Supplementary Material for: Sulfatase 2 Modulates Fate Change from Motor Neurons to Oligodendrocyte Precursor Cells through Coordinated Regulation of Shh Signaling with Sulfatase 1

Sulfatases (Sulfs) are a group of endosulfatases consisting of Sulf1
and Sulf2, which specifically remove sulfate from heparan sulfate
proteoglycans. Although several studies have shown that Sulf1 acts as a
regulator of sonic hedgehog (Shh) signaling during embryonic ventral
spinal cord development, the detailed expression pattern and function of
Sulf2 in the spinal cord remains to be determined. In this study, we
found that Sulf2 also modulates the cell fate change from motor neurons
(MNs) to oligodendrocyte precursor cells (OPCs) by regulating Shh
signaling in the mouse ventral spinal cord in coordination with Sulf1.
In the mouse, Sulf mRNAs colocalize with Shh mRNA and gradually expand
dorsally from embryonic day (E) 10.5 to E12.5, following strong Patched1
signals (a target gene of Shh signaling). This coordinated expression
pattern led us to hypothesize that in the mouse, strong Shh signaling is
induced when Shh is released by Sulf1/2, and this strong Shh signaling
subsequently induces the dorsal expansion of Shh and Sulf1/2 expression.
Consistent with this hypothesis, in the ventral spinal cord of Sulf1
knockout (KO) or Sulf2 KO mice, the expression patterns of Shh and
Patched1 differed from that in wild-type mice. Moreover, the position of
the pMN and p3 domains were shifted ventrally, MN generation was
prolonged, and OPC generation was delayed at E12.5 in both Sulf1 KO and
Sulf2 KO mice. These results demonstrated that in addition to Sulf1,
Sulf2 also plays an important and overlapping role in the MN-to-OPC fate
change by regulating Shh signaling in the ventral spinal cord. However,
neither Sulf1 nor Sulf2 could compensate for the loss of the other in
the developing mouse spinal cord. In vitro studies showed no evidence of
an interaction between Sulf1 and Sulf2 that could increase sulfatase
activity. Furthermore, Sulf1/2 double heterozygote and Sulf1/2 double KO
mice exhibited phenotypes similar to the Sulf1 KO and Sulf2 KO mice.
These results indicate that there is a threshold for sulfatase activity
(which is likely reflected in the dose of Shh) required to induce the
MN-to-OPC fate change, and Shh signaling requires the coordinated
activity of Sulf1 and Sulf2 in order to reach that threshold in the
mouse ventral spinal cord.