- Title
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Essential Role of Nr2f Nuclear Receptors in Patterning the Vertebrate Upper Jaw
- Authors
- Barske, L., Rataud, P., Behizad, K., Del Rio, L., Cox, S.G., Crump, J.G.
- Source
- Full text @ Dev. Cell
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Craniofacial and ocular phenotypes in Nr2f mutants. (A) Nr2f deletion alleles generated in this study. Care was taken to introduce early frame-shift mutations likely abrogating all protein function. The mutation sites in each gene are indicated with arrows. (B) Alcian blue and Alizarin red staining of nr2f1a, nr2f1b, nr2f2, and nr2f5 single mutants at 5 dpf revealed no gross skeletal abnormalities. Combinatorial loss of nr2f1b, nr2f2, and nr2f5 alleles led to fusion of the hyoid joint (black arrow) and reduction of the dorsal hyomandibula cartilage and opercle bone (magenta arrow). Loss of nr2f1a in nr2f5 mutants deficient for nr2f1b and nr2f2 in some cases led to syngnathia (open arrowhead). The full facial skeleton is reduced in quadruple mutants. The probable identities of residual fused cartilages in the quadruple mutant are indicated. M, Meckel’s cartilage; Pq, palatoquadrate; Ch, ceratohyal. (C) First arch origins of the retroarticular process of Meckel’s cartilage in zebrafish. (Left panel) sox10:kikGR+ cells in the center of the first arch were photoconverted from green to red (magenta) fluorescence by selective exposure to UV light at 35 hpf. The right panel shows the same fish at 5 dpf, with residual photoconverted protein (magenta, also see bottom right panel) in cells within the retroarticular process (ra) and jaw joint (jj) region. The boxed area is magnified in the bottom left images. Images are maximum intensity projections except the high magnifications, which are single optical sections. (D) Coloboma in nr2f2; nr2f5 mutants (magenta triangle) was fully penetrant and allowed for straightforward identification of double mutants as early as 30 hpf. Scale bars: B = 50 μm, C = 20 μm, D = 250 μm |
Analysis of osteoblast differentiation, cell death, and proliferation in nr2f5 misexpression embryos. (A) The sox10:DsRed transgene (magenta) and Sp7 antibody (green) label chondrocytes and osteoblasts, respectively. Note the strong Sp7 staining in the second arch-derived branchiostegal ray bone (Br). No ectopic Sp7 staining was detected in the vicinity of the transformed Meckel’s (M) cartilage. Pq, palatoquadrate; Ptp, pterygoid process (out of view in control image). (B) Similar numbers of apoptotic cells (green) were seen in the first two arches of control and sox10:Gal4VP16; UAS:nr2f5 transgenic embryos (nr2f5 OE) at 32 hpf (Mann Whitney U, p > 0.05). Arch NCCs express sox10:DsRed (mCherry antibody staining, magenta), and nuclei were labeled with Hoechst (blue). Dashed lines indicate arch boundaries. (C) nr2f5-misexpressing embryos showed a marginally significant decrease in the number of dividing cells (pHH3+, magenta) in the first two arches relative to controls (Mann Whitney U, p < 0.05). Arch NCCs express fli1a:EGFP (GFP antibody staining, green). The number of embryos evaluated for each genotype is indicated at the bottom left. Scale bars = 20 μm. |
Additional gene expression patterns in nr2f2; nr2f5 mutants. (A) pitx2 and meis2b (magenta) were classified as Nr2f-activated genes by RNAseq but were not consistently altered in nr2f2; nr2f5 mutants. pitx2 was minimally expressed in dlx2a+ arch NCCs (green) but detected in the adjacent ocular mesenchyme (oc). meis2b was mainly confined to the hyoid arch. (B) jag1b expression (magenta) in non-maxillary dorsal cells was largely unaffected in Nr2f mutants. (C) Frontonasal (fn) NCC expression of alx1 and gata3 (magenta) was preserved in nr2f2; nr2f5 mutants. (D) The neurocranium, including the NCC-derived anterior portion, was slightly smaller but otherwise typically formed in nr2f2; nr2f5 mutants. Scale bars: A-C = 20 μm, D = 50 μm. |
Nr2f expression in fish with loss or gain of Edn1. Two-color images corresponding to the Nr2f in situ hybridizations from Fig. 5. The dashed lines in Fig. 5 were drawn based on labeling of all arch NCCs (green) by dlx2a. Nr2f expression (magenta) is elevated to varying degrees in the mandibular domain of edn1 mutants, (white arrows), whereas Edn1 misexpression induced by heat-shocking hsp70l:Gal4; UAS:Edn1 embryos from 20-24 hpf abolished most arch Nr2f expression. Scale bar = 20 μm. |
Scoring system for Meckel’s rescue. The degree of lower jaw rescue in nr2f; edn1 mutants was scored as 1 (typical edn1 mutant) through 5 (typical wild-type) as described. Representative Alcian blue and Alizarin red-stained skeletons and their associated genotypes are shown. |
Reprinted from Developmental Cell, 44(3), Barske, L., Rataud, P., Behizad, K., Del Rio, L., Cox, S.G., Crump, J.G., Essential Role of Nr2f Nuclear Receptors in Patterning the Vertebrate Upper Jaw, 337-347.e5, Copyright (2018) with permission from Elsevier. Full text @ Dev. Cell