Fas2EB112: A Tale of Two Chromosomes

Abstract

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily-conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2^G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2^EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2^EB112 has been used in at least 37 research articles, and Fas2^G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2^G0336) or enhances (Fas2^EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg¹⁴, also a classic null allele.

Figure 1:

Two Fas2^EB112 chromosomes demonstrate different phenotypic severity. A and B) Follicle epithelium mutant for Mannheim Fas2^EB112 demonstrates rare popping out, whereas Bloom. 19A Fas2^EB112 mutant tissue has many popped-out cells. Mutant tissue is marked by the absence of RFP (in magenta). Scale bars = 20μM. C) Quantification of popping out shows that the Bloom. 19A Fas2^EB112 mutant is significantly more severe than other Fas2-disrupting conditions. D, E, F) Fas2 immunoreactivity (measured with the 1D4 antibody) is lost from Fas2^G0336 and Fas2^EB112 clones. Scale bars = 5μM. Significance was determined using an unpaired t-test with Welch’s correction. In this and subsequent figures, significance is indicated as follows: p < 0.05 (*), 0.01 (**), < 0.001 (***), < 0.0001 (****).

Figure 2:

Characterization of E(Fas2)^mut. A) Fas2 is detected at follicle cell-cell borders in E(Fas2)^mut tissue. Scale bar = 5μM. C, D, E) Representative images showing the enhancement of popping out in Fas2-shRNA tissue. Scale bars = 20μM.

Figure 3:

Nrg is not expressed in E(Fas2)^mut tissue. A and B) Anti-Nrg immunoreactivity is lost from follicle cell-cell borders in E(Fas2)^mut clones (marked by the absence of RFP). These images were generated using a rabbit polyclonal antibody that recognizes a C-terminal stretch of Nrg. Two views, sagittal (A) and en face (B) are shown. Discs large (Dlg), which localizes to cell-cell borders in a similar manner to Nrg, is used as a control. Scale bars = 5μM (A) or 20μM (B). C) A mouse monoclonal anti-Nrg antibody also fails to detect signal at follicle cell-cell borders in E(Fas2)^mut clones. Scale bars = 5μM. D and E) Anti-Nrg immunoreactivity is retained at cell-cell borders in Mannheim Fas2^EB112 tissue (D) but lost in Bloom. 19A Fas2^EB112 mutant tissue (E). Scale bars = 5μM. F) Immunoblotting reveals that E(Fas2)^mut and Bloomington Fas2^EB112 chromosomes do not contribute expression of Nrg¹⁶⁷ and Nrg¹⁸⁰ protein isoforms, whereas the Mannheim Fas2^EB112 chromosome does. Nrg¹⁴ is used as a negative control. Expression of Nrg::YFP is stronger when Nrg¹⁶⁷ and Nrg¹⁸⁰ are lost. Amido black staining reveals total protein and is therefore a loading control. Significance was determined using an unpaired t-test.

Figure 4:

E(Fas2)^mut is Nrg¹⁴. A) Expression of Nrg from the second chromosome rescues the viability of E(Fas2)^mut male flies. Balanced mutant females were crossed to males carrying an Nrg genomic rescue insertion on the second chromosome. This strategy allowed for the appearance of Nrg¹⁴ and E(Fas2)^mut male progeny. B) Expression of Nrg from the second chromosome rescues popping out in Bloom. 19A Fas2^EB112 mutant tissue. (The Bloom. Fas2^EB112 data in this figure is also shown in Figure 1C.) Additionally, Nrg knockdown causes the appearance of popped-out cells but this phenotype is not enhanced by E(Fas2)^mut. C) Cumulative plot of broken sequencing reads and their position along the chromosome. Sequencing was performed at 100X coverage and the Y-axis scale is set at 0–100. The X axis (position along the X chromosome) is shown. The central divots indicate small deletions described in the text. D) Polytene X chromosomes from female larvae with one X chromosome from the indicated genotype and the other from a w¹¹¹⁸ male. Loops that indicate the common inversion are highlighted with a red arrow.