Forward genetic approach for behavioral neuroscience using animal models

Abstract

Forward genetics is a powerful approach to understand the molecular basis of animal behaviors. Fruit flies were the first animal to which this genetic approach was applied systematically and have provided major discoveries on behaviors including sexual, learning, circadian, and sleep-like behaviors. The development of different classes of model organism such as nematodes, zebrafish, and mice has enabled genetic research to be conducted using more-suitable organisms. The unprecedented success of forward genetic approaches was the identification of the transcription-translation negative feedback loop composed of clock genes as a fundamental and conserved mechanism of circadian rhythm. This approach has now expanded to sleep/wakefulness in mice. A conventional strategy such as dominant and recessive screenings can be modified with advances in DNA sequencing and genome editing technologies.

Keywords: animal behavior; circadian rhythm; forward genetics; model animal; mutagenesis; sleep.

Figure 1.

(Color online) Major discoveries mainly due to forward genetics. Major mutants and cloned genes related to behaviors are indicated for fruit fly, nematode, zebrafish, and mouse in chronological order. Several technical advances are also indicated. # indicates transcription factors. & indicates channels or transporters. Before 1990, however, the function of the gene was usually unknown at the time of gene identification. TTFL: transcription–translation feedback loop.

Figure 2.

(Color online) Forward genetic screening schemes. A. Dominant screening identifies a heterozygous mutation (m/+) that causes phenotypic changes. B. Recessive screening identifies a homozygous mutation (m/m) that causes phenotypic changes. Compared with dominant screening, recessive screening takes a larger number of generations to identify a mutation. C. Suppressor screening begins with the injection of ENU to the phenodeviants (m/m). The effect of the suppressor mutation on the phenotype was assessed using heterozygous mutant mice (m/+). D. Histogram of a pedigree with a strong dominant phenotype. The two peaks are mice heterozygous for the mutant gene (m/+; red bars) and mice without the mutant gene (+/+; blue bars). E. Histogram of a pedigree with a strong recessive phenotype. The two peaks are mice homozygous for the mutant gene (m/m; red bars) and mice without the mutant gene (+/+; blue bars). Heterozygous mice (m/+) are omitted from this histogram. F. Histogram of heterozygous mutant mice (m/+) with a suppressor mutation (sup/+; green bars) or without the mutation (red bars). G. Histogram of heterozygous mutant mice (m/+) with an enhancer mutation (en/+; purple bars) or without the mutation (red bars).

Figure 3.

(Color online) SIK3 and ion channels related to sleep changes. A. KCNA2 has 6 transmembrane segments and the selective filter formed by a TVGYG sequence. B. CACNA1A is composed of four repeats of 6 transmembrane segments and has a selective filter motif of EEEE. The substitution of F102 to L decreased the time spent in wakefulness in Drowsy mutants. C. NALCN has four repeats of 6 transmembrane domains and has the selective filter motif of EEKE. The substitution of N315 to K caused abnormal REM sleep in Dreamless mutants. D. Wild-type murine SIK3 has a kinase domain and protein kinase A (PKA)-phosphorylation site, S551. E. Slp mutant SIK3 lacks the exon13-encoded region. F. Serine 551 of SIK3 is substituted with alanine. G. Drosophila SIK3 orthologue has a PKA-phosphorylation site, S563, equivalent to murine S551. H. C. elegans SIK3 orthologue, kin-29 has the PKA-phosphorylation site, S517.