Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton

Abstract

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

Fig. 1.

Expression of Tg(Bre:GFP) in the pharyngeal arches. (A-C) GFP fluorescence in living Tg(Bre:GFP) transgenic embryos, lateral views, anterior towards the left. (D-F) Lateral views of the arches at higher magnification. Confocal slices of Tg(Bre:GFP) (D) and Tg(sox10:lyn-tdTomato) (E) double transgenics. The two channels are merged in F, demonstrating direct BMP responses in NC and in the stomodeum (arrow). (G-I) Adjacent transverse sections through the hindbrain and arches stained with anti-GFP (G,I) and anti-pSmad1/5/8 (H), revealing BMP responses in the NC and in surrounding endoderm (en) and ectoderm (ec). (A) 16 hpf. (B) 24 hpf. (C) 48 hpf. (D-F) 28 hpf. (G-I) 30 hpf. cn, commissural neurons; D, dorsal; de, dorsal eye; ec, ectoderm; en, endoderm; I, intermediate; le, lens; nc, neural crest; pa, pharyngeal arches; rp, roofplate; s, somites; V, ventral. Scale bars: 100 μm.

Fig. 2.

Requirements for BMP signaling in pharyngeal cartilage and palate development. (A) Histogram quantifying the frequency of defects in different skeletal elements caused by heat shocking Tg(hs:dnBmpra1-GFP) embryos at different stages between 15 and 36 hpf. (B-G) Alcian Blue stained cartilages of 5 dpf larvae, dissected and flat-mounted, anterior towards the left. (B-D) Pharyngeal cartilages of control (B), moderate (C) and severely affected (D) Tg(hs:dnBmpra1-GFP) transgenics, heat shocked at 16-18 hpf, lateral views. (B′-D′) Diagrams of cartilage elements in arch 1 (mandibular, Mc and Pq) and arch 2 (hyoid, Ch, Ih and Hm) corresponding to cartilages in B-D. Lateral views, anterior towards the left. Colors indicate dorsal (green), intermediate (yellow) and ventral (pink) elements. Arrows indicate dorsal arch 2 elements; asterisks indicate fused joints in arch 1; arrowheads indicate joints in arch 2. (E-G) Neurocranial cartilages of the palate, ventral view: control (E), moderate (F) and severe (G). abc, anterior basicranial commissure; Ch, ceratohyal; e, ethmoid plate; Hm, hyomandibular; Ih, interhyal; Mc, Meckels cartilage; pc, parachordals; Pq, palatoquadrate; ptp, pterygoid process; ra, retroarticular process of Meckel’s cartilage; Sy, symplectic; tr, trabeculae. Scale bars: 100 μm.

Fig. 3.

BMP signaling is required for ventral cell specification in the arches. (A-P) Whole-mount in situ hybridization to detect expression of genes involved in DV arch patterning, lateral views, anterior towards the left in controls (A,C,E,G,I,K,M,O) and Tg(hsp70I:dnBmpra1-GFP) transgenics heat shocked at 16-18 hpf (B,D,F,H,J,L,N,P). dlx3b (A,B), dlx6a (C,D) and hand2 (E,F) expression is reduced at 28 hpf. nkx3.2 (G,H) expression is reduced and jag1b expression (I,J) expands ventrally at 36 hpf (arrows). msxe expression is lost at 30 hpf (K,L). Arches 1 and 2 are numbered. (M-P) Two-color fluorescent in situs to detect dlx3b (M,N, red) or msxe (O,P, red) simultaneously with hand2 (green). Scale bar: 100 μm.

Fig. 4.

Exogenous BMP is sufficient to induce ventral arch identity. (A-D,F-H) Alcian Blue-stained cartilages of 4-5 dpf larvae, dissected and flat-mounted, anterior towards the left. (I-N) Whole-mount in situ hybridization at 30 hpf, lateral views, anterior towards the left. (A,B) Pharyngeal cartilages of a control (A) and an embryo implanted behind the eye at 20 hpf with a bead soaked in 20 μg/μl of human recombinant BMP4/7 heterodimers (B). (C,D) Isolated cartilages of the mandibular (1) and hyoid (2) arches: control (C); BMP4/7 bead-implanted (D). Grey arrowheads in B,D indicate duplicated Mc cartilages. (E) Histogram of phenotype frequency in bead-implanted embryos depending on BMP4/7 concentration. (F-H) Neurocranial cartilages: control (F); BMP4/7 bead-implanted (G); heat-shocked Tg(hsp70I:Gal4;UAS:Bmp4) (H). Black arrowheads indicate ectopic cartilages. (I,J) dlx6a expression slightly expands dorsally (arrow) in response to a BMP4/7-soaked bead (J). (K,L) dlx3b expression does not change when BMP4/7-soaked beads are implanted. (M,N) hand2 expression also expands dorsally (arrow) in response to BMP4/7 protein. Asterisks and blue circles indicate beads. 1, mandibular arch; 2, hyoid arch; Ch, ceratohyal; e, ethmoid plate; Hm, hyomandibular; Mc, Meckel’s cartilage; Mc’, ectopic Meckel’s cartilage; Pq, palatoquadrate; tr, trabeculae. Scale bars: 100 μm.

Fig. 5.

BMPs regulate Edn1 expression. Projections of double fluorescent in situ hybridization, lateral views, anterior towards the left. (A-C) dlx2a (red) and edn1 (green) in wild-type (A), heat shocked Tg(hsp70I:dnBmpr1a-GFP) (B) and heat shocked Tg(hsp70I:Gal4;UAS:Bmp4) (C) embryos. (D-F) dlx2a (red) and bmp4 (green) in wild-type (D), edn1^–/– mutant (E) and heat shocked Tg(hsp70I:Gal4;UAS:Edn1) (F) embryos. Scale bar: 50 μm.

Fig. 6.

Exogenous Edn1 rescues BMP deficiency and vice versa. (A-D,F-K) Alcian Blue stained cartilages of 5 dpf larvae, anterior towards the left. (A-D) Dissected cartilages, lateral views, of a control (A), Tg(hsp70I:dnBmpr1a-GFP) transgenic heat shocked at 16-18 hpf (B) and similarly treated transgenics in which 0.5 μg human recombinant EDN1 protein was microinjected into the arches at 25 hpf (C) and 31 hpf (D). (E) Histogram depicting percentages of Tg(hs:dnBmpra1-GFP)+ embryos with severe ventral arch defects without and with EDN1 protein injected. (F-H) Ventral view of neurocranial cartilage in control (F), dnBmpr+ (G) and dnBmpr+ with 0.5 μg EDN1 injected (H). (I-K) Whole-mount cartilages, ventral views, of non-transgenic wild-type control (I), non-transgenic edn1^–/– mutant (J) and edn1^–/–; Tg(hsp70I:Gal4;UAS:Bmp4) (K) embryos subjected to a 1-minute heat shock at 21 hpf. Arrows show rescued ventral Mc; arrowheads show rescued ventral Ch cartilages; asterisks indicate fused Mc-Pq joints. Ch, ceratohyal; e, ethmoid plate; Hm, hyomandibular; Mc, Meckels cartilage; Pq, palatoquadrate; Sy, symplectic; t, trabeculae. Scale bars: 100 μm.

Fig. 7.

Edn1 rescues ventral patterning in BMP-deficient embryos. (A-O) Whole-mount in situ hybridization to detect expression of genes involved in DV arch patterning at 30 hpf, lateral views, anterior towards the left in controls (A,D,G,J,M), Tg(hsp70I:dnBmpr1a-GFP) transgenics heat shocked at 16-18 hpf (B,E,H,K,N) and heat-shocked transgenics injected with human recombinant EDN1 protein (C,F,I,L,O). hand2 (A-C) expression is unaffected, whereas dlx5a (G-I), dlx6a (J-L) and msxe (M-O) expression are restored by EDN1 protein in heat-shocked transgenics, and dlx3b (D-F) expression is induced throughout the DV extent of the arches. Arches 1 and 2 are numbered. Arrows indicate rescued gene expression. Scale bar: 100 μm.

Fig. 8.

Model. Schematic of the primordia of arches 1 and 2 at 20 hpf (A), 30 hpf (B) and cartilages at 6 dpf (C), lateral views, anterior towards the left. (A) Pharyngeal epithelia (blue) express Bmp2/4/7 and Edn1. Ventral/intermediate (pink/orange stripes) and dorsal (green) domains are indicated within the arch mesenchyme. BMPs induce Edn1, and both induce hand2 expression ventrally and repress jag1b dorsally. (B) Ventral (pink), intermediate (light orange), dorsal (green) and anterior (dark orange) domains are indicated at 30 hpf. BMPs induce hand2 expression as well as Edn1 expression, which in turn induces intermediate domain genes (arrows) but not hand2 at this stage. BMP also promotes trabecular development from the anterior maxillary domain (dark orange). (C) Defects in BMP or Edn1 signaling lead to defects in the corresponding dorsal and ventral cartilages, joints and trabecular cartilages that derive from these different domains.