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. 2016 Jun 1;143(11):2000-11.
doi: 10.1242/dev.129379. Epub 2016 Apr 27.

Bmp Signaling Mediates Endoderm Pouch Morphogenesis by Regulating Fgf Signaling in Zebrafish

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Free PMC article

Bmp Signaling Mediates Endoderm Pouch Morphogenesis by Regulating Fgf Signaling in Zebrafish

C Ben Lovely et al. Development. .
Free PMC article

Abstract

The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face.

Keywords: Bmp signaling; Endoderm morphogenesis; Fgf signaling; Pharyngeal pouches; Zebrafish.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
The reception of Bmp signaling is temporally distinct between the endoderm and CNCCs. (A-K) Confocal images of endoderm (sox17:dsred) and Bmp-responsive cells (BRE:d2GFP) at the indicated stages of zebrafish development. (E-K) Orthogonal (yz-axis) views of A (E,F), B (G,H), C (I,J) and D (K). (L-S) Confocal images of CNCCs (sox10:mRFP) and Bmp-responsive cells (BRE:d2GFP). (P-S) Orthogonal views of L-O. Arrows indicate overlap between transgene expression; arrowheads denote lack of overlap. The endoderm is Bmp responsive by 14 hpf (A-A″) and this overlap persists to 26 hpf (B-D″), but only in the most posterior cells. The ventral CNCCs do not become Bmp responsive until after 18 hpf (L-S). (A-D″,L-O″) Lateral views, anterior to the left. Scale bars: 50 µm.
Fig. 2.
Fig. 2.
Blocking Bmp signaling early disrupts endoderm morphology and craniofacial development. (A,B) Whole-mount images of viscerocranium at 5 days post-fertilization (dpf). Cartilage is blue and bone is red. Arrowheads point to missing cartilage elements and asterisks label the ceratobranchial cartilages. (A) No craniofacial defects are present following DMSO treatment. (B) Dorsomorphin (DM) treatment from 10-18 hpf causes severe craniofacial defects. Ventral views, anterior to the left. Cartilages: Mc, Meckel's; Ch, ceratohyal; Cb's, ceratobranchial; Pq, palatoquadrate; HM, hyomandibular. (C,D) The percentage of viscerocranial defects in embryos treated with Dorsomorphin over multiple time windows. n values for all time points are listed in Table 1. (E,F) Confocal images of control and Dorsomorphin-treated sox17:DsRed embryos. (E′-F″) Imaris surface renderings of E,F. E″,F″ are rotated 45° into the plane of view. (G,H) Confocal images of control or Dorsomorphin-treated sox10:mRFP embryos. Compared with controls (E-E″,G), Dorsomorphin-treated embryos (F-F″,H) had severe defects to both endoderm and CNCC morphology. Lateral views (except for E″,F″, see above), anterior to the left. P, pouch; PA, pharyngeal arch. Scale bars: 100 µm in A; 50 µm in E,G.
Fig. 3.
Fig. 3.
Endoderm requires reception of Bmp signaling for proper development. (A-G) Whole-mount images (ventral view) of 5 dpf viscerocranium. Asterisks label the ceratobranchial cartilages. (D′-E″,G′) Confocal images (lateral view, anterior to the left) of 26 hpf genetic chimeras labeling donor cells with either Alexa 568 dextran (D′,D″), Alexa 488 dextran (E′,E″) or the sox17:dsred transgene (G′). Arrowheads indicate endodermal pouch contribution, whereas arrows indicate endoderm failing to contribute to the pouches; insets show transgene expression with bright-field image. All embryos were heat shocked at 39°C for 1 h at 10 hpf. (A) Heat-shocked hs:dnBmpr1a-GFP embryos have severe craniofacial defects. (B) Control wild-type endoderm transplants do not disrupt craniofacial development. (D-D″) Transplanting wild-type endoderm into a heat-shocked hs:dnBmpr1a-GFP embryo completely rescues craniofacial development (n=15/16). (E-E″, arrows) Heat-shocked hs:dnBmpr1a-GFP donor cells are excluded from the pouches and fail to induce viscerocranial defects in wild-type embryos. (C) No rescue occurs when donor cells fail to populate the endoderm (n=6/6). (F) sox32 mutants lack endoderm and lose all of the viscerocranium. (G,G′) Heat-shocked hs:dnBmpr1a-GFP endoderm does not rescue sox32 mutants (n=6/6). Cartilages: Mc, Meckel's; Ch, ceratohyal; Cb's, ceratobranchial; Pq, palatoquadrate; HM, hyomandibular. Scale bars: 100 µm in A-G; 50 µm in D′-E″,G′.
Fig. 4.
Fig. 4.
Endoderm specification is maintained in Dorsomorphin-treated embryos. Images of 26 hpf control or Dorsomorphin-treated embryos. (A,B) foxa1, (C,D) foxa2, (E,F) pdgfaa and (G,H) pdgfab are all expressed in Dorsomorphin-treated embryos. foxa1 expression is expanded, whereas foxa2 and pdgfab expression is reduced, and pdgfaa expression is normal. Pouch morphology is disrupted in all Dorsomorphin-treated embryos. (A-D) ventral views, (E-H) lateral views, anterior to the left. Scale bars: 50 µm.
Fig. 5.
Fig. 5.
Blocking Bmp signaling does not alter endodermal cell number. (A-F′,M) Confocal images of sox17:dsred transgenic embryos labeled for cell death at 18 and 26 hpf. Similar, low levels of apoptosis are present across all groups (18 hpf, f=0, P=1; 26 hpf, f=0.3, P=0.6, one-way ANOVA, n=4 embryos). (G-L′,N) Confocal images of sox17:dsred transgenic embryos labeled for cell proliferation at 18 and 26 hpf. No differences in the level of proliferating endoderm cells are apparent (18 hpf, f=0.2, P=0.8; 26 hpf, f=0.1, P=0.8, one-way ANOVA, n=4 embryos). Arrows indicate overlap between the transgene and marker expression. Lateral views, anterior to the left. (O) Total endoderm cell numbers were counted via FACS and no statistically significant differences between 26 hpf Dorsomorphin-treated, untreated or DMSO-treated embryos were observed (f=1.8, P=0.2, one-way ANOVA, n=8 samples, 10 embryos per sample). Error bars indicate mean+s.e.m. Scale bar: 50 µm.
Fig. 6.
Fig. 6.
Bmp signaling regulates pouch morphogenesis. Still images from confocal time-lapse movies of sox17:dsred; sox10:EGFP double-transgenic embryos. (A,A′,F,F′) 18 hpf embryos at the initiation of the movie (T0). (A-E′) Pouches (arrows) form in a sequential fashion to segregate the pharyngeal arches (arrowheads) in control embryos. (F-J′) In Dorsomorphin-treated embryos, pouch formation and arch segmentation fail. Lateral views, anterior to the left. Scale bar: 50 µm.
Fig. 7.
Fig. 7.
Fgf signaling activity requires Bmp. (A,B) Endodermal pouches express the Fgf signaling target etv4 at 30 hpf in (A) control but not (B) Dorsomorphin-treated embryos. Lateral views, anterior to the left. (C-H) Expression of the Fgf receptors fgfr1b, fgfr3 and fgfr4 at 18 hpf in control and Dorsomorphin-treated embryos. Only fgfr4 is expressed in the pharyngeal tissues and is reduced in Dorsomorphin-treated embryos (G,H, arrows). (C-F) Dorsal views, (G,H) ventral views, anterior to the left. Scale bars: 50 µm.
Fig. 8.
Fig. 8.
Blocking Bmp reduces the Fgf signaling response in the forming pouch. Still images from confocal time-lapse movies of sox17:dsred; dusp6:d2GFP (Fgf-responsive cells) double-transgenic embryos. (A-A″,F-F″) Embryos at 18 hpf, the initiation of the movie (T0). Arrows indicate overlap between transgene expression, whereas arrowheads indicate lack of overlap. (F-J″) Dorsomorphin-treated embryos show a reduced Fgf signaling response in the endoderm and pouches fail to form, in contrast to controls (A-E″). Lateral views, anterior to the left. Scale bar: 50 µm.
Fig. 9.
Fig. 9.
Bmp and Fgf signaling synergistically interact to achieve proper craniofacial development. Whole-mount images of 5 dpf viscerocranium. (A-C) No gross craniofacial defects are present in wild-type or heterozygous controls (A), wild-type embryos treated with suboptimal doses of Dorsomorphin (B) or untreated fgf8a mutants (C). (D) Treating fgf8a mutants with suboptimal doses of Dorsomorphin causes severe craniofacial defects. (E,F) Similarly, wild-type or smad5 heterozygous embryos do not display any craniofacial defects (E), whereas these embryos treated with suboptimal doses of SU5402 (Pan-Fgf inhibitor, 10-18 hpf) display minor hyomandibular defects (F, arrow). (G) Untreated smad5 mutants have characterized craniofacial defects (Swartz et al., 2011). (H) Suboptimal SU5402-treated smad5 mutants display severe craniofacial defects, including loss of ceratobranchial cartilages (arrowhead). (I-L) Severe craniofacial defects mirroring those of SU5402-treated smad5 mutants (see H) are present in smad5; fgf8a compound mutants (L) and not any other allelic combination (I-K). Ventral views, anterior to the left. Cartilages: Mc, Meckel's; Ch, ceratohyal; Cb's, ceratobranchial; Pq, palatoquadrate; HM, hyomandibular. Scale bar: 100 µm.

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