‐injected 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 pdgf‐b morphants. C: Statistical analysis of data shown in B; graph reporting the percentage of embryos with reduced cdh6 expression (stage 20: Co MO 11%, pdgf‐b MO 88%; stage 25: Co MO 5%, pdgf‐b MO 80%). D: WISH on pdgf‐b MO‐ and Co MO‐injected 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%, pdgf‐b MO 82%; stage 25: Co MO 4%, pdgf‐b MO 75%). The injected side is marked with an asterisk (*). MO, morpholino; N, number of independent experiments; n, number of embryos qRT‐PCR, real‐time RT‐PCR; WISH, whole‐mount in situ hybridization
Figure 6
Control and functional rescue experiments to verify pdgf‐b MO specificity. A: Rescue experiment on embryos co‐injected with pdgf‐b MO and PDGF‐B mRNA showing expression of cdh6 and cdh11 at neurula and tail bud stages. Black dashed line shows the middle line of the embryo. Pdgf‐b MO and mRNA co‐injection restored cadherins expression. B: Graph indicating the mean percentage of stage‐20 embryos with reduced cadherins expression level (cdh6: pdgf‐b MO 88%, pdgf‐b MO + PDGF‐B mRNA 45%; cdh11: pdgf‐b MO 82%, pdgf‐b MO + PDGF‐B mRNA 47%). Rescue percentage is indicated. C: TUNEL assay performed on pdgf‐b 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
