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dermatan sulfate glycosyltransferase genes are essential for craniofacial development

['Judith Habicher', 'Department Of Organismal Biology', 'Uppsala University', 'Uppsala', 'Department Of Cellular', 'Computational', 'Integrative Biology', 'Cibio', 'University Of Trento', 'Trento']

Date: 2022-04

Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a -/- fish develop distinct craniofacial defects while the chsy1 -/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a -/- ;chsy1 -/- adults and csgalnact1a -/- ;csgalnact2 -/- larvae. While csgalnact1a -/- ;csgalnact2 -/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1 -/- , csgalnact1a -/- and csgalnact1a -/- ;csgalnact2 -/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.

The components of the extracellular matrix are crucial for interactions and communication between cells during animal development and disease progression. One major component of the extracellular matrix is chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans, which support and modify cell functions and tissue homeostasis. The biosynthesis of CS/DS is complex and no genetic models have been developed to specifically reduce CS/DS in the zebrafish model organism. We have used CRISPR/Cas9 technology to knock out key CS/DS biosynthesis genes. We find that knocking out single genes rarely causes major effects on zebrafish morphology and viability, but by combining several knockout alleles we could observe malformations in the zebrafish craniofacial skeleton. In addition, one combination of alleles was embryonic lethal. Our findings describe the role of CS/DS in the development of the head skeleton and give insights in the regulation of genes involved in CS/DS biosynthesis. The zebrafish mutants generated in this study can be used as tools to further study human diseases caused by mutations in CS/DS biosynthesis enzymes.

Funding: This study was financed by funding to SB and GV from National Human Genome Research Institute (1ZIAHG000183), to JL and LW from SciLifeLab ( www.scilifelab.se ), to JH, JL, DS and LW from the Department of Organismal Biology at Uppsala University ( www.uu.se ) and to LK, AG, and TD from the Foundation for Proteoglycan Research and the Department of Medical Biochemistry and Microbiology at Uppsala University ( www.uu.se ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Zebrafish lines with mutations in HS glycosyltransferases, which reduce HS but not CS/DS accumulation, also affect craniofacial cartilage formation and in addition pectoral fin development and axon sorting [ 11 ]. No genetic knockout of CS/DS biosynthesis enzymes or core proteins has yet been reported in zebrafish. In mouse Csgalnact1 -/- and Chsy -/- animals have been reported to show skeletal phenotypes [ 24 , 25 ]. Morpholino knockdown in zebrafish is a transient alternative to genetic knockouts, but the outcome can differ significantly [ 26 ], either because of morpholino off target effects [ 27 ] or due to genetic compensation [ 28 ]. This highlights the need of genetic mutants to complement knockdown experiments [ 29 ]. In this study we investigate the role of CS/DS in zebrafish development and the importance of individual biosynthesis enzymes. We hypothesized that knockout of one or several glycosyltransferase genes would decrease CD/DS production and affect zebrafish development, allowing us to study the specific role of CS/DS in zebrafish development. For this purpose, we have generated loss-of-function alleles in genes encoding for zebrafish CS/DS biosynthetic enzymes. The generated genetic mutants revealed a functional redundancy for enzymes in CS/DS biosynthesis and by combining different null-alleles, we were able to create a set of lines differing in CS/DS production and displaying varying phenotypes.

The introduction of random mutations in the zebrafish genome has for a long time been used for forward genetic screens, where the mutagens can introduce loss-of-function mutations. A number of genetic mutants with defective GAG biosynthesis have been identified using this approach. Loss-of-function mutations in ughd, uxs1, xylt1, fam20b, b4galt7, b3gat3, genes for precursor transport and synthesis of the shared linkage structure of HS and CS/DS GAGs ( Fig 1 , indicated by #) typically develop abnormal jaw and pharyngeal cartilage structures [ 22 , 23 ], see also recent review [ 12 ]), highlighting the importance for these genes in skeletal development.

Zebrafish (Danio rerio) belonging to the teleost lineage, is a well-established animal model broadly used in biomedical research because of its genetic tractability. Teleosts underwent an additional round of whole genome duplication, compared to mammals and while most of the duplication was eventually lost through “rediplodization”, roughly 20% of zebrafish genes still have two copies in the genome. Zebrafish orthologues of mammalian HS and CS/DS biosynthetic enzymes have been previously identified ( Fig 1 ) [ 11 , 12 ]. In some cases, two gene copies of GAG biosynthetic enzymes are retained (indicated by blue bars in Fig 1 ). In zebrafish the CS/DS biosynthetic enzymes are spatially and temporally regulated during embryonic development, indicating that tissue specific GAG structures exist [ 13 – 21 ].

HS and CS/DS chains are attached to serine (Ser) residues of the core protein. The first four monosaccharides form the linkage region of both HS and CS/DS. Extl3 initiates HS polymerization, while Csgalnact1a and Csgalnact2 perform this function in CS/DS polymerization. Elongation of HS is carried out by Ext enzymes, while Chsy and Chpf enzymes polymerize the CS/DS chain. The first modification of HS is carried out by Ndst enzymes, replacing an N-acetyl groups of GlcNAc residues with an N-sulfate group. Glcea and Glceb epimerize GlcA into IdoA in HS, while Dse, Dsela and Dselb are responsible for this modification in DS. Hs2st enzymes add a sulfate groups to the IdoA C-2 position of HS and Hs6st and Hs3st enzymes add sulfate groups to the GlcNAc or GlcNS residues. Ust adds a sulfate group to the hexuronic acid C-2 position of CS/DS while Chst11, Chst12a, Chst12b, Chst13 and Chst14 are GalNAc 4-O-sulfotransferases while Chst3a, Chst3b, Chst7 and Chst15 are GalNAc 6-O-sulfostransferases. Blue bars indicate two or three zebrafish genes orthologous to a single mammalian gene. Ndst3 in zebrafish is a single gene orthologous to two mammalian genes (NDST3 and NDST4). # Indicates previously published mutant zebrafish alleles while * indicates the targeted mutations reported in this study. Xyl: xylose, Gal: galactose, GlcNAc: N-acetylglucosamine, IdoA: iduronic acid, GlcA: glucuronic acid, GalNAc: N-acetylgalactosamine.

A complex biosynthetic machinery with a variety of enzymes is required to polymerize and modify CS/DS chains in vertebrates ( Fig 1 ). Initiation of the CS/DS chain, on the tetrasaccharide linkage region which is shared between HS and CS/DS, is carried out by Csgalnact1 and Csgalncat2, while further polymerization is performed by Chsy and Chpf enzymes ( Fig 1 ). Then the CS/DS chain is modified by epimerization and sulfation resulting in generation of binding sites for a vast number of proteins, including cytokines and chemokines, growth factors and morphogens, fibrous proteins like collagens, signaling receptors and cell adhesion proteins [ 8 – 10 ].

CS/DS proteogylcans are essential components of cartilage and bone tissues with important roles in development and function of the skeleton [ 2 , 3 ]. The pharyngeal cartilage is derived from migrating neural crest cells and produces an extracellular matrix rich in proteoglycans [ 4 ]. Most of these cartilage structures undergo endochondral ossification [ 5 , 6 ]. Mutations in the human genes encoding CS/DS biosynthesis enzymes cause a number of genetic disorders, often causing skeletal dysmorphism and growth retardation [ 7 ].

Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans are heavily glycosylated proteins, crucial for animal development and homeostasis. They consist of long, unbranched, sulfated glycosaminoglycans (GAGs), covalently attached to core proteins via serine residues. GAGs are composed of repeating disaccharide units of an amino sugar and an hexuronic acid or galactose [ 1 ]. The GAG chains are synthesized in the Golgi apparatus, and the mature proteoglycans are secreted into the extracellular matrix, stored in secretory granules or are incorporated into the plasma membrane. In animals, GAGs are highly abundant and produced by most cells, where a single core protein may contain more than one type of GAG chain [ 1 ].

Results and discussion

Mutagenesis of key CS/DS biosynthetic enzymes To reduce CS/DS biosynthesis in zebrafish we generated loss-of-function alleles in a number of genes encoding enzymes with key roles in CS/DS biosynthesis. We selected a subset of CS/DS modifying enzymes and glycosyltransferases for targeted mutation where we from previous studies expected a key function in CS/DS biosynthesis [12] and where suitable CRISPR targets could be found. We have previously developed protocols for high-throughput CRISPR/Cas9 modification of the zebrafish genome [30,31]. CRISPR targets were designed in the early part of the coding region of the CS/DS glycosyltransferases csgalnact1a, csgalnact2, chsy1, chpfa and the CS/DS sulfotransferases ust, chst3a, and chst7. Using this method, we isolated 20 zebrafish alleles with frame shifts in the coding sequence (Table 1) in seven different genes related to CS/DS biosynthesis (Table 1). PPT PowerPoint slide

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TIFF original image Download: Table 1. Genomic and amino acid sequence for identified loss-of-function alleles. CRISPR target sequences are underlined and the PAM site is colored blue. Aberrant protein sequence is colored in red. Stop codon is indicated by a star (*). § (position of amino acid sequence interruption)/(total number of amino acids in protein). https://doi.org/10.1371/journal.pgen.1010067.t001

Some GAG biosynthetic enzymes are not critical for zebrafish development To find genes with key roles in CS/DS biosynthesis we screened for morphological defects in homozygous mutants in larvae at 6 days post fertilization (dpf) and in adults (Table 2). With the exception of csgalnact1a-/- (discussed below), they all displayed overall normal morphology (Table 2). From these results we conclude that defective gene function of single GAG biosynthetic enzymes typically allows for normal zebrafish development. Given the many enzymes that can both polymerize and modify the CS/DS molecule (Fig 1), this finding suggests widespread redundancy among CS/DS biosynthesis genes and might explain why no loss-of-function alleles in genes encoding for CS/DS biosynthesis enzymes have ever been identified in forward genetic screens, particularly since most screens focused on larval phenotypes. PPT PowerPoint slide

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TIFF original image Download: Table 2. Homozygous mutants survive into adulthood. List of identified alleles and observed general morphology phenotypes at 6 dpf and in adults. (-) = not determined. *In this paper referred to as csgalnact1a-/-, csgalnact2-/- and chsy1-/-, respectively. https://doi.org/10.1371/journal.pgen.1010067.t002

csgalnact1a-/- and chsy1-/- develop malformations in the craniofacial cartilage elements already at larval stages The early skeletal structures in the zebrafish larvae are mainly cartilage. In order to visualize the skeleton already at developmental stages we performed alcian blue staining on csgalnact1a-/-, chsy1-/- and their respective control larvae. Data from optical tomography where then used to generate maximum projections of average patterns resulting in an average 3D representation from 8–10 individuals of each genotype (Fig 4A–4F). These representations show that pharyngeal cartilage structures of mutant larvae at 9 dpf develop all major elements but that in particular csgalnact1a-/- develop a malformed morphology (Fig 4C and 4D). By combining the 3D average structures of mutants with the controls, a rigid alignment showed the altered morphology more clearly as an overlay of mutant (magenta) and control (green) larvae was generated (Fig 4G–4J). The chsy1-/- larvae developed a milder phenotype (Fig 4I–4J) compared to csgalnact1a-/- larvae (Fig 4G–4H). The pharyngeal skeleton in chsy1-/- larvae was smaller compared to control larvae and even further reduced in csgalnact1a-/- larvae (Fig 4K). The mild skeletal phenotype of chsy1-/- larvae was further analyzed in 40 dpf juveniles. The total body length and the head of chsy1-/- juveniles were reduced compared to control fish, reminiscent of the facial dysmorphism and short stature in human patients [39–42]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Malformations in the craniofacial cartilage structures. Maximum projections of average patterns generated from control, csgalnact1a-/- and chsy1-/- alcian blue stained larvae at 9 dpf show the pharyngeal cartilage structures in a ventral view (A-F). Maximum projections for each mutant (magenta) aligned to the control (green) are displayed using a rigid transformation (G-J). Measurements of the length and width of the head skeleton of 9 dpf larvae were performed on maximum projection images as shown in the image to the right (K) and plotted as a factor of control larvae (n = 8 for all genotype groups) (K). csgalnact1a-/- and chsy1-/- larval head skeleton is significantly smaller compared to control (K). Measurements of the standard body length (1) and different other measurements of the head (2–5) are indicated on images of 40 dpf old juvenile fish (L). chsy1-/- juveniles are significantly shorter and have a smaller head compared to control larvae (chsy1-/- n = 17, control n = 27) (L). Statistical significance is indicated by * for p-values <0.05 and ** for p-values <0.005. https://doi.org/10.1371/journal.pgen.1010067.g004

Mutations in different CS/DS glycosyltransferases result in similar effects on transcriptome composition We next investigated how a reduction in CS/DS biosynthesis affected the transcriptome in zebrafish larvae. We chose 6 dpf old larvae depleted of Chsy1, larvae depleted of Csgalnact1a and larvae depleted of Csgalnact1a and Csgalnact2 to include phenotypes ranging from mild to severe (Figs 4 and 5) for the transcriptome analysis. In this study we defined differentially expression of a gene as a two-fold decrease or increase in gene expression (p<0.05). With this definition we detected 98 differentially expressed genes in larvae depleted of Chsy1 and 72 genes in Csgalnact1a depleted larvae. Approximately half of the genes were the same in the two groups (Fig 8A). Although both Chsy1 or Csgalnact1a are involved in CS/DS synthesis, we conclude that effects on the transcriptome of the larva in the respective mutants were not identical. The differences might be due to differential expression in the developing embryo since csgalnact1a expression is restricted mainly to developing cartilage structures while chsy1 is more broadly expressed [38] and this difference is also manifested in the differences in skeletal phenotype (Figs 3 and 4). In larvae depleted of both Csgalnact1a and Csgalnact2, 173 genes were differently expressed compared to the control which was in line with the stronger and lethal phenotype (Fig 5). 18 genes were differently expressed in all three groups. In total 263 genes were differently expressed in at least one of the three mutant lines and the similarity is illustrated by heatmap analysis (Fig 8B). We grouped and colored these genes according to increase or decrease in transcription which revealed an overall striking similarity in the trend of change in expression between the mutant lines (i.e. if the expression of a gene is significantly changed in one mutant line, it is almost always changed in the same direction in the two other mutant lines) (Fig 8B). We conclude that the changes in transcriptome composition was similar but not identical in the three investigated cohorts of larvae. PPT PowerPoint slide

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TIFF original image Download: Fig 8. The area proportional venn diagram (A) shows differentially expressed genes in csgalnact1a-/-*, csgalnact1a-/-;csgalnact2-/- and chsy1-/- larvae compared to control larvae. The heat map (B) shows all genes with a two-fold increase (red) or decrease (blue) in the csgalnact1a-/-, csgalnact1a-/-;csgalnact2-/- or chsy1-/- larvae. * 50% of the csgalnact1a-/- individuals lack one functional allele of chsy1. https://doi.org/10.1371/journal.pgen.1010067.g008

Mutations in csgalnact1a, csgalnact2 or chsy1 genes do not activate compensatory transcription of CS/DS glycosyltransferase genes The fully viable and fertile adult chsy1-/- phenotype was unexpected given the severe embryonic phenotype reported for chsy1 morpholino knockdowns and distinct skeletal phenotype in patients with mutations the human ortholog CHSY1 [38,39]. The common observation of stronger morpholino knockdown phenotypes as compared to genetic knockout phenotypes has been discussed in recent years. One explanation may be that many reported morpholino phenotypes are at least in part a result of off-target effects [26]. Another possibility may be genetic compensation in mutants triggered by mutant mRNA degradation (i.e. nonsense-mediated decay) which induces compensatory transcription of genes with sequence similarities (i.e. paralogues) [28,45]. Candidates for genetic compensation in chsy1-/- mutants would be other genes in the GAG biosynthesis machinery with high sequence similarity (Fig 1). However, all GAG glycosyltransferase genes identified on the microarray (chsy1, chpf2, chpfa, chsy3, csgalnact1a and csgalnact2) were expressed similarly in chsy1-/- and control larvae, indicating that the induced frameshift mutations do not increase the degradation of glycosyltransferase mRNA (S2 Table). The same is true for csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- mutants (S2 Table). We further note that the reduced CS/DS biosynthesis in chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae does not affect transcription of any GAG sulfotransferases, epimerases, sugar transporters and degrading enzymes. Thus, in the three mutant lines investigated, we found no evidence for genetic compensation due to mRNA decay. Our data further indicates that no mechanism exists to increase transcription of GAG biosynthesis genes as a consequence of missing CS/DS glycosyltransferases or decreased CS/DS accumulation.

[END]

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