In vivo functional analysis of the human NF2 tumor suppressor gene in Drosophila.

The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes

In Vivo Functional Analysis of the Human <i>NF2</i> Tumor Suppressor Gene in Drosophila

The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes.</p

Ectopic expression of hNF2 isoforms 1 and 2 has different effects on Drosophila wing development.

<p>Two transgenic copies of each isoform were expressed in the dorsal half of the developing wing blade under the control of the <i>apterous</i>-Gal4 driver. The apterous-Gal4 driver itself has little or no effect on wing development (A–B), while expression of isoform 1causes some ectopic vein formation, particularly along the second and fifth wing veins (C–D). Expression of isoform 2 (E–F) causes more severe ectopic vein formation that includes veins 2, 3 and 5. In addition, expression of this isoform causes a downward cupping of the wing blade (data not shown).</p

qPCR analysis of effect of hNF2 expression on endogenous Merlin expression.

1<p>Fold expression change compared to control genotype, <i>ApGal4/+.</i></p

Mutations in <i>hNF2</i> alter the subcellular localization of the Merlin protein expressed in Drosophila.

<p>Confocal optical sections of wing imaginal epithelia, either cutting deeply into the epithelium (‘Cross section’) or tangentially through the apical surface of the epithelium, are presented for each allele or isoform. A–B) NF2 isoform 1 is primarily localized to the apical membrane (arrows) of these polarized epithelial cells, and adopts a punctate appearance at the apical cortex that is not clearly associated with cell boundaries (see inset at right). In addition, some punctate staining is observed basally (arrowheads in A). C–D) In contrast isoform 2, while still primarily apical (arrows, C), appears associated with cell boundaries (outlining the periphery of the apical ends of cells in D) and does not form punctae. The localizations of the K413E (E–F) and L64P (G–H) point mutations appear similar to that of isoform 1. The Δ2-3 deletion allele (I-J) appears localized to the lateral cell cortex, somewhat similar to isoform 2, but less strongly apically localized (arrows mark apical; also note staining throughout the basolateral domain in I). NF2^1-356 (K–L) is substantially cytoplasmic and fails to accumulate apically while NF2^351-595 (M–N) shows some apical enhancement (arrows in M). Both truncations display substantial cytoplasmic localizations, as indicated by the tangential views (compare L and N to J).</p

Genetic rescue by hNF2 alleles.

1<p>Average of three or more independent lines.</p>2<p>Using two of the insertions tested in the one-dose experiments.</p

The <i>NF2^L64P</i> mutation affects subcellular localization in a temperature sensitive fashion.

<p>Subcellular localizations of expressed NF2 isoform 1 (A–B) and the L64P mutation (C–D) are compared in wing imaginal discs kept at 25°C (A,C) or 37°C (B,D) for two hours prior to dissection. While the L64P mutant protein shows normal apical, punctate localization at 25°C (C), it is much more cytoplasmic and evenly distributed at 37°C (D). In contrast, the higher temperature has no apparent effect on subcellular distribution of the wild type isoform 1 protein, which was apical and punctate at both temperatures (A–B). Insets in B and D show higher magnification views of representative areas.</p

Diagrams of hNF2 proteins used in this study.

<p>The known protein domains, including the FERM domain, the coiled-coil domain, and the C-terminal FERM binding domain, are indicated as shown. All constructs are N-terminally tagged with the FLAG epitope. Details of the mutant alleles are provided in the text.</p