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

Abstract 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/Nf2cKO ). 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/Nf2cKO 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 Nf2cKO in NC‐derived tongue mesenchyme. Compared to Cre −/Nf2fx / fx littermates, Wnt1‐Cre/Nf2cKO 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 Nf2cKO 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 Nf2cKO tongue.


| INTRODUC TI ON
The tongue development requires a proper regulation of its molecular mechanisms to attain its stereotypical shape, while developmental defects including microglossia, macroglossia and aglossia 1 can hamper the normal function of the tongue, for example taste sensing, speaking and food processing. It has been shown that molecular signalling pathways and their interactions play important roles in the proper formation of the tongue. [2][3][4][5][6][7][8] However, our current understanding of tongue development in relation to molecular signalling pathways is far from complete.
In this study, we report the important role of Neurofibromin 2 (Nf2) in the neural crest (NC)-derived tongue mesenchymal cells in regulating mouse tongue shape and size which is distinct from that in other organs.
In mice, the tongue forms from four branchial arches (BAs) 1-4, that is tongue primordia. Among these four BAs, BAs 1-2 give rise to the anterior two thirds of the tongue -the oral tongue, and BAs 3-4 give rise to the posterior third of the tongue -the pharyngeal tongue. 9 The tongue emerges as three lingual swellings, two lateral and one posterior (tubercula impar) on the floor of the mandible at embryonic day (E) 11.5. 10 These swellings fuse and grow into a spatulate tongue at E12.5. Thereafter, the tongue organ continues to grow and various types of cells are differentiated and highly organized, including formed appendages such as taste papillae and taste buds.
Nf2 is considered as a tumour suppressor gene that activates the Hippo signalling pathway through its gene product known as merlin. 11 Under normal cellular conditions, merlin recruits mammalian sterile 20-like protein kinase (Mst1/2), a large tumour suppressor (Lats1/2) and adaptor protein Salvador (Salv) to activate the Hippo signalling pathway by phosphorylating the transcriptional activator Yes-associated protein (Yap). 12,13 Phosphorylation causes Yap to remain in the cytoplasm and prevents the transcription of proliferation-specific genes. 14 On the other hand, the absence of Nf2 results in the lack of merlin, hence prevents Yap from phosphorylation and promotes the nuclear translocation of Yap to transcribe cell proliferation-specific genes. 14

Deficiencies in Nf2 function and
Hippo signalling activity lead to excess cell proliferation, organ overgrowth, tumourigenesis 15,16 and metastasis, 17 which are often promoted by the nuclear translocation of Hippo signalling repressor form, Yap protein. [18][19][20] However, our results indicated paradoxically distinct roles that give rise to a large population, if not all, of the lingual mesenchyme. 6,22 To understand where Nf2 plays its regulatory role during tongue development, we examined the distribution of Nf2 and Yap proteins. At E18.5, Nf2 immunosignals were extensively distributed in the tongue mesenchyme ( Figure 1A), but not apparent in the tongue epithelium ( Figure 1A). Yap immunoproducts were broadly detected in the mesenchyme including the papilla core region subjacent to the tongue epithelium and bright signals were observed in the deeper layers of the mesenchyme of E18.5 tongue ( Figure 1B). The concurrent distribution of Nf2 (bright) and Yap (faint) immunosignals was evident in the mesenchyme including the mesenchymal zone immediately under the epithelium (arrowheads in Figure 1A,B).
Wnt1-Cre has been widely used to label NC cell lineage 4 and labelled cells are largely distributed in the tongue mesenchyme, 6,23,24 though rare labelled cells have also been found in the tongue epithelium. 6 We have previously reported that Wnt1-Cre-labelled NC-derived cells populate the mesenchyme of tongue primordium -all four branches 1-4 at E10.5. 2 To confirm Wnt1-Cre-driven genetic alterations in neural crest-derived tongue mesenchyme at later stages of tongue development, E18.5 Wnt1-Cre/RFP tongues were analysed and we found that RFP + cells were dominantly detected in the tongue mesenchyme and no RFP + cells were seen in the tongue epithelium in the examined serial sections ( Figure 1C).

| Wnt1-Cre induces deletion of Nf2 in the tongue mesenchyme and reduced Nf2 transcripts in the epithelium
Nf2 in situ hybridization analyses revealed that mRNA transcripts were broadly distributed in both epithelium and mesenchyme of Cre − /Nf2 fx/fx littermate control tongues at all three stages tested (E12.5, E15.5 and E18.5) (Figure 2A, B). In addition, our RNA sequencing data also showed that Nf2 mRNA transcripts were present in both epithelium and mesenchyme in E12.5, E14.5 and postnatal day 1 (P1) tongues with a significant difference at E12.5 when tongue mesenchyme had a higher level of Nf2 mRNA transcripts compared to the epithelium (p < 0.05 in Figure 2C). In Wnt1-Cre/Nf2 cKO mutants, Nf2 transcripts were significantly reduced in both epithelium and mesenchyme at all three stages tested (Figure 2A,B). Quantitative RT-PCR analyses confirmed the significantly low Nf2 expression in both epithelium and mesenchyme of E12.5 Wnt1-Cre/Nf2 cKO mutant tongues compared to that of Cre − /Nf2 fx/fx littermate controls (p < 0.05 in Figure 2D).

| Nf2 cKO in neural crest-derived tongue mesenchyme leads to macroglossia at early but microglossia at late stages of tongue development
To understand the roles of Nf2 in the tongue development, phenotypic analyses were performed in Wnt1-Cre/Nf2 cKO mutant tongues and Cre − /Nf2 fx/fx littermate controls at multiple embryonic stages.
At E11.5 when tongue swellings emerged from the branchial arches, tongue swellings were clearly seen in Cre − /Nf2 fx/fx littermate controls  Figure 3A). As a result of the collective changes in the tongue length and width, E15.5-E16.5 Wnt1-Cre/Nf2 cKO mutants F I G U R E 1 Single-plane laser scanning confocal images of E18.5 tongue sections immunoreacted with Nf2 (A, green), Hippo signalling transcription factor Yap (B, green) or epithelial cell marker E-cadherin (C, green). RFP + cells in C were labelled by Wnt1-Cre. Sections were counterstained with DAPI (blue). Arrowheads in A and B point to the mesenchymal layer immediately under the epithelium. Dashed lines separate the epithelium from the underlying mesenchyme. Scale bars: 50 μm had a relatively smaller tongue (i.e. microglossia, Figure 3A) compared to the Cre − /Nf2 fx/fx littermates ( Figure 3A). At E18.5, microglossia phenotype in the Wnt1-Cre/Nf2 cKO mutants was evident compared to Cre − /Nf2 fx/fx littermates ( Figure 3A).

| Region-and stage-specific alterations of Hippo signalling in Wnt1-Cre/Nf2 cKO mouse tongues
Nf2 is a known cell proliferation suppressor and often acts via Hippo-YAP signalling to regulate organ size. 13,25 To understand the potential cause of the alteration of tongue shape and size, we

| Distinct alterations of cell proliferation in different regions of tongue primordium (branchial arches) in mesenchymal Nf2 cKO mice
To understand the potential cause of the unproportioned alterations in tongue shape and size at early stages, we examined the Nf2 expression, Hippo signalling activity and cell proliferation in E10.5 branchial arches (BAs) 1-4 of Wnt1-Cre/Nf2 cKO mutants and Nf2 in situ hybridization revealed that in Cre − /Nf2 fx/fx littermate controls Nf2 mRNA transcripts were abundantly distributed in all four BAs that will give rise to the developing tongue ( Figure 8A). In Wnt1-Cre/Nf2 cKO mutants, Nf2 transcripts were absent in the corresponding regions of all four BAs ( Figure 8B). Similar to Cre − /Nf2 fx/fx littermate controls ( Figure 8C), all four BAs (n = 3) were developed in the Wnt1-Cre/Nf2 cKO mutants ( Figure 8D). However, the distance between the lateral edges of each BA was significantly smaller in Wnt1-Cre/Nf2 cKO mutants ( Figure 8D,E, p < 0.05) compared to those of Cre − /Nf2 fx/fx littermate controls ( Figure 8C).

| DISCUSS ION
Our study demonstrated that the absence of Neurofibromin 2 suggesting that the effects of altered Nf2/Hippo signalling activity are distinct in different organs.

| Stage-and tongue region-specific regulation of Hippo signalling and cell proliferation in response to mesenchymal Nf2 deletion
As aforementioned, Nf2/Hippo signalling serves as a cell proliferation suppressor. 51 To understand the stage-specific alterations of

| Mesenchymal deletion of Nf2 alters the development of overlying tongue epithelium
It is intriguing that although the genetic deletion of Nf2 driven by Wnt1-Cre is largely tongue mesenchyme-specific, 6

| Animals use and tissue collection
The use of animals was approved by the Institutional Animal Care

| Immunohistochemistry on sections
Tongue tissues were carefully dissected from the mandible and   Quantitative RT PCR was conducted using the cDNA to analyse the expression of Yap target genes using the primers in Table 2.

| In situ hybridization
Changes of gene expression levels in tongue epithelium and mesenchyme of Wnt1-Cre/Nf2 cKO mutant and Cre − /Nf2 fx/fx littermate control groups were presented as mean ± standard deviation (X ± SD;

| Statistical analysis
Student's t-test was used to analyse the statistical significance of differences between Wnt1-Cre/Nf2 cKO mutants and Cre − /Nf2 fx/fx littermate controls for the indices below: the oral tongue length, anterior and posterior tongue widths, Western blot band intensities of Yap, p-Yap, and Gapdh. Two-way analyses of variance (ANOVA) followed by Fisher's LSD analyses were used to compare BrdU + cells per unit area (mm 2 ) in individual BAs and tongues between Wnt1-Cre/Nf2 cKO mutants and Cre − /Nf2 fx/fx littermate controls. A p-value <0.05 was taken as statistically significant.

| CON CLUS ION
Our data indicate a region-and stage-specific role of Nf2 in NCderived tongue mesenchyme in regulating Hippo signalling and cell proliferation, which is in distinction from many other organs.

| NF2/Hippo signalling shapes tongue organ
Schematic diagram to represent the stage-and region-specific roles

CO N FLI C T O F I NTE R E S T
All authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.