Submandibular Parasympathetic Gangliogenesis Requires Sprouty-Dependent Wnt Signals from Epithelial Progenitors

Abstract

Parasympathetic innervation is critical for submandibular gland (SMG) development and regeneration. Parasympathetic ganglia (PSG) are derived from Schwann cell precursors that migrate along nerves, differentiate into neurons, and coalesce within their target tissue to form ganglia. However, signals that initiate gangliogenesis after the precursors differentiate into neurons are unknown. We found deleting negative regulators of FGF signaling, Sprouty1 and Sprouty2 (Spry1/2DKO), resulted in a striking loss of gangliogenesis, innervation and keratin 5-positive (K5+) epithelial progenitors in the SMG. Here we identify Wnts produced by K5+ progenitors in the SMG as key mediators of gangliogenesis. Wnt signaling increases survival and proliferation of PSG neurons and inhibiting Wnt signaling disrupts gangliogenesis and organ innervation. Activating Wnt signaling and reducing FGF gene dosage rescues gangliogenesis and innervation in both the Spry1/2DKO SMG and pancreas. Thus K5+ progenitors produce Wnt signals to establish the PSG-epithelial communication required for organ innervation and progenitor cell maintenance.

Endothelial cell regulation of salivary gland epithelial patterning

“…Reciprocal interactions between the developing vasculature and epithelium are likely to be multifactorial, similar to the contribution of innervation to gland development (Knox et al, 2010;Nedvetsky et al, 2014;Knosp et al, 2015). The fact that our assays with IGFBP2 and IGFBP3 show modest effects on SMG morphogenesis and differentiation is consistent with the concept that there are likely to be numerous endothelial-produced factors that impact parenchymal development.…”

“…Loss of VEGFR2/CD31 + endothelial cell function would then be expected to increase canonical Wnt signaling to the epithelium, prevent proacinar differentiation, downregulate Kit expression and accelerate ductal formation. Wnt signaling is regulated by balancing activation of Wnt receptors by ligands at many levels, and several reports indicate that the Wnt family has complex roles in SMG development (Haara et al, 2011;Musselmann et al, 2011;Patel et al, 2011;Knosp et al, 2015;Maimets et al, 2016;Matsumoto et al, 2016). Thus, VEGFR2/CD31 + endothelial cells could act in many ways to modulate mesenchymal Wnt signaling to the epithelium, and might confer this regulation in a spatially restricted manner during branching morphogenesis.…”

The Wnt-Myb pathway suppresses KIT expression to control the timing of salivary proacinar differentiation and duct formation

“…At the early stage of SMG development (E12-E15), WNT/β-catenin-dependent signaling is active in mesenchyme around end buds and around the parasympathetic ganglion, and after E15 WNT/β-catenin signaling downregulates in mesenchyme and is concomitantly upregulated in epithelium of SMG main ducts (Knosp et al, 2015;Patel et al, 2011). Opposing reports exist for the role of the WNT signaling pathway in salivary gland development.…”

“…However, WNT signaling activity is important for suppressing premature end bud differentiation into proacini, and for promoting undifferentiated cell proliferation at an early developmental stage until the E15 canalicular stage in vivo, which corresponds to SMG rudiments at day 4. WNT signaling maintains proximal ductal progenitor cells (Knosp et al, 2015). In addition, of the distal progenitor markers Sox10, Myc, Krt14 and Kit, all of which are increased by FGF signaling, WNT signaling increased the former three markers but decreased KIT.…”

Regulatory Mechanisms Driving Salivary Gland Organogenesis

“…Sproutys are key intracellular modulators of FGF signaling and act as negative-feedback antagonists (Tang et al, 2011). The genetic deletion of Spry1 and Spry2 ( Spry1/2DKO ) from the SMG epithelium resulted in a striking loss of the PSG and SMG innervation (Figure 2), resulting in reduced branching morphogenesis (Knosp et al, 2015). During PSG formation it was shown that increasing FGF signaling reduced the expression of the Wnt signals in the epithelium.…”