In C. elegans, the sole insulin/IGF-1 receptor DAF-2 has multiple prominent functions in development, metabolism, reproduction and lifespan regulation. Its functional importance has attracted intense research interest since 1992. However, after 25 years and hundreds of research articles, the expression pattern of
daf-2 remains unknown, let alone the expression patterns and functions of individual isoforms. The only exception is the
daf-2c isoform, which was characterized and found to be a neuronal isoform that regulates learning (Ohno H, Science. 2014; Tomiako M, Nat Commun. 2016). Taking advantage of the recently developed CRISPR/Cas9 gene editing technology, we sought to dissect the functions and expression patterns of
daf-2 in an isoform-specific manner. To visualize the expression of
daf-2 mRNAs, we knocked in a tandem GFP::mNeonGreen tag with two nuclear localization sequences and a SL2-type ICR (inter-cistronic region) sequence in front of the coding sequence of each predicted isoform of
daf-2, except the c isoform. To visualize the expression of DAF-2 proteins, we fused a tandem GFP::mNeonGreen fluorescent protein tag to the DAF-2 C-terminus, which is shared by all six protein-coding isoforms, and then introduced frame-shifts to knock out specific isoforms individually or in combination. The function of DAF-2 appears intact with the C-terminal GFP::mNeonGreen tag, for this knock-in strain is phenotypically indistinguishable from wild type in development and lifespan. From the strains described above, we detected the DAF-2 protein on the surface of early embryonic cells starting from the two-cell stage and on the surface of adult neurons, germ cells, oocytes, and vulval cells. The
daf-2 mRNA was detected in two additional tissues-intestine and hypodermis. With respect to the isoforms, we found that the DAF-2a protein was expressed exclusively in the germ line, although expression of
daf-2a mRNA was detected in the intestinal and hypodermal cells. Deletion of
daf-2a reduced brood size and greatly extended lifespan to a level that is more than half way between wild type and the
daf-2(
e1370) mutant. The DAF-2f protein was expressed exclusively in a subset of neurons. This isoform was critically important for dauer formation, and its contribution to lifespan regulation will be analyzed after we overcome a technical issue. Expression of the d and e isoforms were not detected. Expression of the g isoform, which encodes a truncated DAF-2 protein lacking the extracellular and trans-membrane regions, was found in vulval cells only. These isoforms appeared to make no or little contribution to dauer formation, reproduction, and lifespan regulation.