The role of Rspondins in left-right asymmetry and beyond

R-Spondins (RSPO1-4) are a family of four secreted proteins implicated in development and cancer, which we have discovered in 2004 as Wnt agonists. RSPOs function by protecting WNT receptors from ubiquitination and degradation by the E3 ubiquitin ligases ZNRF3/RNF43. RSPOs became famous as a key ingredient to maintain organoid cultures where they stimulate stem cell growth. We are investigating the molecular mechanism of RSPO signaling as well as its biological roles during embryonic development. Recently, we found a pivotal role of RSPO2 in development of the Left-Right (LR) body axis.

How animals establish the LR axis, is a fundamental problem in biology, as it serves as the basis for the formation of all subsequent LR-asymmetric structures. LR asymmetry is regulated by the left-right organizer (LRO) where LR symmetry breaking takes place. Defective LRO formation results in heterotaxy (HTX), congenital diseases ranging from organ malformation to misarrangement of organs across the LR axis, notably congenital heart defects. The LRO harbors motile cilia, which rotate clockwise to generate a leftward fluid flow.

We discovered in Xenopus embryos, that rspo2 acts in the LRO as a LR morphogen. Rspo2 is expressed in the LRO and it is necessary and sufficient for organ laterality and LR specification. Specifically, Rspo2 acts as sinistralizing (determining Left-specific cell fate) signal and operates upstream of the well-established Nodal-Pitx2 cassette, which is essential for sinistral fate. Surprisingly, in LR patterning Rspo2 acts as an FGF receptor antagonist. Rspo2 via its TSP1 domain binds Fgfr4 and promotes its membrane clearance by ZNRF3-dependent endocytosis. Concordantly, FGF signaling acts dextralizing (determining Right-specific cell fate) and forms a MAPK signaling gradient across the LRO. This FGF signaling gradient is high on the dextral- and low on the sinistral side. We are currently analyzing the LR signaling cascades downstream of RSPO2 and FGF as well as the evolutionary conservation of this mechanism in mouse.

We recently showed that RSPO2 and RSPO3 are also BMP (bone morphogenetic protein) antagonists. Specifically, RSPO2 is a high affinity ligand for the BMP receptor BMPR1A. RSPO2 forms a ternary complex between BMPR1A and the E3 ligase ZNRF3, which triggers endocytosis and degradation of the BMP receptor. We showed that Rspo2 antagonizes BMP signaling during embryonic axis formation in Xenopus. Moreover, we found that this novel function of RSPO2 as BMP antagonist also plays a critical role in acute myeloid leukemia (AML): AML cells secrete RSPO2 to promote their self-renewal and prevent cell differentiation. RSPO2 expression is generally elevated in AML patients with poor prognosis and inhibiting RSPO2 prolongs survival in AML mouse xenograft models.

Thus, RSPOs are multimodal modulators of growth factor signaling pathways, i.e. they can affect several receptors families (Wnt (ON) – BMP (OFF) – FGF (OFF)). Hence, we suspect even more target pathways and investigate the full repertoire of RSPO target receptors.