Deletion of Nf2 in neural crest‐derived tongue mesenchyme alters tongue shape and size, Hippo signalling and cell proliferation in a region‐ and stage‐specific manner

OBJECTIVES: The mammalian tongue develops from the branchial arches (1–4) and comprises highly organized tissues compartmentalized by mesenchyme/connective tissue that is largely derived from neural crest (NC). This study aimed to understand the roles of tumour suppressor Neurofibromin 2 (Nf2) in NC‐derived tongue mesenchyme in regulating Hippo signalling and cell proliferation for the proper development of tongue shape and size. MATERIALS AND METHODS: Conditional knockout (cKO) of Nf2 in NC cell lineage was generated using Wnt1‐Cre (Wnt1‐Cre/Nf2(cKO) ). Nf2 expression, Hippo signalling activities, cell proliferation and tongue shape and size were thoroughly analysed in different tongue regions and tissue types of Wnt1‐Cre/Nf2(cKO) and Cre (‐)/Nf2 ( fx/fx ) littermates at various stages (E10.5–E18.5). RESULTS: In contrast to many other organs in which the Nf2/Hippo pathway activity restrains growth and cell proliferation and as a result, loss of Nf2 decreases Hippo pathway activity and promotes an enlarged organ development, here we report our observations of distinct, tongue region‐ and stage‐specific alterations of Hippo signalling activity and cell proliferation in Nf2(cKO) in NC‐derived tongue mesenchyme. Compared to Cre (−)/Nf2(fx) (/) (fx) littermates, Wnt1‐Cre/Nf2(cKO) depicted a non‐proportionally enlarged tongue (macroglossia) at E12.5–E13.5 and microglossia at later stages (E15.5–E18.5). Specifically, at E12.5 Nf2(cKO) mutants had a decreased level of Hippo signalling transcription factor Yes‐associated protein (Yap), Yap target genes and cell proliferation anteriorly, while having an increased Yap, Yap target genes and cell proliferation posteriorly, which lead to a tip‐pointed and posteriorly widened tongue. At E15.5, loss of Nf2 in the NC lineage resulted in distinct changes in cell proliferation in different regions, that is, high in epithelium and mesenchyme subjacent to the epithelium, and lower in deeper layers of the mesenchyme. At E18.5, cell proliferation was reduced throughout the Nf2(cKO) tongue.