The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

Purpose

Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants.

Methods

We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality.

Results

Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants.

Conclusion

GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.