Dynamic Expression of Genes Involved in Proteoglycan/Glycosaminoglycan Metabolism during Skin Development

Abstract

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

During various cell signaling processes, glycosaminoglycans (GAGs), such as heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), play a role in binding, guiding, and modulating signaling molecules, e.g., growth factors and morphogens [1–3]. In skin this role can be illustrated by the importance of GAGs in adult wound healing [2, 4] and in the extracellular matrix architecture formed during dermal development [5, 6]. A further example to illustrate the importance of GAGs comes from mice overexpressing heparanase, an enzyme involved in the degradation of HS, showing accelerated hair growth [7], indicating its involvement in hair follicle morphogenesis and homeostasis. Other observations show that HS is involved in hair follicle cycling, sebaceous gland morphogenesis, and homeostasis [8]. Finally, HS and heparanase influence wound healing in adult mice by enhancing keratinocyte migration and stimulating blood vessel maturation [9]. Taken together, GAGs play an important role in skin healing and development and this prompted us to evaluate the expression of GAG related genes during (embryonic) development in skin.

Inhibition of the expression of genes coding for enzymes involved in GAG modification reactions clearly indicates the importance of GAGs during organogenesis [10], especially with respect to growth factor handling. For example, mice deficient in Ndst1 (N-deacetylase sulfotransferase isoenzyme 1) die neonatally due to several defects in which defective sonic hedgehog (Shh) signaling is implicated [11, 12]; mice deficient in Hs2st (heparan sulfate 2-O sulfotransferase) or Glce (glucuronic acid epimerase) display renal agenesis [13, 14], whereas mice deficient in Hs6st1 (heparan sulfate 6-O sulfotransferase isozyme 1) show aberrant signaling of VEGF (vascular endothelial growth factor) and impaired lung development [15]. A skin phenotype of the above mouse models, however, has not been reported.

In general, it is thought that specific modifications of the GAG chain are involved in the binding and modulation of signaling molecules resulting in cell-type and/or tissue specific reactions [2, 3]. GAG mimetics like the RGTAs (regenerating agents) have been used to treat skin disorders and improve skin healing [16, 17]. To obtain insight in GAG metabolism during skin development we studied the expression of GAG related genes covering six functional classes ranging from the synthesis of precursor molecules to the synthesis and degradation of GAGs. In addition, we probed the expression of a number of (GAG binding) signaling molecules.

2. Materials and Methods

An overview of the experimental setup on the gene expression during murine skin development is given in Figure 1.

[figure omitted; refer to PDF]

2.1. Animals for the Study on Skin Development

NIH guidelines for the care and use of laboratory animals (NIH publication 85–23 Rev. 1985) were followed. The study was approved by the Ethics Committee of the Radboud university medical center (DEC 2005-111, project: 81027). C57BL6/j mice were obtained from Elevage Janvier (Le Genest Saint Isle, France). Mice aged 90 days (90 days post birth [P90]) were used for timed mating and dorsal skin was collected at 14 days (E14) and 16 days after conception (E16). At E14 hair follicle development is initiated, and at E16 this process is almost completed in combination with a stratified epidermis and organized dermis [18, 19]. For the RNA samples of E14, dorsal skin of seven embryos from one female was pooled and used for RNA isolation. Skin was isolated at E14 by snap freezing the whole embryo in liquid nitrogen followed by scraping the skin layer in a cryomicrotome with a scalpel to minimize contamination with other embryonic tissues (skin is very thin at this time point). Samples were stored at -80°C. RNA samples for E16 were taken from two females, collecting dorsal skin form 7 embryos each. In addition, skin from 1-day old pups (P1) and adult mice (P90) was collected. At P1 skin is more organized and has been exposed to air [18, 19]. For the two dorsal skin samples for P1, three pups from two females were taken per sample. Two adult three-month old mice were used for the two dorsal skin samples at P90. Samples for RNA isolation for E16, P1, and P90 were collected by removing dorsal skin and snap freezing it in liquid nitrogen and storage at -80°C.

2.2. Tissue of Genetically Modified Mice

Skin samples of glucuronic acid epimerase (Glce) knockout mice (E18.5 for expression analysis; E17.5 and E18.5 for histological analysis) and of heparanase overexpression (Hpse) mice (P70) were provided by Prof. Dr. Jin-Ping Li (Department of Medical Biochemistry and Microbiology, University of Uppsala, Sweden) and Prof. Dr. Israel Vlodavsky (Vascular and Cancer Biology Research Center Rappaport Faculty of Medicine and Research Institute Technion-Israel Institute of Technology, Israel), respectively [7, 20]. For RNA isolation two wildtype and two mutant mice were used of both mouse models.

2.3. RNA Isolation, Real Time Quantitative PCR, and Microarray Analysis

Frozen samples were grinded in a micro-dismembrator (Sartorius, Bunnik, The Netherlands) and RNA was isolated using the TRIZOL-method (Invitrogen, Paisley, UK) in combination with RNeasy Mini kit with DNAse step (Qiagen, Hilden, Germany). RNA quality was assessed using the Bioanalyzer system (Agilent Technologies, Amstelveen, The Netherlands). The RNA integrity numbers (RIN, 27) were 8.8±0.25 (technical replicate N=2), 8.0±0.35, 8.5±0.55, and 7.3±0.2 for E14, E16, P1, and P90 (biological replicates N=2), respectively. The same procedure was used for the RNA isolation for the Glce knockout mouse and Hpse overexpression mouse. The RIN was 6.5±0.51 for Glce-/- samples and 8.0±0.48 for Glce+/+ and 6.3±0.3 and 7.7±0.6 for HPA-TG and HPA-WT, respectively (all biological replicate N=2).

Gene Chip Mouse exon 1.0 ST Arrays (Affymetrix, High Mycombe, UK) were used to analyze gene expression for E14, E16, P1, and P90 using 1 μg of RNA per chip. Expression data were preprocessed to check sensitivity and specificity of the results based on Kadota et al. (2009) as shown in Uijtdewilligen et al. (2016) [18, 21]. Gene level expression data were calculated for the CORE transcripts (probe sets supported by RefSeq mRNAs) using Affymetrix Expression Console software with quantile normalization (all arrays are considered to have an equal intensity distribution), GC-content background correction (probes with high GC-content hybridize better, corrected for with built-in probes with different known GC-contents) and summarization with the RMA algorithm [22]. Data were imported into GeneSpring GX 7.3 (Agilent Technologies), duplicates were averaged, and the expression of each transcript was normalized to the median per array.

Real Time-Quantitative PCR (qPCR) was performed using custom designed Taqman Low Density arrays (TLDA) (Applied Biosystems, Nieuwerkerk aan de IJssel, the Netherlands) containing probes against genes involved in GAG metabolism and GAG binding proteins (Supplementary data Table 1). Glce and Hpse samples were analyzed using qPCR using custom designed TLDA with an adapted design containing additional GAG related genes (Supplementary data Table 2).

For the TLDA cards, 100 ng cDNA in Taqman Universal PCR Master Mix (Applied Biosystems) was loaded on the TLDA card per slot and run on a 7900HT Fast Real Time PCR System (Applied Biosystems). Expression was analyzed based on the threshold cycle (Ct) which was obtained using the SDS 2.3 software and RQ Manager 1.2 of Applied Biosystems using the combined expression data of the tested TLDA cards. In Microsoft Excel the reference genes for ΔCt calculation were checked for stability of expression by analyzing the results of the reference genes across all used TLDA cards and selecting the reference genes with the smallest deviation across the cards tested. Subsequently ΔCt values were calculated using a reference gene with the smallest difference between the average Ct found for the gene of interest and for the reference gene. The obtained ΔCt values were further processed using the $2^{-\Delta \Delta \mathrm{C}\mathrm{t}}$ method using P90 as a calibration point in case of the developmental study and the wild type background (C57BL6 mice) data in case of the two mouse models [23].

2.4. Statistical Methods

Statistical significance of the exon array data was analyzed using ANOVA and Benjamini-Hoghberg multiple testing correction [24]. Statistical significance of the TLDA card data was tested with an unpaired T-test (2-tailed) using Microsoft Excel. Data with a statistical threshold of p<0.10 and a fold threshold of >2.0 were considered statistically significant.

3. Results

Genes involved in glycosaminoglycan (GAG) synthesis, modification, and degradation were studied during skin development at 14 and 16 days after conception (E14 and E16, respectively) and at one day after birth (P1) and compared to mature skin of a 3 month old mouse (P90). In addition, two mouse models, a Glce knockout mouse (E18.5) and an Hpse overexpression mouse model (P70), were analyzed. Taqman Low Density Array (TLDA) cards were designed to contain genes involved in GAG metabolism (Supplementary data Tables 1 and 2). The expression data obtained using TLDA cards and exon arrays were screened for genes with 2-fold differential expression at a statistical threshold of p<0.10 (Tables 1, 2, 3, and 4, Supplementary data Tables 4, 5, and 6).

Table 1

Comparison of the number of differentially expressed genes during skin development in mice (p<0.10, fold>2.0) based on real-time qPCR and on exon array analysis.

Total genes	System		E14 vs. P90			E16 vs. P90			P1 vs. P90
			Down	Up		Down	Up		Down	Up
Production of precursors
43	TLDA		1	4		3	2		1	2
43	Exon		2	6		2	0		1	0
	Overlap		0	2		2	0		1	0
Core proteins
14	TLDA		2	3		1	1		2	2
14	Exon		3	2		1	1		0	2
	Overlap		2	2		0	1		0	2
Preparation of linkage region
8	TLDA		0	1		0	1		0	1
8	Exon		0	2		0	0		0	0
	Overlap		0	0		0	0		0	0
Glycosaminoglycan chain polymerisation
13	TLDA		1	4		1	2		0	2
13	Exon		1	2		0	0		0	0
	Overlap		0	0		0	0		0	0
Glycosaminoglycan chain modification
32	TLDA		1	9		0	5		0	8
32	Exon		1	3		0	3		0	1
	Overlap		1	2		0	1		0	0
Glycosaminoglycan chain degradation
19	TLDA		2	2		3	1		0	1
19	Exon		3	1		1	1		2	0
	Overlap		2	1		1	1		0	0
Growth factors
37	TLDA		0	13		3	8		1	11
37	Exon		2	14		3	10		1	4
	Overlap		0	10		2	8		0	4

$\ast$ P values for the exon array measurements were calculated using Benjamini–Hochberg multitesting correction. P values for the TLDA assay were calculated using an unpaired T-test.

Overlap refers to genes differentially expressed in both TLDA card and exon array.

Table 2

Differentially expressed GAG related genes during skin development in mice in comparison to mature skin (p<0.10) based on real-time qPCR.

			E14 vs. P90			E16 vs. P90			P1 vs. P90
Gene symbol	Full gene name and probe set		P-value	Relative change		P-value	Relative change		P-value	Relative change
Production of precursors
Galk1 ‡	Galactokinase 1		0.029	5.897		0.057	3.867		0.074	3.043
	Mm00444182_m1
Galt ‡	Gal-1-P-Uridylyltransferase		0.042	0.573		0.027	0.497		0.832	0.964
	Mm00489459_g1
Gfpt1 ‡	Glu-Fru-6-P-Transaminase 1		0.002	2.378		0.160	1.273		0.282	1.481
	Mm00600127_m1
Gfpt2 ‡	Glu-Fru-6-P-Transaminase 2		0.269	0.772		0.079	0.494		0.356	0.780
	Mm00496565_m1
Hk2 ‡	Hexokinase 2		0.027	0.652		0.253	1.390		0.586	0.904
	Mm00443385_m1
Pgm3 ‡	Phosphoglucomutase 2		0.058	1.876		0.296	1.249		0.098	1.470
	Mm00459270_m1
Pgm5 ‡	Phosphoglucomutase 5		0.002	4.053		0.033	2.792		0.019	2.515
	Mm00723432_m1
Slc13a5 ‡	Solute Carrier Family 13 Member A5		Not detected			Not detected			Not detected
	Mm00523288_m1
Slc26a9 ‡	Solute Carrier Family 26 Member A9		Not detected			0.036	0.264		0.023	0.318
	Mm00628490_m1
Slc35a3 ‡	Solute Carrier Family 35 Member a3		0.008	0.444		0.060	0.567		0.455	0.811
	Mm00523288_m1
Core proteins
Cd44	CD44 Molecule		0.018	0.536		0.171	0.637		0.223	1.385
	Mm01277164_m1
Gpc2 ‡	Glypican 2		0.005	11.686		0.638	1.294		0.369	1.249
	Mm00549650_m1
Gpc3 †	Glypican 3		<0.001	6.765		0.019	3.734		0.013	8.044
	Mm00516722_m1
Gpc6 ‡	Glypican 6		0.027	2.880		0.275	1.376		0.130	1.926
	Mm00516235_m1
Hspg2 ‡	Perlecan		0.030	1.504		0.064	1.959		0.007	3.325
	Mm01181179_g1
Sdc1 ‡	Syndecan 1		0.012	0.293		0.107	0.313		0.069	0.403
	Mm00448918_m1
Sdc4 †	Syndecan 4		0.002	0.201		0.039	0.240		0.065	0.471
	Mm00488527_m1
Preparation of linkage region
B3gat1	β-1,3-Glucuronyltransferase 1		Not detected			Not detected			Not detected
	Mm00661499_m1
B3gat2	β-1,3-Glucuronyltransferase 2		Not detected			Not detected			Not detected
	Mm00549042_m1
B4galt2 ‡	β-1,4-Galactosyltransferase 2		0.023	4.562		0.059	2.679		0.038	3.384
	Mm00479556_m1
Glycosaminoglycan chain polymerisation
Chpf ‡	Chondroitin Polymerizing Factor		<0.001	2.991		0.059	1.865		0.050	2.401
	Mm01262239_g1
Chsy1 ‡	CS Synthase 1		0.013	3.900		0.024	2.282		0.072	2.229
	Mm01319178_m1
Chsy3 ‡	CS Synthase 3		0.026	4.006		0.075	2.184		0.101	2.013
	Mm01545329_m1
Csgalnact1 ‡	CS-GalNAc-transferase 1		0.099	0.496		0.098	0.475		0.634	1.128
	Mm00555164_m1
Extl1 ‡	Exostoses (multiple)-like 1		Not detected			Not detected			Not detected
	Mm00621977_s1
Extl2 ‡	Exostoses (multiple)-like 2		0.007	2.043		0.660	1.220		0.106	1.765
	Mm00469621_m1
Has2 ‡	Hyaluronan Synthase 2		0.188	1.933		0.235	1.705		0.410	1.577
	Mm00515089_m1
Glycosaminoglycan chain modification
Chst11 ‡	Chondroitin 4-O-Sulfotransferase 1		0.002	3.000		0.087	1.195		0.087	1.957
	Mm00517563_m1
Chst14 ‡	Dermatan 4 Sulfotransferase 1		0.026	2.459		0.156	1.513		0.203	1.707
	Mm00511291_s1
Chst2 ‡	Carbohydrate Sulfotransferase 2		0.014	3.664		0.010	3.145		0.006	2.773
	Mm00490018_g1
Chst3 ‡	Chondroitin 6-O-Sulfotransferase 1		0.041	3.241		0.152	1.941		0.028	3.550
	Mm00489736_m1
Chst8 ‡	GalNAc-4-O-Sulfotransferase 1		0.089	2.587		0.221	0.591		0.139	2.280
	Mm00558321_m1
Hs3st1 ‡	HS 3-O-sulfotransferase		0.051	1.796		0.039	1.937		0.027	2.809
	Mm01964038_m1
Hs3st3b1‡	HS 3-O-sulfotransferase 3b1		0.004	3.204		0.028	2.511		0.002	2.629
	Mm00479621_m1
Hs3st6 ‡	HS 3-O-sulfotransferase 6		0.006	0.208		0.089	0.664		0.041	1.765
	Mm01299930_m1
Hs6st2	HS 6-O-sulfotransferase 2		Not detected			Not detected			Not detected
	Mm00479296_m1
Ndst1 ‡	N-deacet./N-sulfotrans. 1		0.118	1.487		0.140	1.449		0.054	2.202
	Mm00447005_m1
Ndst2 ‡	N-deacet./N-sulfotrans. 2		0.008	1.347		0.001	2.017		0.002	2.021
	Mm00447818_m1
Ndst3 ‡	N-deacet./N-sulfotrans. 3		0.004	4.708		0.041	7.910		0.004	12.034
	Mm00453178_m1
Sulf1 ‡	Sulfatase 1		0.004	4.644		0.079	2.674		0.089	2.077
	Mm00552283_m1
Sumf2 ‡	Sulfatase modifying factor 2		0.008	2.657		0.104	2.023		0.038	1.857
	Mm01197721_m1
Glycosaminoglycan chain degradation
ArsJ ‡	Arylsulfatase J		0.013	7.805		0.010	12.075		0.014	7.146
	Mm00557970_m1
ArsK ‡	Arylsulfatase K		0.306	0.801		0.059	0.466		0.143	0.678
	Mm00513099_m1
Galns ‡	Galactosamine (N-Acetyl)-6-Sulfatase		<0.001	2.648		0.066	1.674		0.091	1.584
	Mm00489575_m1
Hpse ‡	Heparanase		0.044	0.304		0.342	1.450		0.169	0.578
	Mm00461768_m1
Hyal1 ‡	Hyaluronoglucosamini-dase 1		0.001	0.198		0.008	0.288		0.006	0.607
	Mm00476206_m1
Sgsh ‡	N-Sulfoglucosamine Sulfohydrolase		0.055	0.647		0.002	0.435		0.644	0.897
	Mm00450747_m1
Growth factors
Areg	Amphiregulin		Not detected			Not detected			0.656	0.834
	Mm00437583_m1
Bmp3 ‡	Bone morphogenetic growth factor 3		0.007	4.270		0.004	7.240		0.002	11.628
	Mm00557790_m1
Bmp5 ‡	Bone morphogenetic growth factor 5		0.022	12.051		0.303	1.788		0.587	1.300
	Mm00432091_m1
Ctgf ‡	Connective tissue growth factor		0.668	1.079		0.011	0.232		0.076	0.580
	Mm01192931_g1
Fgf10 ‡	Fibroblast growth factor 10		0.063	1.649		0.050	1.801		0.093	2.262
	Mm00433275_m1
Fgf13 ‡	Fibroblast growth factor 13		0.002	3.205		0.059	1.750		0.181	1.544
	Mm00438910_m1
Fgf2 ‡	Fibroblast growth factor 2		0.277	0.651		0.166	0.539		0.382	1.446
	Mm01285715_m1
Fgf20	Fibroblast growth factor 20		Not detected			Not detected			Not detected
	Mm00748347_m1
Fgf22 ‡	Fibroblast growth factor 22		Not detected			0.386	0.632		0.060	0.614
	Mm00445749_m1
Fgf7 ‡	Fibroblast growth factor 7		0.045	0.606		0.002	0.394		0.087	0.630
	Mm00433291_m1
Fgf8	Fibroblast growth factor 8		Not detected			Not detected			Not detected
	Mm00438921_m1
Figf ‡	C-fos induced growth factor		0.081	1.397		0.030	1.787		0.545	0.834
	Mm01131929_m1
Gdf10 ‡	Growth differentaition factor 10		0.015	3.181		0.454	1.143		0.166	1.519
	Mm03024279_s1
Hbegf ‡	Heparin-binding epidermal growth factor		Not detected			0.015	0.347		0.016	0.423
	Mm00439305_g1
Hdgf †	Hepatoma-derived growth factor		0.257	1.221		0.737	0.911		0.975	1.008
	Mm00725733_s1
Igf1 ‡	Insulin-like growth factor 1		0.320	1.207		0.217	0.705		0.364	0.790
	Mm00439560_m1
Igf2 †	Insulin-like growth factor 2		<0.001	592.335		0.002	338.094		0.001	416.096
	Mm00439565_g1
Nog ‡	Noggin		0.054	2.945		0.019	2.935		0.021	3.067
	Mm01297833_s1
Pdgfa ‡	Platelet-derived growth factor a		0.021	3.005		Not detected			0.016	3.669
	Mm01205760_m1
Pdgfb ‡	Platelet-derived growth factor b		0.321	1.098		0.033	1.468		0.010	2.096
	Mm01298578_m1
Pdgfc ‡	Platelet-derived growth factor c		Not detected			0.016	2.362		Not detected
	Mm00480205_m1
Pdgfd ‡	Platelet-derived growth factor d		Not detected			0.288	0.709		0.139	1.644
	Mm00546829_m1
Shh	Sonic hedgehog		Not detected			Not detected			Not detected
	Mm00436527_m1
Tgfb1 ‡	Transforming growth factor beta 1		0.027	0.540		0.488	0.817		0.157	1.372
	Mm01178820_m1
Tgfb2 ‡	Transforming growth factor beta 2		0.039	2.697		0.757	1.081		0.127	1.809
	Mm01321739_m1
Tgfb3 ‡	Transforming growth factor beta 3		0.033	2.420		0.094	1.854		0.034	2.517
	Mm01307950_m1
Vegfa ‡	Vascular endothelial growth factor a		0.394	1.108		0.112	1.613		0.461	1.358
	Mm01281447_m1
Vegfb	Vascular endothelial growth factor b		Not detected			Not detected			Not detected
	Mm00442102_m1
Vegfc ‡	Vascular endothelial growth factor c		0.024	1.839		0.303	1.250		0.015	1.996
	Mm00437313_m1
Wnt10b ‡	Wingless-related integration site 10b		0.180	5.829		0.122	9.688		0.105	11.748
	Mm00442104_m1
Wnt16 ‡	Wingless-related integration site 16		0.066	2.094		0.016	4.809		0.014	4.362
	Mm00446420_m1
Wnt2 ‡	Wingless-related integration site 2		0.054	3.144		0.090	3.555		0.074	3.760
	Mm00470018_m1
Wnt2b	Wingless-related integration site 2b		Not detected			Not detected			Not detected
	Mm00437330_m1
Wnt3a ‡	Wingless-related integration site 3a		0.394	1.610		0.441	1.520		0.840	1.106
	Mm00437337_m1
Wnt6 ‡	Wingless-related integration site 6		0.015	11.709		0.018	10.400		0.016	10.758
	Mm00437353_m1
Wnt7a	Wingless-related integration site 7a		Not detected			Not detected			Not detected
	Mm00437355_m1
Wnt7b ‡	Wingless-related integration site 7b		0.003	4.180		0.056	6.076		0.003	4.181
	Mm00437357_m1

Numbers in italic are significant (p<0.10); numbers in bold are >2-fold differentially expressed. Gene symbols indicated with a †-symbol are normalized using GAPDH as a reference gene. Gene symbols indicated with a ‡-symbol are normalized using TBP as a reference gene. Genes, for which a signal was not or only partly detected at a given time point or multiple time points and therefore a fold change and/or p value could not be calculated based on the available data, are given as “not detected.” Gene symbols for which all time points were classified as “not detected” do not show a symbol for the used reference gene due to lack of data for a calculation.

Table 3

Differentially expressed GAG related genes during skin development in mice in comparison to mature skin (p<0.10) based on gene Chip Mouse Exon 1.0 ST Arrays.

			E14 vs. P90			E16 vs. P90			P1 vs. P90
Gene symbol	Full gene name and probe set		Stepup P-value	Fold change		Stepup P-value	Fold change		Stepup P-value	Fold change
Production of precursors
Galk1	Galactokinase 1		0.030	4.242		0.126	2.483		0.231	2.194
	6792485
Galt	Gal-1-P-Uridylyltransferase		0.664	0.814		0.231	1.864		0.665	1.340
	6912944
Gfpt1	Glu-Fru-6-P-Transaminase 1		0.020	2.110		0.077	1.682		0.801	1.081
	6947679
Gfpt2	Glu-Fru-6-P-Transaminase 2		0.176	0.610		0.087	0.426		0.229	0.547
	6780767
Hk2	Hexokinase 2		0.003	0.451		0.040	1.375		0.039	0.655
	6954982
Pgm3	Phosphoglucomutase 2		0.083	2.193		0.676	1.178		0.654	1.286
	6997513
Pgm5	Phosphoglucomutase 5		0.058	2.338		0.104	2.209		0.291	1.696
	6872290
Slc13a5	Solute Carrier Family 13 Member A5		0.003	2.588		0.059	1.322		0.576	1.075
	6789531
Slc26a9	Solute Carrier Family 26 Member A9		0.012	0.227		0.090	0.471		0.091	0.392
	6753079
Slc35a3	Solute Carrier Family 35 Member A3		0.105	0.767		0.596	0.925		0.572	0.890
	6908510
Core proteins
Cd44	CD44 Molecule		0.009	0.370		0.144	0.728		0.873	0.955
	6889258
Gpc2	Glypican 2		Not measured			Not measured			Not measured
Gpc3	Glypican 3		0.003	4.339		0.013	3.305		0.015	4.721
	7016826
Gpc6	Glypican 6		0.019	2.767		0.109	1.747		0.193	1.640
	6821985
Hspg2	Perlecan		0.309	1.183		0.075	1.554		0.042	2.230
	6917933
Sdc1	Syndecan 1		0.031	0.380		0.067	0.413		0.103	0.424
	6793226
Sdc4	Syndecan 4		0.017	0.341		0.094	0.536		0.215	0.621
	6892905
Preparation of linkage region
B3gat1	β-1,3-Glucuronyltransferase 1		0.008	3.467		0.256	1.302		0.828	0.929
	6987632
B3gat2	β-1,3-Glucuronyltransferase 2		0.009	3.241		0.286	1.269		0.690	1.129
	6748174
B4galt2	β-1,4-Galactosyltransferase 2		0.039	1.726		0.053	1.856		0.176	1.477
	6924869
Glycosaminoglycan chain polymerisation
Chpf	Chondroitin Polymerizing Factor		0.071	1.679		0.182	1.459		0.309	1.403
	6759816
Chsy1	CS Synthase 1		Not measured			Not measured			Not measured
Chsy3	CS Synthase 3		0.429	1.153		0.056	1.769		0.849	1.058
	6861281
Cs-galnact1	CS-GalNAc-transferase 1		0.137	0.512		0.202	0.541		0.974	0.975
	6983073
Extl1	Exostoses (multiple)-like 1		0.025	0.407		0.149	0.625		0.144	0.505
	6926017
Extl2	Exostoses (multiple)-like 2		0.085	1.702		0.819	1.066		0.518	1.272
	6900659
Has2	Hyaluronan Synthase 2		0.095	2.107		0.157	1.969		0.537	1.403
	6854042
Glycosaminoglycan chain modification
Chst11	Chondroitin 4-O-Sulfotransferase 1		0.049	1.922		0.465	1.208		0.239	1.531
	6769366
Chst14	Dermatan 4 Sulfotransferase 1		0.151	1.491		0.506	1.191		0.773	1.122
	6880476
Chst2	Carbohydrate Sulfotransferase 2		0.035	2.302		0.072	2.160		0.207	1.682
	6997990
Chst3	Chondroitin 6-O-Sulfotransferase 1		0.885	1.034		0.108	1.545		0.162	1.530
	6774295
Chst8	GalNAc-4-O-Sulfotransferase 1		0.945	1.007		0.569	1.054		0.420	1.108
	6966453
Hs3st1	HS 3-O-sulfotransferase 1		0.074	1.658		0.105	1.688		0.119	1.824
	6937654
Hs3st3b1	HS 3-O-sulfotransferase 3b1		0.683	1.178		0.253	1.634		0.511	1.432
	6788991
Hs3st6	HS 3-O-sulfotransferase 6		0.019	0.406		0.291	0.771		0.721	1.130
	6849317
Hs6st2	HS 6O-sulfotransferase 2		0.004	6.417		0.012	5.609		0.041	3.080
	7016808
Ndst1	N-Deacetylase and N-Sulfotransferase 1		0.090	1.363		0.056	1.649		0.067	1.763
	6865926
Ndst2	N-Deacetylase and N-Sulfotransferase 2		0.822	1.044		0.059	1.697		0.083	1.733
	6823122
Ndst3	N-Deacetylase and N-Sulfotransferase 3		0.173	2.472		0.111	3.856		0.122	4.860
	6908958
Sulf1	Sulfatase 1		0.010	4.522		0.051	2.546		0.232	1.627
	6747641
Sumf2	Sulfatase modifying factor 2		Not measured			Not measured			Not measured
Glycosaminoglycan chain degradation
ArsJ	Arylsulfatase J		0.003	2.498		0.006	3.164		0.031	1.702
	6901136
ArsK	Arylsulfatase K		0.368	0.784		0.102	0.543		0.514	0.779
	6814451
Galns	Galactosamine (N-Acetyl)-6-Sulfatase		0.087	1.671		0.322	1.311		0.786	1.115
	6985943
Hpse	Heparanase		0.021	0.267		0.540	1.244		0.095	0.360
	6940363
Hyal1	Hyaluronoglucosamini-dase 1		0.003	0.154		0.011	0.222		0.041	0.402
	6992224
Sgsh	N-Sulfoglucosamine Sulfohydrolase		0.017	0.458		0.052	0.537		0.201	0.699
	6792702
Growth factors
Areg	Amphiregulin		0.016	0.157		0.043	0.200		0.333	0.550
	6932394
Bmp3	Bone morphogenetic growth factor 3		0.280	1.392		0.067	2.381		0.100	2.365
	6932718
Bmp5	Bone Morphogenetic growth factor 5		0.001	7.247		0.141	1.191		0.688	1.057
	6990569
Ctgf	Connective tissue growth factor		0.148	0.729		0.025	0.366		0.068	0.485
	6766623
Fgf10	Fibroblast growth factor 10		0.138	1.521		0.057	2.250		0.094	2.142
	6810592
Fgf13	Fibroblast growth factor 13		0.012	2.998		0.075	1.824		0.296	1.391
	7017134
Fgf2	Fibroblast growth factor 2		0.201	0.664		0.279	0.696		0.952	0.967
	6896850
Fgf20	Fibroblast growth factor 20		0.659	1.228		0.125	2.483		0.777	1.220
	6981854
Fgf22	Fibroblast growth factor 22		0.005	0.329		0.972	0.994		0.097	0.646
	6769141
Fgf7	Fibroblast growth factor 7		0.531	0.740		0.078	0.286		0.212	0.418
	6880900
Fgf8	Fibroblast growth factor 8		0.492	0.883		0.655	1.085		0.682	0.895
	6873363
Figf	C-fos induced growth factor		0.051	1.734		0.062	1.909		0.548	0.832
	7015007
Gdf10	Growth differentiation factor 10		0.028	2.334		0.757	1.086		0.894	1.060
	6818153
Hbegf	Heparin-binding epidermal growth factor		0.037	0.551		0.063	0.547		0.087	0.530
	6864680
Hdgf	Hepatoma-derived growth factor		0.104	1.246		0.575	1.070		0.409	1.147
	6899028
Igf1	Insulin-like growth factor 1		0.235	0.631		0.173	0.537		0.398	0.641
	6769597
Igf2	Insulin-like growth factor 2		0.001	59.615		0.002	55.864		0.002	52.364
	6972317
Nog	Noggin		0.275	2.605		0.517	1.773		0.677	1.712
	6790670
Pdgfa	Platelet-derived growth factor a		0.028	2.013		0.035	2.347		0.057	2.338
	6942654
Pdgfb	Platelet-derived growth factor b		0.037	0.704		0.199	1.197		0.157	1.298
	6837144
Pdgfc	Platelet-derived growth factor c		0.019	2.152		0.035	2.207		0.902	1.042
	6898686
Pdgfd	Platelet-derived growth factor d		0.925	0.952		0.191	0.504		0.790	1.205
	6986677
Shh	Sonic hedgehog		0.448	0.595		0.308	2.117		0.152	4.571
	6936889
Tgfb1	Transforming growht factor beta 1		0.106	0.552		0.241	0.653		0.975	0.981
	6959236
Tgfb2	Transforming growht factor beta 2		0.057	2.441		0.448	1.335		0.264	1.802
	6764953
Tgfb3	Transforming growth factor beta 3		0.026	2.386		0.238	1.408		0.100	2.077
	6802449
Vegfa	Vascular endothelial growth factor a		0.779	0.875		0.721	1.174		0.940	1.061
	6855659
Vegfb	Vascular endothelial growth factor b		0.008	1.461		0.031	1.318		0.060	1.277
	6871273
Vegfc	Vascular endothelial growth factor c		0.088	2.120		0.477	1.316		0.316	1.700
	6976200
Wnt10b	Wingless-related integration site 10b		0.629	1.280		0.120	2.812		0.240	2.325
	6838399
Wnt16	Wingless-related integration site 16		0.358	1.180		0.026	2.402		0.093	1.710
	6944581
Wnt2	Wingless-related integration site 2		0.057	3.047		0.082	3.260		0.122	3.219
	6951974
Wnt2b	Wingless-related integration site 2b		0.009	2.189		0.024	2.030		0.077	1.590
	6907887
Wnt3a	Wingless-related integration site 3a		0.457	1.199		0.103	1.728		0.684	1.159
	6788662
Wnt6	Wingless-related integration site 6		0.011	3.166		0.032	2.668		0.064	2.352
	6750567
Wnt7a	Wingless-related integration site 7a		0.060	2.117		0.131	1.865		0.395	1.456
	6955539
Wnt7b	Wingless-related integration site 7b		0.072	2.297		0.050	3.623		0.237	1.912
	6837582

Numbers in Italic are significant (p<0.10); numbers in bold are >2-fold differentially expressed. Genes indicated as “not measured” represent genes for which probes were not available on the used exon array version.

Table 4

Differentially expressed genes in C5 epimerase (Glce) knockout mouse (p<0.10) based on real-time qPCR.

Gene symbol	Full gene name and probe set		P-value	Relative change
Production of precursors
Gnpnat1	Glucosamine-Phosphate N-Acetyltransferase 1		0.033	0.468
	Mm00834602_mH
Slc2a4	Solute Carrier Family 2 Member 4		0.086	2.526
	Mm01245507_g1
Core proteins
Acan	Aggrecan		0.005	0.242
	Mm00545807_m1
Aspn	Asporin		0.010	0.382
	Mm00445945_m1
Glycosaminoglycan chain polymerisation
Csgalnact	CS N-Acetylgalactosaminyltransferase 2		0.049	0.431
2	Mm00513340_m1
Glycosaminoglycan chain modification
Glce	Glucuronic Acid Epimerase		0.013	0.079
	Mm00473667_m1

Numbers in Italic are significant (p<0.10); numbers in bold are >2 fold differentially expressed.

All genes were normalized using 18S RNA as a reference gene.

In Tables 1–3 and Supplementary data Table 3, an overview is given of the differentially expressed genes applying TLDA cards and exon arrays. In all categories of genes involved in GAG metabolism, i.e., production of precursor molecules, core proteins, synthesis of linkage region, polymerization, modification and degradation of the GAG chain, and differences in expression were found (Tables 1–3). This indicates a highly dynamic expression pattern during skin development. Some isoenzymes were upregulated, whereas other isoenzymes were downregulated, further stressing metabolic complexity. This is, for instance, the case with GFPT1 and 2, both rates limiting enzymes involved in the production of hexosamines, and the isoenzymes HS 3-O sulfotransferase 6 and 3b1.

With respect to the core proteins, differential expression was found for both HS and CS/DS proteoglycans. Differential expression was found for two of the four syndecans, viz. Sdc1 and Sdc4, three of the six glypicans, viz. Gpc2, Gpc3, and Gpc6, and Hspg2 (Tables 2 and 3). The syndecans were downregulated, while the glypicans were upregulated, indicating an embryonic role for glypicans as described in literature [25, 26]. Hspg2, a secreted HS presenting proteoglycan coding for perlecan [2], was found to be upregulated (Tables 2 and 3). Based on the exon array the CS/DS core protein of versican (Vcan) was upregulated at all time points (Supplementary data Table 5).

The upregulated expression of genes involved in the synthesis of the linkage region may signal increased GAG synthesis during development since after the formation of the linkage region the GAG chain is formed. For HS polymerization differential expression was found for, e.g., Extl1 and Extl2. Extl1 showed downregulation at E14 while Extl2 was upregulated, and both enzymes are involved in the initiation and elongation of the HS chain [2].

During and after synthesis of the glycosaminoglycan chain, disaccharide units within the chain are specifically modified. These modifications determine which effector molecules can bind to the chain and thus play a role in cell signaling [1, 3]. Upregulated expression was found for three of the four N-deacetylase/N-sulfotransferases (Ndst; TLDA cards, Table 2), especially isoenzyme Ndst3. Upregulation was also found for two out of seven genes coding for 3-O-sulfotransferases (Hs3st1 and Hs3st3b1), involved in 3-O sulfation of GlcNS and GlnNAc residues, whereas one was downregulated (Hs3st6).

The GlcNS and GlcNAc residues can also be 6-O sulfated by 6-O-sulfotransferases (Hs6st) [2] and selectively desulfated extracellularly by two sulfatases (Sulf1 and Sulf2) aided by two cofactors (Sumf1 and Sumf2) [2, 27]. Hs6st2 was upregulated at all time points (Table 3). Sulf1 was upregulated during embryonic development, whereas Sumf2 was upregulated at E14 (Tables 2 and 3). These results indicate that specific expression of GAG modifying enzymes may play a role in specific cellular signaling during skin development.

Within the class of genes encoding for GAG chain degradation enzymes, two genes were differentially expressed. Heparanase expression was downregulated at E14 and P1 (Tables 2 and 3), whereas N-sulfoglucosamine sulfohydrolase (Sgsh) was downregulated during embryonic development at E16 (Table 2) and at E14 (Table 3).

In addition to genes involved in GAG metabolism, the TLDA card contained 37 genes encoding growth factors, which were also present in the microarray (Tables 2 and 3). Differential expression was found by both TLDA card and microarray analysis for 10, 9 and 4 growth factors at E14, E16 and P1 respectively. Examples are insulin-like growth factor 2 (Igf2), wingless-related integration site 6 (Wnt6), and Wnt7b. Igf2 was dramatically upregulated at all time points, as expected based on previous research [18]. Wnt6 was also upregulated at all time points, while Wnt7b was upregulated only during embryonic development.

Next to their expression during development, gene expression of GAG-associated genes was studied in a Glce (glucuronyl epimerase) knockout mouse model and a heparanase overexpression mouse model using TLDA cards. In the Glce knockout mice six genes were differentially expressed (Table 4). Three of them are involved in CS and DS proteoglycans and were downregulated, i.e., aggrecan (Acan), asporin (Aspn), and chondroitin sulfate N-acetylgalactosaminyltransferase 2 (Csgalnact2). Up/downregulation was not found for HS related genes, except for Glce, which was downregulated as expected. For the heparanase overexpression mouse model, in which a human heparanase was overexpressed [7], the results showed only one gene to be differentially expressed, i.e., aggrecan (Acan) which was 2.5-fold upregulated. The complete results of both the Glce knockout mouse and the Hpse overexpression mouse are given in Supplementary data Table 6.

4. Discussion

GAGs play a regulating role during embryonic development of various organs [1–3]. Therefore, we examined the expression of genes involved in GAG metabolism during skin development using custom designed Taqman Low Density Arrays (TLDA card) and exon arrays. To structure the data we studied gene expression in six functional classes, viz. the production of precursor molecules, the synthesis of core proteins and the linkage region, and the synthesis, modification, and degradation of the GAG chain proper. In addition we studied a number of growth factors, since GAGs are involved in their regulation including growth factor diffusion and signaling [3, 28].

With respect to core proteins, the heparan sulfate proteoglycans syndecan and glypican showed notable differential expression (Tables 2 and 3). Glypicans play an important role in development and cell signaling [12, 26, 29], and we found upregulation of 3 out of 6 glypican core proteins. Gpc3 was upregulated during embryonic development and one day postbirth, suggesting that this glypican has a role during skin development. A possible function of Gpc3 in skin has been suggested for the Gpc3-null mouse, which showed pigmentation defects [30]. Humans deficient in Gpc3 suffer from the Simpson-Golabi-Behmel syndrome (SBGS). Based on the symptoms of SGBS and the phenotype found for the Gpc3-null mice, it has been suggested that Gpc3 is involved in the regulation of hedgehog signaling [31], a signaling pathway involved in hair follicle development [32]. Surprisingly, the Gpc3-null mice did not show a defect in appendage formation [30], indicating a functional but not essential role. Further research is needed to elucidate the role of Gpc3 and the two other differentially expressed glypicans, i.e., Gpc2 and Gpc6.

Syndecans are described to take part in adult wound healing [33]. We found downregulation of the core proteins of two syndecans during embryonic development, which could indicate that these proteoglycans do not play a major general role during skin development. Specific roles, such as the involvement of Sdc1 in hair follicle development, as described on basis of immunohistochemical data [34], can, however, not be excluded.

In the class of GAG chain polymerization, we found differential expression of genes encoding for the initiation of HS or CS/DS synthesis. HS chain polymerization is initiated by the addition of GlcNAc by Extl2 [35] or Extl3 [36], while CS/DS chain polymerization is initiated by the addition of GalNAc by Csgalnact1 [37]. Extl2 was upregulated during early skin development (Table 2), while Csgalnact1 was downregulated (Supplementary data Table 5), which suggests that during early skin development HS production is stimulated in comparison to CS/DS production.

Enzyme mediated chemical modifications of the GAG chains result in the creation of specific binding sites for effector molecules [38]. Enzymes forming the class of N-deacetylase/sulfotransferases (Ndst’s) are initiating elements in this respect. Especially Ndst3 was upregulated, being one of four enzymes responsible for the removal of the acetyl group from the N-acetylated glycosamine and for the addition of a sulfate group. The additional expression of Ndst3 in combination with Ndst1 and Ndst2 points to the fine tuning of HS chains for specific recognition of ligands. Ndst3 has a higher deacetylation activity in comparison to the N-sulfotransferase activity, while Ndst1 and Ndst2 have a slightly higher N-sulfotransferase activity [39]. In addition, the data on the expression of heparan sulfate 3-O sulfotransferases (Hs3sts) [40] and heparan sulfate 6-O-sulfotransferases (Hs6sts) [41] suggest dynamic and specific modification of HS chains.

Three genes encoding for enzymes involved in HS and CS/DS degradation were differentially expressed, one of them being Hpse (heparanase). Hpse is downregulated at E14 and at P1, but not at E16 at which time point hair follicle development is taking place. Hpse has been reported to be involved in this process [42, 43].

Glycosaminoglycans are involved in growth factor regulation during developmental processes [1, 2]. We therefore studied 37 growth factors implied in skin development. A number of genes encoding growth factors were differentially expressed during development and the data are in line with earlier results for, e.g., Igf2 [18], Wnt6, and Wnt7b [44]. Although speculative, the dynamics in GAG structure may be correlated with the dynamics of growth factors.

Next to skin development we also studied gene expression in skin of a Glce (glucuronyl epimerase) knockout mouse and an Hpse (heparanase) overexpression mouse [7, 13]. In the Glce knockout mice relatively few genes were differentially expressed, suggesting that skin is relatively unaffected by the lack of Glce in line with the observation that skin in these mice is phenotypically normal [20]. The skin phenotype of the Hpse overexpression mouse shows accelerated hair growth [7]. Gene expression analysis of this model showed only one differentially expressed gene (aggrecan). These results may touch upon the regulation of translation of mRNAs coding for GAG related enzymes. Enzymes involved in the synthesis and modification of GAGs as well genes coding for (some) growth factors share a common alternative translation mechanism via IRES sites [45, 46]. In general mRNAs are translated by the ribosomal scanning mechanism which scans for short leader sequences of 50 to 70 nucleotides [46, 47]. The leader sequences of the HS modifying enzymes and growth factors are characterized by long but structured sequences, which are not recognized by the ribosomal scanning mechanism [46, 47]. Within these sequences internal ribosomal entry sites (IRES) allow alternative translation, e.g., under stress conditions [47]. This indicates that in addition to mRNA levels an additional control mechanism on the translational level may be present. In addition, other types of regulatory levels are known including the interaction of biosynthetic enzymes with each other and the (possible) presence of large biosynthetic complexes (GAGosomes) [48]. This makes the regulation of GAG biosynthesis very complex, gene expression being only a part of it.

Taken together, it is concluded that a highly dynamic expression of genes involved in GAG metabolism and in GAG binding growth factors is associated with skin development. This indicates the importance of fine tuning of GAG structures during developmental processes. Further studies should focus on the biochemical analysis of the GAGs chains themselves.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

P. J. E. Uijtdewilligen wrote the main manuscript text and prepared Figure 1, Tables 1–4, and Supplementary data Tables 1–6. P. J. E. Uijtdewilligen, E. M. Versteeg, and E. M. A. van de Westerlo were responsible for the performance of the genetic analysis using RNA isolation, real time quantitative PCR, and microarray analysis. P. J. E. Uijtdewilligen, J. van der Vlag and T. H. van Kuppevelt were responsible for the design of the Taqman Low Density Array as described in Supplementary data 1-2. W. F. Daamen and T. H. van Kuppevelt were involved in study design, manuscript text, and design of the figures. All authors have given approval for the final version of the manuscript.

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