LRP2 Gives Patched a Hand

Science Signaling  21 Feb 2012:
Vol. 5, Issue 212, pp. ec55
DOI: 10.1126/scisignal.2002975

Without appropriate signaling by the Sonic hedgehog (SHH) pathway, the forebrain does not develop correctly; in humans, this leads to holoprosencephaly. Loss or reduced activity of Megalin, also known as lipoprotein receptor–related protein 2 (LRP2), produces phenotypes similar to holoprosencephaly in humans. LRP2 binds two morphogens implicated in forebrain development, BMP4 and SHH. Christ et al. set out to determine which of these binding partners was important for the effects of LRP2 in early forebrain specification. LRP2 was detected at embryonic day 7.5 (E7.5) at the apical side of the developing neural plate and became progressively restricted to the midline by E10.5. In situ hybridization at E8.0 for Shh and Six3, which mutually regulate each other’s expression, showed that Six3 was reduced in the prospective forebrain area of lrp2-knockout mice. Although Shh was present in the prechordal plate that specifies the rostral diencephalon ventral midline (RDVM), which are the cells that will become the forebrain, it was not present in the RDVM. By E9.5, an increase in Bmp4, which is repressed by SHH, was detectable in the dorsal forebrain of the lrp2 knockouts, suggesting that an increase in BMP4 signaling was a secondary consequence of loss of function of its negative regulator SHH at a previous stage in development. Analysis of the caudal neural tube showed no alterations in SHH, suggesting that LRP2 may be a specific co-receptor important for forebrain development but not for SHH signaling during spinal cord development. SHH colocalized with LRP2 and with the SHH receptor Patched1 on the apical surface of the neuroepithelial cells of the RDVM, whereas the SHH signal was absent from this region in the brains of the knockout mice. Because LRP2 was associated with clathrin-coated pits, the authors analyzed the ability of this receptor to promote SHH endocytosis in explants. Explants from wild-type and lrp2 knockout mice showed an appropriate response to a Hedgehog signaling pathway agonist that bypasses Patched1, indicating that the pathway was functional downstream of the receptors. Binding of exogenously applied recombinant forms of SHH and internalization of Patched1 in response to these ligands in the explants from the lrp2 knockout mice were compromised, whereas the wild-type explants exhibited robust binding and colocalization of LRP2 and SHH and of Patched1 and SHH in intracellular compartments, suggesting that LRP2 contributes to the uptake of SHH. Introduction of a truncated form of LRP2 into transfected cells (truncation was necessary to enable production of the protein, which is very large) enhanced the induction of an SHH reporter in response to exogenously applied SHH. Thus, LRP2 appears to deliver SHH to the cells that will become the forebrain and thereby facilitate SHH signaling in this tissue during the early stages of brain development.

A. Christ, A. Christa, E. Kur, O. Lioubinski, S. Bachmann, T. E. Willnow, A. Hammes, LRP2 is an auxiliary SHH receptor required to condition the forebrain ventral midline for inductive signals. Dev. Cell 22, 268–278 (2012). [Abstract]