Reisoli E et al. (2010), Serotonin 2B receptor signaling is required for...

XB-ART-41818

Development 2010 Sep 01;13717:2927-37. doi: 10.1242/dev.041079.

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Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus.

Reisoli E , De Lucchini S , Nardi I , Ori M .

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Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

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Species referenced: Xenopus laevis
Genes referenced: dlx2 efnb2 gal.2 hand2 hoxa2 htr2b nkx3-1 nkx3-2 pcgf2 plcb3 slc12a3 snai2 sox9 tbx2 znf469
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	Fig. 1. Skeletal and muscular alteration in 5-HT2B-overexpressing tadpoles (stage 49). (A) Dorsal view of a 5-HT2B-overexpressing embryo. (B,D) Alcian Blue-stained whole-mount (B) and flat-mount (D) skeleton of injected embryos. Red arrows indicate the ectopic cartilage and black arrows indicate the reduction of the quadrate (Q) and subocular (So) cartilages. (C,E) Scheme of the contribution of cranial NCC streams to the neurocranium and pharyngeal skeleton of Xenopus laevis as reported by Pasqualetti et al. (Pasqualetti et al., 2000) (C) and in 5-HT2B-overexpressing embryos (E). (F) Flat-mount of an injected embryo double stained for cartilage (blue) and muscles (brown). (G) Scheme of muscular alterations of the 5-HT2B-overexpressing embryo shown in F. On the injected side, the orbitohyoideus muscle (Oh) is anchored to the ectopic cartilage (red arrow in F,G) instead of to the reduced quadrate (white arrow in F,G). Note the disorganization of the levatores arcuum branchialum muscles I-II (Lab I-II) on the injected side. C, ceratohyal; Et, ethmoidal plate; G, genohyoideus; Ha, hyoangularis; Ih, interhyoid muscle; Im, intermandibular; Ir, infrarostral; M, Meckel's; Qa, quadratoangularis; Lm, levator mandibulae; Sr, subarcuales rectus.
	Fig. 2. 5-HT2B overexpression does not influence NCC specification and migration. The injected side, visualized by n-β-gal staining (red), is always on the right. (A,B) Frontal view of a neurula stage Xenopus embryo hybridized with XSlug (A) and XEphrinB2 (B) probes. (C-D′) Lateral view of tailbuds hybridized with XSox9 (C,C′) and XHoxa2 (D,D′) probes. MCS, mandibular crest stream (branchial arch I); HCS, hyoid crest stream (branchial arch II); BCS, branchial crest stream (branchial arches III-IV).
	Fig. 3. 5-HT2B overexpression results in ectopic Xbap and XHand2 mRNA expression. The injected side, visualized by n-β-gal staining (red), is always on the right. Lateral view (A,A′,C,C′,E,E′,F,F′) and vibratome sections (A′,B,C′,D,E′) of stage 37 Xenopus embryos. (A-A′) Xbap mRNA ectopic expression in the mandibular arch (arrow in A′,A′). (B) Double ISH showing Xbap ectopic expression (dark blue) in a subdomain (arrow) of the XSox9-expressing cells (light blue). (C-C′) XHand2 ectopic expression in the dorsal domain of the posterior branchial arches (arrow in C′,C′). (D) Double ISH showing Xbap (dark blue) and XHand2 (light blue). On the injected side, only Xbap is ectopically expressed in the mandibular arch (arrow). (E-E′) XHoxa2 expression in the second branchial arch is normal. (F,F′) XMel1 expression in third and fourth branchial arches is normal. h, heart.
	Fig. 4. 5-HT2B influences cranial NCC development in a cell-autonomous manner. (A) Scheme of the cranial NCC transplantation assay. (B,C) Lateral view of stage 30 transplanted and wild-type embryos, respectively. (B) GFP fluorescence in transplanted cranial NCCs. (C) Cranial NCCs visualized by the XDll4 probe in a wild-type embryo. (D,E) Horizontal vibratome sections of Xenopus tadpole. The red staining identifies transplanted NCCs. (E) Magnification of the second pharyngeal arch of a transplanted embryo stained for n-β-gal. (F) Flat-mount preparation of a stage 49 transplanted embryo double stained for cartilage (blue) and GFP immunoreactivity (brown). Skeletal elements derived from the transplanted cranial NCCs are visualized by the presence of GFP immunoreactivity. Note the reduced quadrate (red arrow) and the presence of ectopic cartilage (black arrow).
	Fig. 6. Skeletal and muscular connectivity alterations in 5-HT2B morphants. (A) Flat-mount preparation of an Alcian Blue-stained 5-HT2B-MO-injected Xenopus embryo. (A′,A′) Magnification of the jaw joint region of the control (A′) and injected (A′) side of the embryo in A. Note the lack of the jaw joint (red arrow in A′) compared with the uninjected side (arrow in A′), the reduction of the quadrate (Q; black arrow in A′), and the absence of the ventral cartilaginous muscular process of the Meckel's cartilage, which is present on the control side (arrowhead in A′ and A′, respectively). (B) Flat-mount preparation of a 5-HT2B-MO-injected embryo double stained for cartilage and muscle. (B′,B′) Magnification of the jaw joint region of the uninjected (B′) and injected (B′) sides of the embryo in B. Note the abnormal development of the hyangularis (Ha) and quadratoangularis (Qa) muscles (arrow in B′).
	Fig. 7. 5-HT2B loss of function results in the downregulation of Xbap expression in postmigratory NCCs. The injected side, visualized by n-β-gal staining (red), is always on the right. (A,A′,B,B′,C,C′) Lateral view of stage 37 Xenopus embryos hybridized with Xbap (A,A′), XHoxa2 (B,B′) and XHand2 (C,C′) probes. (A′) Frontal view of the embryo in A. (A′′′,B′,C′) Vibratome coronal sections across the line in A, B and C, respectively. (A-A′′′) The Xbap expression is reduced on the injected side of stage 37 5-HT2B morphants (arrows in A′, A′′′) as compared with the control side. XHoxa2 (B-B′) and XHand2 (C-C′) expression is unaffected. (D,D′) Lateral view of a whole-mount stage 28 5-HT2B morphant hybridized with XSox9 probe.
	Fig. 8. Phospholipase C beta 3 acts downstream of 5-HT2B to regulate the jaw joint formation. Flat-mount preparations of Alcian Blue-stained injected Xenopus embryos. (A-B′) PLC-MO morphants. (A′,A′,B′,B′) Magnification of the jaw joint region of the embryos in A,B. Note the lack of the jaw joint (red arrow in A′,B′) and of the ventral cartilaginous muscular process of the Meckel's (arrowhead in A′), as compared with the control side (arrowhead in A′). (B′) A strong phenotype PLC morphant presenting the loss of the jaw joint (arrow) and of the distal portion of the Meckel's (squared bracket). (C,E) Embryos injected with subthreshold levels of either 5-HT2B-MO (C) or PLC-MO (E) develop a normal visceral skeleton. (D) The co-injection of subthreshold levels of 5-HT2B-MO plus PLC-MO causes loss of the jaw joint (red arrow). (F,G) The co-injection of PLC-MO together with 5-HT2B mRNA results in 79% normal embryos (F) and 21% presenting the ectopic cartilage (G). (H) In 5-HT2B overexpression embryos, the ectopic cartilage is always bigger than that originating in the 5-HT2B mRNA+PLC-MO injected embryos (compare red arrow in G to black arrow in H).
	dlx2 (distal-less homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, head region only, lateral view, anterior left, dorsal up.
	Fig. S1. 5-HT2B mRNA expression in migrated cranial NCCs. In situ hybridization with the 5-HT2B probe. (A) Lateral view of a wild-type stage 28 Xenopus embryo. 5-HT2B mRNA is expressed in NCCs that have migrated into the four branchial arches and in the heart primordia (arrow). (B) Cryostat coronal section of a wild-type stage 37 embryo. 5-HT2B mRNA is present in scattered cells of the retina and of the neural tube (arrows). (B′) Magnification of the region highlighted in B. 5-HT2B mRNA is expressed in the cement gland (cg), in the ventricular region of the ventral diencephalon (arrowhead) and in the region surrounding the oral cavity (arrow).
	Fig. S2. Muscular alterations in 5-HT2B-overexpressing tadpoles (stage 49). (A,B) 5-HT2B-overexpressing embryos double stained for cartilage (blue) and muscle (brown) in whole-mount (dorsal view, A) and flat-mount (B) preparations. Note the muscular alterations on the injected side of the embryo: the levatores arcuum branchialum muscles I-II (Lab I-II) are disorganized (red arrows in A) and the orbitohyoideus muscle (Oh) is anchored to the ectopic cartilage (red arrow in B) instead of to the reduced quadrate, as on the wild-type side.
	Fig. S3. Co-injection of 5-HT2B mRNA with 5-HT2B-MO1 rescues both the skeletal and molecular phenotypes. (A) Dorsal view of a stage 49 Xenopus embryo co-injected with 5-HT2B mRNA and 5-HT2B-MO1. The injected side of the embryo is visualized by the GFP fluorescence. (B) Flat-mount preparation of the skeleton of an embryo co-injected with 5-HT2B mRNA and 5-HT2B-MO1 and stained with Alcian Blue. The injected side of the embryo is normal. (C,C′) Lateral views of a stage 37 embryo co-injected with 5-HT2B mRNA and 5-HT2B-MO1 and hybridized with the Xbap probe. There are no differences in the Xbap mRNA expression on the wild-type (C) and injected (C′) sides of the embryo.
	Fig. S4. Xbap and XHand2 expression is downregulated on the injected side of PLC morphants. (A-C′) The injected side of each embryo, visualized by n-β-gal staining (red), is always on the right. (A,A′,B,B′) Lateral view of stage 37 PLC morphants hybridized with Xbap (A,A′) and XHand2 (B,B′) probes. (A′′,B′′) Frontal view of the embryos in A,A′ and B,B′, respectively. (C,C′) Lateral views of a stage 37 embryo injected with PLC-control-MO and hybridized with the Xbap mRNA probe. There are no differences in the Xbap expression on the wild-type (C) and injected (C′) sides of the embryo. (D) Dorsal view of a PLC morphant. The left, injected side is visualized by GFP fluorescence. (E) Flat-mount preparation of the embryo in D stained with Alcian Blue. Note that the injected side of the embryo is normal.
	Frontal view of a neurula stage Xenopus embryo hybridized with efnb2 probes.
	Anterior view of a late neurula stage Xenopus embryo, showing gene expression for efnb2 (ephrin B2), assayed via in situ hybridization, in rhombomere r2 and rhombomere r4, as well as eye primordium (optic field).