The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state “catch or present” model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS–Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

The signaling activities of multiple developmental ligands require sulfated heparan sulfate (HS) proteoglycans as coreceptors. QSulf1 and its mammalian orthologs are cell surface HS 6- O -endosulfatases that are expressed in embryonic mesodermal and neural progenitors and promote Wnt signal transduction. In this study, we have investigated the function of QSulf1 in fibroblast growth factor (FGF) signaling, which requires 6-O-sulfated HS for FGF receptor (FGFR) dimerization and tyrosine kinase activation. Here, we report that QSulf1 inhibits FGF2- and FGF4-induced mesoderm formation in the Xenopus embryo and FGF-dependent angiogenesis in the chicken embryo through 6-O-desulfation of cell surface HS. QSulf1 regulates FGF signaling through inhibition of HS-mediated FGFR1 activation by interfering with FGF–HS–FGFR1 ternary complex formation. Furthermore, QSulf1 can produce enzymatically modified soluble heparin that acts as a potent inhibitor of FGF2-induced angiogenesis in the chicken embryo. QSulf1, therefore, has dual regulatory functions as a negative regulator of FGF signaling and a positive regulator of Wnt signaling. Therefore, QSulf1 provides another reagent to produce enzymatically modified heparin compounds, in vivo and in vitro , to modulate cellular signaling in stem cell-based therapies to promote tissue regeneration and in cancer therapies to control cell growth and block angiogenesis.

Heparan sulfate (HS) plays an essential role in extracellular signaling during development. Biochemical studies have established that HS binding to ligands and receptors is regulated by the fine 6-O-sulfated structure of HS; however, mechanisms that control sulfated HS structure and associated signaling functions in vivo are not known. Extracellular HS 6-O-endosulfatases, SULF1 and SULF2, are candidate enzymatic regulators of HS 6-O-sulfated structure and modulate HS-dependent signaling. To investigate Sulf regulation of developmental signaling, we have disrupted Sulf genes in mouse and identified redundant functions of Sulfs in GDNF-dependent neural innervation and enteric glial formation in the esophagus, resulting in esophageal contractile malfunction in Sulf1^-/-;Sulf2^-/- mice. SULF1 is expressed in GDNF-expressing esophageal muscle and SULF2 in innervating neurons, establishing their direct functions in esophageal innervation. Biochemical and cell signaling studies show that Sulfs are the major regulators of HS 6-O-desulfation, acting to reduce GDNF binding to HS and to enhance GDNF signaling and neurite sprouting in the embryonic esophagus. The functional specificity of Sulfs in GDNF signaling during esophageal innervation was established by showing that the neurite sprouting is selectively dependent on GDNF, but not on neurotrophins or other signaling ligands. These findings provide the first in vivo evidence that Sulfs are essential developmental regulators of cellular HS 6-O-sulfation for matrix transmission and reception of GDNF signal from muscle to innervating neurons.

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

In the embryonic chick ventral spinal cord, the initial emergence of oligodendrocytes is a relatively late event that depends on prolonged Sonic hedgehog (Shh) signaling. In this report, we show that specification of oligodendrocyte precursors (OLPs) from ventral Nkx2.2-expressing neural progenitors occurs precisely when these progenitors stop generating neurons, indicating that the mechanism of the neuronal/oligodendroglial switch is a common feature of ventral OLP specification. We further show that an experimental early increase in the concentration of Shh is sufficient to induce premature specification of OLPs at the expense of neuronal genesis indicating that the relative doses of Shh received by ventral progenitors determine whether they become neurons or glia. Accordingly, we observe that the Shh protein accumulates at the apical surface of Nkx2.2-expressing cells just before OLP specification, providing direct evidence that these cells are subjected to a higher concentration of the morphogen when they switch to an oligodendroglial fate. Finally, we show that this abrupt change in Shh distribution is most likely attributable to the timely activity of Sulfatase 1 (Sulf1), a secreted enzym that modulates the sulfation state of heparan sulfate proteoglycans. Sulf1 is expressed in the ventral neuroepithelium just before OLP specification, and we show that its experimental overexpression leads to apical concentration of Shh on neuroepithelial cells, a decisive event for the switch of ventral neural progenitors toward an oligodendroglial fate.

Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1^CreERT2/+ mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms’ tumor 1 (Wt1) gene expression. We visualized WT1⁺ mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin⁺ fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific.