Loss of Hs3st3a1 or Hs3st3b1 enzymes alters heparan sulfate to reduce epithelial morphogenesis and adult salivary gland function

Abstract

Heparan sulfate 3-O-sulfotransferases generate highly sulfated but rare 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes but that Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. To analyze their in vivo functions, we generated both Hs3st3a1^-/- and Hs3st3b1^-/- single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1^-/- mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1^-/- HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. Understanding how 3-O-sulfation regulates myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration.

Keywords: Basement membrane; HS-3-O-sulfotransferase; Heparan sulfate; Proteoglycan; Salivary gland development; fgf10.

Figure 1.. DotPlot visualization of HS biosynthetic gene expression from scRNA-seq analysis of SMG development.

Embryo day(E)12 SMG, E14 SMG, E16 SMG, Postnatal day (P)1 SMG and adult SMGs. Cell identities listed on y-axis, showing unbiased gene expression per cell cluster identified by log Fold Change; HS biosynthetic genes (features) are listed along the x-axis. Dot size reflects the percentage of cells in a cluster expressing each gene; dot color reflects expression level. Epithelial clusters expressing both Hs3st3a1 and Hs3st3b1 are highlighted with a red box.

Figure 2.. Generation of both Hs3st3a1 and Hs3st3b1 knockout mice.

Schematic and genotyping of the wild-type Hs3st3a1 (A-B) or Hs3st3b1 (C-D) locus, targeting vector and genotyping. A lacZ reporter with a floxed neomycin selection marker replaces most of the coding sequence of the gene. (B and D) Hs3st3a1 and Hs3st3b1 knockout mice genotyping. PCR amplified the WT allele (lower band) and detected the targeting vector (Neomycin cassette- upper band). (E) Data shown are the number of mice of a given genotype for each strain. Chi square analysis shows no significant difference from the expected numbers (F-G) Whole mount X-gal staining in SMG and kidney of E13 Hs3st3a1 KO (F) and Hs3st3b1 KO (G) embryos. Whole-mount X-gal staining was performed on E14 embryos showing SMG and kidneys with positive beta galactosidase staining. Scale bar; 100 μm. (H) Section of X-gal staining of Hs3st3b1 KO SMG in P1 SMG showing positive beta galactosidase staining in cells surrounding the acinar cells (AC). Black arrows point to myoepithelial cells.

Figure 3.. There is reduced FGF10-dependent epithelial morphogenesis in both Hs3st3a1 and Hs3st3b1 KO SMG epithelia.

(A) Images of isolated epithelia from E13 SMGs treated with 400 ng/ml FGF10 for 24 hr. (B) Morphogenic index (number of endbuds × duct length × endbud width, in AU) decreases in Hs3st3a1 and Hs3st3b1 KO epithelia compared to the WT. Error bars represent ±SEM. ANOVA; * p<0.05 and *** p < 0.001, compared to WT. (C) Deletion of Hs3st3a1 decreases expression of Krt19. Deletion of Hs3st3b1 does not affect expression of other genes tested. Gene expression was measured by qPCR and normalized to Rps29 and WT epithelia (dotted line). Error bars: SEM. Unpaired t-test; * p<0.05 and *** p < 0.001.

Figure 4.. Branching morphogenesis of embryonic E13 SMGs from Hs3st3b1, but not Hs3st3a1, KO mice is reduced in ex vivo culture.

(A) Light micrographs of SMGs isolated from E13 embryos and cultured ex vivo for 48 hours. (B) Quantification of the number of endbuds (expressed as a ratio of the number at 48h/the number at 1h) of WT, HET and KO SMGs. Error bars; SEM. One-way ANOVA compared to WT, * p < 0.05, ** p < 0.01. (C) Gene expression changes was normalized to WT control (dotted line) and Rps29. Error bars: SEM. One-way ANOVA compared to WT, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5.. 3-O-sulfation of HS in the basement membrane of embryonic SMGs is reduced in Hs3st3b1 KO but FGF10/FGFR2b complex binding is not altered.

(A) Representative single confocal images of HS4C3V (green), perlecan (red), E-cadherin (magenta) and nuclei in blue. Scale bar: 5 μm. (B) Quantification of fluorescence intensity normalized to total nuclei staining and expressed as a fold change compared to WT. At least five SMGs from three independent experiments were imaged and used for quantification. Error bars: SEM. One-way ANOVA compared to WT, * p < 0.05, ** p < 0.01 and *** p < 0.001. (C) Representative images of single confocal sections showing FGF10-FGFR2b-Fc complex (green), perlecan (red), E-cadherin (magenta) and nuclei (blue). Scale bar: 5 μm. (D) Quantification of fluorescence intensity normalized to total nuclei staining and expressed as a fold change compared to WT. At least five SMGs from three independent experiments were imaged and used for quantification. Error bars: SEM. One-way ANOVA compared to WT, * p < 0.05, ** p < 0.01 and *** p < 0.001. White arrows point to basement membrane.

Figure 6.. Adult Hs3st3a1 and Hs3st3b1 KO SMGs are similar in size and histology as the WT but show differences in HS biosynthetic enzyme transcripts.

(A) Comparison of the weights of the adult SMGs from males and females show no difference. Weights of SMGs are normalized to the weight of the mouse and then normalized to that of the wildtype and expressed as a %. (B) H&E staining of the adult SMG show no gross histological differences between the WT, Hs3st3a1 KO and Hs3st3b1 KO in both male and female. Black asterisks label granular ducts, black arrows point to ducts, and red arrows point to acinar cells. (C-D) Gene expression of HS biosynthetic enzymes and markers of different cell types in adult female SMGs. Gene expression was normalized to wildtype control (dotted line) and Rps29. Error bars: SEM. Unpaired t-test compared to WT, * p < 0.05, ** p < 0.01 and *** p < 0.001. (E) Representative maximum projection images of confocal sections from WT, Hs3st3a1 KO and Hs3st3b1 KO female SMGs showing KRT5 (green), SMA (red), MUC10 (cyan) and nuclei (gray). Scale bar: 10 μm. (F) Quantification of fluorescence intensity normalized to total nuclei staining and expressed as a fold change compared to WT. At least three SMGs with a minimum of 5 different positions were imaged and used for quantification. Error bars: SEM. One-way ANOVA compared to WT, not significant.

Figure 7.. Composition of total GAGs, HS disaccharides and HS staining in adult SMGs.

Analysis of total GAG (A) and HS disaccharide (B) composition between WT, Hs3st3a1 KO and Hs3st3b1 KO SMGs. All data were averaged. Error bars: SD. One-way ANOVA compared to WT, * p < 0.05. N = 4 for each group. (C) Representative images of single confocal sections from WT, Hs3st3a1 KO and Hs3st3b1 KO SMGs showing 10E4 (green), ColIV (red), and nuclei (blue). (D) Representative images of single confocal sections from WT, Hs3st3a1 KO and Hs3st3b1 KO SMGs showing FGF10-FGFR2b-Fc complex (green), ColIV (red), and nuclei (blue). (E-F). Scale bar: 10 μm. Quantification of 10E4 (E) and FGF10/FGFR2b-Fc (F) fluorescence intensity normalized to total nuclei staining and expressed as a fold change compared to WT. Three SMGs were imaged and used for quantification. Error bars: SEM. One-way ANOVA compared to WT, * p < 0.05 or not significant.

Figure 8.. Hs3st3a1 and Hs3st3b1 KO mice drink significantly more water than the wildtype mice, but saliva flow and salivary proteins are not affected.

(A) Female adult mice were tested for 1 h using the Lickometer with free access to water and the number of licks are expressed as a percentage to their respective WT controls for each mouse strain. Data is presented as mean of four mice per groups that was tested 5 different times. Error bars: SD. One-way ANOVA compared to WT, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. (B) Salivary flow rates in adult female mice were determined as described in Materials and Methods. Whole saliva was collected after pilocarpine stimulation. Saliva flow normalized to the WT and shown as %. Mean ± SEM. Dots represent saliva measurements of individual mice. Unpaired t-test for FGF10 HET, ** P < 0.01, as compared to FGF10 WT. One-way ANOVA for Hs3st3a1 KO and Hs3st3b1 KO compared to WT, not significant (C) Quantification of protein concentration in saliva from female mice assessed using BCA assay. Graph shows Mean ± SEM. Dots represent measurements of individual mice. No significant differences detected compared to WT littermates.