Precise mapping of one classic and three novel GluRIIA mutants in Drosophila melanogaster

Abstract

Mutation of the Drosophila melanogaster GluRIIA gene or pharmacological agents targeting it are commonly used to assess homeostatic synaptic function at the larval neuromuscular junction (NMJ). The commonly used mutation, GluRIIA ^SP16 , is a null allele created by a large and imprecise excision of a P-element which affects GluRIIA and multiple upstream genes. Here we mapped the exact bounds of the GluRIIA ^SP16 allele, refined a multiplex PCR strategy for positive identification of GluRIIA ^SP16 in homozygous or heterozygous backgrounds, and sequenced and characterized three new CRISPR-generated GluRIIA mutants. We found the three new GluRIIA alleles are apparent nulls that lack GluRIIA immunofluorescence signal at the 3 ^rd instar larval NMJ and are predicted to cause premature truncations at the genetic level. Further, these new mutants have similar electrophysiological outcomes as GluRIIA ^SP16 , including reduced miniature excitatory postsynaptic potential (mEPSP) amplitude and frequency compared to controls, and they express robust homeostatic compensation as evidenced by normal excitatory postsynaptic potential (EPSP) amplitude and elevated quantal content. These findings and new tools extend the capacity of the D. melanogaster NMJ for assessment of synaptic function.

Figure 1. Identification of exact bounds of the GluRIIA ^SP16 lesion and three new CRISPR-generated GluRIIA mutants.

(A) The GluRIIA gene region is shown with the span of the GluRIIA ^SP16 lesion and the location of the CRISPR target site in exon 6 of GluRIIA . (B) Representative traces of EPSP and mEPSP electrophysiology. Horizontal and vertical scale bars for EPSP (mEPSP) traces are 50 ms (1000ms) and 10 mV (1 mV) respectively. (C) mEPSP amplitude, (D) mEPSP frequency, (E) EPSP amplitude, and (F) quantal content were assessed at the NMJ of wandering 3 ^rd instar animals. (C-F) Data plotted are medians with 25%-75% interquartile ranges. Each symbol represents a single muscle (n ≥ 8 muscles per condition). * = p < 0.05 by Kruskal-Wallis with Dunn’s correction for multiple comparisons of GluRIIA mutant lines to w ¹¹¹⁸ controls. (G) Larval NMJs were stained with HRP (top) to detect the motor nerve and anti-GluRIIA antibodies (bottom). G luRIIA ³⁰⁴ , GluRIIA ³⁰⁶ , and GluRIIA ⁷⁰⁴ failed to generate signal for GluRIIA. (H) The reference genome (chr2L: 5,556,421 - 5,556,460) with CRISPR-target site (blue), were aligned to DNA sequencing reads. Red symbols (*) and text in CRISPR-generated lines denote deleted and inserted nucleotides respectively.