Wnts in development : focus on midbrain

Abstract

Studies in the field of embryonic development reveal the temporal and spatial requirement of specific genes/proteins in the formation of a given tissue. The specific knowledge of which genes/proteins regulate development of the ventral midbrain (VM) provides a guideline for the in vitro differentiation of specific cell types therein, such as dopaminergic (DA) neurons. Large-scale in vitro production of DA neurons is a prerequisite for cell replacement therapies in Parkinson s disease (PD, a neurodegenerative disease in which DA neurons are lost). Ideally, this differentiation could be accomplished by direct application of extracellular proteins. One family of signaling proteins, which may fill such a function, is the Wnt family of proteins.

Previous results from our laboratory demonstrated a role for Wnt1 and Wnt5a in the development of DA neurons in vitro, while multiple other laboratories had shown that Wnt1 was required for midbrain development in vivo.

In this thesis work, we investigated signaling through Wnt5a and LRP6 (a Wnt receptor thought to bind Wnt1/Wnt3a, but not Wnt5a), as well as the roles of Wnt1, Wnt5a, LRP6, sFRP1 and sFRP2 (secreted proteins which reportedly bind Wnts to antagonize their function) in the development of the midbrain and DA neurons.

The extracellular domain of LRP6 bound Wnt5a, and inhibited Wnt5a or Wnt11 signaling. LRP6 loss-of-function (LOF) studies, in mouse and Xenopus, revealed typical convergent extension (CE) defects which were rescued by Wnt5a or Wnt11 LOF. Conversely, Wnt5a or Wnt11 gain-of-function (GOF) were rescued by LRP6 GOF. Thus, LRP6 acts as a negative regulator of Wnt5a/Wnt11 signaling in vivo. In a DA neuron cell line, overexpression of LRP6 decreased active Rac1 levels, while Wnt5a activated Rac1 and was required for Wnt5a-induced DA neuron differentiation. Despite the capacity of Wnt5a to induce DA neurons in vitro, Wnt5a-/- mice did not display an overall loss of DA neurons. Instead, a transient increase in proliferating progenitors and Nurr1+ (NR4A2+) precursors led to a bottleneck in differentiation, such that the ratio of differentiated cells was lower in Wnt5a-/- mice. The Wnt5a-/- VM displayed typical convergent extension (CE) defects, with a flattened medial ventricular zone, and a laterally expanded Shh domain. This CE defect resulted in an anteroposterior shortening and lateral expansion of the DA neuron nucleus. Examination of sFRP1-/-;sFRP2-/- mice revealed a phenocopy of this CE defect, but no similarity in effects on proliferation or NR4A2+ cell numbers, indicating that CE and differentiation may be separately regulated. In support of this, neither sFRP1 nor sFRP2 induced DA neurons in primary VM cultures.

The Wnt1-/- midbrain displayed a medial loss of floorplate DA markers, including Lmx1a. The DA population was instead bisected and displaced laterally, forming bilateral populations in the basal plate region, with ~10% remaining DA neurons. Haploinsufficiency of LRP6, or ablation of Wnt5a, in the Wnt1-/- background was sufficient to reduce the number of DA neurons by an additional 70% compared to Wnt1-/- alone. Thus, Wnt5a may partially compensate loss of Wnt1, while the mild Wnt5a-/- DA phenotype may be a result of robust compensation by Wnt1.

Finally, we developed an improved VM neurosphere DA differentiation protocol combining Wnt3a and Wnt5a treatments. Sequential treatment with Wnt3a and Wnt5a robustly increased the number of DA neurons, compared to either Wnt alone.

In sum, the data presented in this thesis describes multiple Wnt ligand and receptor interactions, in regulation of diverse processes such as CE or DA differentiation. Hopefully, these insights into Wnt signaling, especially in the realm of DA neurogenesis, will contribute to improved therapies for PD.