UDP xylose synthase 1 is required for morphogenesis and histogenesis of the craniofacial skeleton - PubMed (original) (raw)

. 2010 May 15;341(2):400-15.

doi: 10.1016/j.ydbio.2010.02.035. Epub 2010 Mar 11.

Amy Singer, Gabriel A Smith, Zachary A Wood, Yi-Lin Yan, Xinjun He, Samuel J Polizzi, Julian M Catchen, Adriana Rodriguez-Mari, Tor Linbo, David W Raible, John H Postlethwait

Affiliations

UDP xylose synthase 1 is required for morphogenesis and histogenesis of the craniofacial skeleton

B Frank Eames et al. Dev Biol. 2010.

Abstract

UDP-xylose synthase (Uxs1) is strongly conserved from bacteria to humans, but because no mutation has been studied in any animal, we do not understand its roles in development. Furthermore, no crystal structure has been published. Uxs1 synthesizes UDP-xylose, which initiates glycosaminoglycan attachment to a protein core during proteoglycan formation. Crystal structure and biochemical analyses revealed that an R233H substitution mutation in zebrafish uxs1 alters an arginine buried in the dimer interface, thereby destabilizing and, as enzyme assays show, inactivating the enzyme. Homozygous uxs1 mutants lack Alcian blue-positive, proteoglycan-rich extracellular matrix in cartilages of the neurocranium, pharyngeal arches, and pectoral girdle. Transcripts for uxs1 localize to skeletal domains at hatching. GFP-labeled neural crest cells revealed defective organization and morphogenesis of chondrocytes, perichondrium, and bone in uxs1 mutants. Proteoglycans were dramatically reduced and defectively localized in uxs1 mutants. Although col2a1a transcripts over-accumulated in uxs1 mutants, diminished quantities of Col2a1 protein suggested a role for proteoglycans in collagen secretion or localization. Expression of col10a1, indian hedgehog, and patched was disrupted in mutants, reflecting improper chondrocyte/perichondrium signaling. Up-regulation of sox9a, sox9b, and runx2b in mutants suggested a molecular mechanism consistent with a role for proteoglycans in regulating skeletal cell fate. Together, our data reveal time-dependent changes to gene expression in uxs1 mutants that support a signaling role for proteoglycans during at least two distinct phases of skeletal development. These investigations are the first to examine the effect of mutation on the structure and function of Uxs1 protein in any vertebrate embryos, and reveal that Uxs1 activity is essential for the production and organization of skeletal extracellular matrix, with consequent effects on cartilage, perichondral, and bone morphogenesis.

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Figures

Fig. 1

Fig. 1

Craniofacial and skeletal phenotypes of zebrafish larvae. Ventral and lateral views of live (A, B, E, F, I, J, M, N) and Alcian blue-, Alizarin red-stained (C, D, G, H, K, L, O, P) animals. Compared to wild types (A, B), mutant animals (E,F moww60 allele; I, J hi3357 allele) had reduced lower jaws (arrows) at 5 dpf. Reduced lower jaw (arrow) in moww60/hi3357 double heterozygotes (M, N) showed failure of complementation. Alcian blue and Alizarin red staining for cartilage (blue) and bone (red) revealed the lack of cartilage and reduced bones in mutants (G, H, K, L) compared to wild type (C, D) at 7 dpf. Nomarski optics on dissected pharyngeal skeletons suggested that mutant cartilages (P) condensed in the same areas as wild types (O), but did not secrete Alcian blue-positive matrix. Abbreviations: cb1-5, ceratobranchials 1 to 5; ch, ceratohyal; cl, cleithrum; ep, ethmoid plate; hs, hyosymplectic; m, Meckel’s cartilage; op, opercle; pq, palatoquadrate; ps, parasphenoid.

Fig. 2

Fig. 2

Mapping, cloning, and sequencing of mow. (A) LG9 from the HS panel above (Woods et al., 2005) and the interval mapped on the mow mapping panel below. Distances are in centiMorgans (cM). (B) Phylogenetic tree of proteins retrieved by BLASTP search of NCBI, aligned by Clustal-X, and analyzed by neighbor joining showed that the zebrafish protein disrupted by moww60 falls in the Uxs1 clade, well-separated from the next most closely related protein, TGDS. Sequences and sequence identities listed in Materials and methods. (C) Nucleotide sequence comparison of uxs1 from homozygous moww60 mutants to wild-type siblings and reference sequence NM_173242 revealed a G-to-A replacement at nucleotide position 1283 (arrow). (D) Amino acid sequence alignment of the portion of Uxs1 corresponding to the nucleotides shown in part C for zebrafish (WT), zebrafish (moww60) with the arginine (R) to histidine (H) substitution at position 233, human, chicken, frog Xenopus tropicalis, sea squirt Ciona intestinalis (translated from genomic sequence, CINT1.95:scaffold_505), fruit fly Drosophila melanogaster (CG7979-PA), fungus Cryptococcus neoformans (AAM22494) and mustard plant Arabidopsis thaliana (NP_190920). Unless otherwise noted, sequences were the same as those used in panel B. (E) Structure of the zebrafish uxs1 gene, showing the location of the hi3357 viral insert in exon 1 (Amsterdam et al., 2004; Golling et al., 2002; Nissen et al., 2006) and the position of the moww60 nucleotide substitution in exon 9.

Fig. 3

Fig. 3

Effects of the moww60 mutation on Uxs1 function and structure. (A) Capillary zone electrophoresis chromatogram of enzyme reactions catalyzed by wild-type human UXS1 (lower black trace) or mutated UXS1 corresponding to the zebrafish moww60 (R233H, upper red trace). Peaks represent absorbance at 260 nm and are on the same scale, but offset vertically for ease of comparison using Plot (

http://plot.micw.eu

). UXS1 with the moww60 mutation produced no detectable enzyme product, UDP-xylose, after an 18 h incubation with substrate (UDP-glucuronate) at 37 °C but the wild-type enzyme converted nearly all of the substrate into product. (B) Ribbon drawing of the crystal structure of dimerized human UXS1 (PDB entry 2B69), with different monomers colored orange and purple. NAD (red and blue) and the side chains corresponding to zebrafish R233 (green and blue) are depicted as sticks. (C, D) Conserved hydrogen bonding and salt bridge interactions (dashed pale blue lines) revealed the structural consequences of the zebrafish R233H substitution (D) at the dimer interface, compared to wild-type (C). The histidine (red sticks in D) was modeled in several of its common rotomeric states to illustrate the unfavorable contacts it introduces and its inability to satisfy the electrostatic interactions of the R233 guanidinium. Depiction of R233 (green sticks) provided as a frame of reference. Numbering corresponds to zebrafish residues. Figure generated using Pymol (DeLano, 2002).

Fig. 4

Fig. 4

uxs1 expression during zebrafish embryogenesis (A) RT-PCR for uxs1 transcript in animals of indicated ages, along with β-actin positive controls. Maternal uxs1 mRNA was detected at the 1–2 cell stage and detection decreased at sphere-dome stage. Zygotic uxs1 expression appeared to increase gradually and was maintained at least through 5 dpf. (B) Whole-mount in situ hybridization of a one-cell embryo revealed transcript in the fertilized egg. (C, D) Whole-mount (C) and section (D) of 24 hpf embryos illustrated general expression of uxs1 in brain and craniofacial mesenchyme, as well as in the yolk syncytial layer. The dashed line in panel C indicates the plane of section in panel D. (E) Lateral view of whole-mount 2 dpf embryo showed widespread uxs1 expression in the craniofacial region. (F–H) Horizontal sections of 3 dpf (F) and 5 dpf (G,H) animals. Expression of uxs1 became localized to layers of the retina, brain, and cartilages of the pharyngeal arches. Levels of uxs1 transcript were severely reduced or absent in pharyngeal regions of uxs1hi3357 embryos. High magnification of 5 dpf ceratohyals shows uxs1 expression in both chondrocytes (c) and perichondral cells (pc) of wild types (G’), but low transcript levels in uxs1hi3357 embryos (H’). Abbreviations: bp, basal plate; c, chondrocyte; cb1-5, ceratobranchials 1-5; ch, ceratohyal; e, eye; f, fin bud; fb, forebrain; hb, hindbrain; hs, hyosymplectic; mb, midbrain; no, notochord; pc, perichondrium; pq, palatoquadrate; y, yolk; ysl, yolk syncytial layer.

Fig. 5

Fig. 5

Cellular visualization of cartilage and bone morphologies in wild-type and uxs1 mutant larvae. (A–H) Optical sections of live Alizarin red-stained Tg(fli1:EGFP)y1 larvae, ventral views, at 4 dpf and the same individuals at 7 dpf. (E–H) Focus on the ceratohyal. In wild types (A, B, E, F), chondrocytes stacked and were lined with a flattened layer of perichondral cells (white arrow in F). Ossification centers stained with Alizarin red, reflecting perichondral bone formation in the ceratohyal and hyosymplectic and intramembranous ossification in the dentary and maxilla. In homozygous uxs1hi3357 animals (C, D, G, H); however, chondrocytes were disorganized, the perichondral sheath did not align properly (white arrow in H), and Alizarin red-positive ossification centers (dentary, maxilla, and ceratohyal) were severely reduced in perichondral and intramembranous sites. Abbreviations: ch, ceratohyal; de, dentary; hs, hyosymplectic; m, Meckel’s cartilage; max, maxilla; pq, palatoquadrate.

Fig. 6

Fig. 6

Proteoglycan detection in wild-type and uxs1 mutant skeletons. (A–F) Whole-mount wheat germ agglutinin (WGA) staining to visualize _N_-acetylglucosamine, ventral views. (G–J) Whole-mount immunostaining against heparan sulfate (G, H) and chondroitin sulfate (I, J) proteoglycans, ventral views. Dissected pharyngeal cartilages revealed reduced WGA staining in uxs1hi3357 mutants (B, D), compared to wild-type siblings (A, C) at 5 dpf. Higher magnification of ceratohyal regions also showed that WGA-positive material was not deposited normally in mutants (D), compared to organized deposition in wild types (C). Dissected pectoral fins showed that both endoskeletal disc and actinotrichia had less WGA staining and fewer actinotrichia in uxs1hi3357 mutants (F), compared to wild-type siblings (E) at 5 dpf. Immunodetection of heparan sulfate demonstrated that HSPGs were localized to pharyngeal domains in wild type (G), but HSPGs were not detectable in homozygous uxs1hi3357 animals (H). Similarly, immunodetection of chondroitin sulfate was abundant in wild-type cartilages (I), but was absent in uxs1 mutants (J). Abbreviations: at, actinotrichia; cb1-5, ceratobranchials 1-5; ch, ceratohyal; ed, endoskeletal disc; m, Meckel’s cartilage; pq, palatoquadrate.

Fig. 7

Fig. 7

Collagen detection in wild-type and uxs1 mutant skeletons. (A–D) Whole-mount (A, B) and horizontal section (C, D) in situ hybridization for col2a1a gene expression; (E, F) whole-mount immunostaining for Col2 protein; (G–L) whole-mount (G–J) and horizontal section (K, L) in situ hybridization for col10a1 gene expression. Expression of col2a1a increased in developing cartilage of uxs1hi3357 mutants in lateral views of the head at 3 dpf (B) and ceratohyal sections at 5 dpf (D), compared to wild-type siblings (A, C). Longer substrate developing times demonstrated that col2a1a levels are high in cartilage of both wild type and mutant heads at 3 dpf (A’, B’). In contrast, although Col2a1 protein was easily detected in wild type cartilages (E) in ventral view at 5 dpf, it was not detected in mutant cartilages (F). Lateral (G, H) and ventral (I, J) whole-mount views showed that domains of col10a1 gene expression were greatly reduced in regions of endochondral and intramembranous skeletal elements in 5 dpf uxs1 mutants (H, J), compared to wild types (G, I). In situ hybridization on histological sections of the ceratohyal at 5 dpf illustrated reduced perichondral staining of col10a1 in mutants (L), compared with wild types (K). Also, chondrocyte expression of col10a1 was absent in mutants, although wild-type ceratohyal chondrocytes strongly expressed col10a1. Abbreviations: bsr, branchiostegal ray; cb1-5, ceratobranchials 1-5; ch, ceratohyal; cl, cleithrum; de, dentary; ent, entopterygoid; ep, ethmoid plate; f, fin; hm, hyomandibular; hs, hyosymplectic; m, Meckel’s cartilage; max, maxilla; op, opercle; pq, palatoquadrate; ps, parasphenoid.

Fig. 8

Fig. 8

Detection of molecular regulators of skeletogenesis in uxs1 mutant cartilage. (A–N) In situ hybridization on horizontal sections through the ceratohyal for sox9a (A–D), sox9b (E, F), runx2a (G, H), runx2b (I–L), and erm (M, N). Wild-type chondrocytes in the mid-diaphyseal region showed decreased sox9a expression from 3 dpf (A) to 5 dpf (C) as they matured. Not only did chondrocytes of uxs1hi3357 mutants fail to show this down-regulation over time (B, D), but in addition, sox9a expression overall was much higher in mutants compared to wild types. Expression of sox9b was absent in wild-type chondrocytes at 3 dpf (E), but transcripts were detected in uxs1 mutants (F). runx2a expression was obvious in perichondrium of wild types at 3 dpf (G), but was absent in uxs1 mutants (H). runx2b expression was found in perichondrium of wild types at 3 dpf (I) and 5 dpf (K), but was not easily detected in perichondrium of mutants at these timepoints (J, L). In addition, chondrocyte expression of runx2b was much higher in uxs1 mutants compared to wild types at 3 and 5 dpf. Expression of the FGF-responsive gene erm was found in just a few wild-type chondrocytes at 3 dpf (M), whereas erm transcripts were at high levels in all uxs1 mutant chondrocytes (N). Abbreviations: ch, ceratohyal; md, mid-diaphyseal region; pe, perichondrium.

Fig. 9

Fig. 9

Markers of Hedgehog signaling during uxs1 mutant endochondral ossification. (A–J) In situ hybridization on horizontal sections through 5 dpf ceratohyal for ihha (A, B), ihhb (C, D), ptc1 (E, F), ptc2 (G, H), and gli3 (I, J). Wild-type expression of ihha (A) and ihhb (B) appeared similar to that observed in uxs1hi3357 mutant chondrocytes (B, D). Compared to wild-type perichondrium (E, G, I), uxs1 mutant perichondrium demonstrated increased expression of ptc1 (F), ptc2 (H), and gli3 (J).

Fig. 10

Fig. 10

The role of Uxs1 and the extracellular matrix in skeletogenesis. (A) Uxs1 converts UDP-glucuronic acid (open circles) to UDP-xylose (closed triangles), which serves as the linker sugar between the protein core (thick red and blue lines) and subsequent glycosaminoglycan deposition (thin red and blue lines) for heparan sulfate and chondroitin sulfate proteoglycans. Endoplasmic reticulum and secretory organelles are omitted for simplicity. (B) Model for signaling roles of proteoglycans as they mediate interactions between chondrocytes and perichondrium during endochondral ossification. (1) Throughout chondrocyte development, proteoglycans negatively regulate growth factor signaling, such as FGF, that serves to promote sox9 expression, which would otherwise drive col2 transcription. In a similar fashion in maturing chondrocytes, proteoglycans inhibit runx2 expression, which would otherwise drive expression of markers of chondrocyte maturation, such as ihh. (2) When Ihh is produced by maturing chondrocytes, proteoglycans negatively regulate its action on perichondral pre-osteoblasts by ligand sequestration.

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