Giannetti K et al. (2016), Platelet derived growth factor B gene expressio...

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Int J Dev Biol 2016 Jan 01;604-6:175-9. doi: 10.1387/ijdb.160045mo.

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Platelet derived growth factor B gene expression in the Xenopus laevis developing central nervous system.

Giannetti K , Corsinovi D , Rossino C , Appolloni I , Malatesta P , Ori M .

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Platelet-derived growth factor B (PDGF-B) belongs to the mitogen and growth factor family and like the other members it has many roles in cell differentiation, proliferation and migration during development, adult life and in pathological conditions. Among them it has been observed that aberrant PDGF signalling is frequently linked to glioma development and progression, and Pdgf-b over-expression in mouse neural progenitors leads to the formation of gliomas. Despite this evidence, the mechanisms underlying PDGF-B driven tumorigenesis and its role during brain development are not fully understood. In order to contribute to clarifying possible new roles of pdgf-b signalling, we present here the embryonic gene expression pattern of pdgf-b, so far unknown in early vertebrate development. By using Xenopus laevis as a model system we performed qRT-PCR and whole mount in situ hybridization. Pdgf-b mRNA is expressed in discrete regions of the developing central nervous system, in the cranial nerve placodes and in the notochord. We also compared the gene expression of pdgf-b with that of its receptor pdgfr-α suggesting so far unsuspected roles for this signalling pathway during the development of specific embryonic structures.

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Species referenced: Xenopus laevis
Genes referenced: egr2 odc1 pdgfa pdgfb rax slc12a3 twist1

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	Fig. 2. The temporal expression pattern of pdgfb revealed by qRT-PCR. Expression levels (y axis) of pdgf-b (red) and its receptor pdgfr-a (blue) at different developmental stages (x axis) were analyzed by qRT-PCR. The data are an average of three independent experiments, the values were normalized to odc and presented as mean ± s.d. Expression of pdgf-b mRNA reaches its maximum level as maternal transcript (stage 3), then drastically decreases during gastrulation (stage 10,5). A slight increase is observed during early neurulation (stage 13), however the expression becomes more robust at late neurula stage (stage 20) to decrease again in tailbud stages. Pdgfr-a,is barely detectable at stage 3 and then rise firmly already during gastrulation, reaching its maximum level at stage 20.
	Fig. 3 (right). Pdgf-b spatial expression pattern during early stages of development in Xenopus laevis embryos. (A) Pdgf-b maternal transcript is distributed in the animal pole (arrow) at four cell stage (stage 3), lateral view. (B) Lateral view of a stage 10,5 embryo showing the pdgf-b mRNA localization in the animal pole of the embryo (arrow). (B’) Bisected gastrula showing the pdgf-b expression confined within the deep layer of the ectodermal blastocoel roof (arrowheads). (A)WISH performed using a pdgf-b sense riboprobe on a stage 3 embryo, lateral view. (B) Stage 10,5 lateral view WISH using the pdgf-b sense riboprobe: no signal is detected in the animal pole. (B’*) Bisected gastrula showing no unspecific labelling of the blastocoel cavity using the pdgf-b sense riboprobe. (C) Late neurula,(stage 20) embryo frontal view. Pdgf-b mRNA is localized in the CNS (midbrain– hindbrain region: black and red arrowheads: the most ventral anterior region of neural tube: yellow arrow), the placode of the facial nerve (black arrow) and the notochord (inset). (D) Stage 20 frontal view double WISH: pdgf-b signal revealed in purple (black and red arrowheads), engrailed2 labelling the midbrain-hindbrain boundary and rombomere 1, in light blue. The two signals overlaps at the level of rhombomere 1. (E) Stage 20 frontal view double WISH: pdgf-b signal in purple (black and red arrowheads), krox20 labelling rhombomeres 3 (R3) and 5 (R5), in light blue. The two signals overlaps faintly at the level of rhombomere 3. (F) Double WISH using twist, a NCC marker in light blue. NCC are in close proximity of the pdgf-b expression domain but never overlap it. (G) Double WISH using pdgfr-a in light blue. Pdgfr-a is expressed in migrating NCC. (H) Frontal view of an embryo processed with double WISH using rx1, a marker for the eyefield (dashed oval), revealing a partial overlap of the pdgf-b signal (yellow arrow) with an anterior and ventral domain of the eyefield.
	Fig. 4. Pdgf-b expression spatial distribution in Xenopus laevis embryo at tailbud stage.(A)Pdgf-b mRNAis present in the hindbrain (R1 and R3), the most ventral anterior region of the developing CNS (yellow arrow), the placode of the facial nerve (black arrow) and the notochord (inset, green arrowhead). (B-E) Double WISH was performed using different markers (the color code is described in Fig.3) at tailbud stage (stage 25). (B) Lateral view of an embryo processed by double WISH using twist as NCC marker. (C) Pdgfr-a is expressed by NCC, in close proximity with the pdgf-b mRNA expression domain. (D-D’) Frontal and lateral views of the same embryo labelled to visualize rx1, a retinal marker (dashed oval identifies the eyefield) and pdgf-b. (E) A sagittal view of a half embryo bisected along the antero-posterior axis shows the expression of pdgf-b in the ventral telencephalon.