The Merlin lab uses laboratory raised and wild populations of monarch butterflies to study the genetic, neural and evolutionary bases of migration and clockwork mechanisms in animals. Current projects focus on:
1. Identifying the genetic and epigenetic basis of migratory physiology and behavior
The genetic and epigenetic basis of migration, a seasonal adaptation used by animals to escape from unfavorable environments, have remained largely unknown. The Eastern North American monarch butterfly is uniquely suited to study the genetics of migration because: 1) its yearly migratory cycle involves several generations of migratory and non-migratory forms equipped with the same genetic make up, 2) migratory monarchs have a remarkable repertoire of seasonal physiological and behavioral traits that are quantifiable and activated when day length decreases in the fall (i.e., oriented flight and reproductive diapause), 3) the monarch genome is sequenced, and 4) we can now introduce targeted mutations in vivo.
Our current approaches to illuminate the molecular program underlying migratory behavior and physiology focus on identifying 1) the mechanisms of circadian clock control of the photoperiodically-induced migration, and 2) the epigenetic mechanisms governing the migratory switch. To tackle these questions, we use integrated approaches that combine genome-wide profiling of gene expression and of active gene cis-regulatory elements (using RNA-seq and DNase-seq), reverse-genetics (using CRISPR-mediated gene targeting) and physiology and behavior.
Our ultimate goal is to characterize candidate genes and regulatory regions using reverse-genetic tools to determine how specific genes contribute to the awe-inspiring yearly migration of monarch butterflies.
Top: Monarch migratory routes. Orange arrow:
Fall migration; Green arrows: Spring remigration. Map from MonarchWatch. Bottom: Fall southward migration
coincides withdecreasing daylengths. Data from
2. Determining the mechanisms of circadian repression in the animal clockwork and their evolution
Circadian clocks are cell-autonomous timekeeping systems that allow a wide array of organisms to anticipate daily changes in the environment. Such anticipation enables the coordination of physiological, metabolic and behavioral processes so that they occur at the appropriate time of day. Because the clock is central for monarch migration, we have a keen interest in understanding the molecular logic underlying the butterfly circadian timekeeping mechanism.
The intracellular molecular mechanisms driving ~24hour rhythms in animals rely on transcriptional/translational feedback loops. Interestingly, the monarch clockwork has been found to be a hybrid form of the Drosophila and the mammalian circadian clocks. In mammals, the heterodimeric bHLH-PAS-domain containing transcription factors CLOCK (CLK):BMAL1 activate the transcription of period 1 and period 2 (pers), and cryptochrome 1 and cryptochrome 2 (crys), which upon translation, translocate back into the nucleus and inhibit transcription through interactions with the BMAL1 C-terminus. In Drosophila, CLK:CYCLE (CYC) drive rhythmic transcription of period (per) and timeless (tim), but after translation, PER interacts with CLK to inhibit CLK:CYC-mediated transcription. Studies conducted by the Reppert lab led to the surprising discovery that the monarch clock possesses mammalian-like features, i.e the presence of a vertebrate-like cryptochrome (insect CRY2), which functions as the main transcriptional repressor of the molecular oscillator.
Our lab is currently manipulating clock genes in vivo to further dissect circadian repressive mechanisms in the monarch clockwork and to understand how insect clocks have evolved in different lineages.
Circadian clock mechanisms rely on a transcriptional/translational feedback loop in which repressors inhibit their own transcription on a 24hr basis. The monarch butterfly core circadian clockwork is an hybrid between the Drosophila and the mammalian clockworks.
3. Expanding the genome editing toolbox in the monarch butterfly
Representation of genome editing technologies to manipulate genes in vivo in non-model insects. Top: Transcription Activator-Like Effector Nucleases (TALENs); Bottom: RNA-guided Nuclease (CRISPR/Cas9). From Reppert, Guerra and Merlin, Annual Reviews of Entomology, in press.
To effectively use the monarch as a model system to study the genetic and neural basis of migratory behavior, it was necessary to develop efficient genetic tools. In vivo targeted mutagenesis has previously been realized in the monarch using Zinc-Finger Nucleases - the only approach available to create custom site-specific mutations at the time. This work established an embryo injection strategy for robust nuclease delivery to target the monarch germline, but improving targeting efficieny is still required to make reverse genetics amenable to routine use in the lab.
Genomic engineering has been revolutionized by the discovery of new classes of engineered endonucleases, Transcription Activator-Like Effector Nucleases (TALENs) and the bacterial CRISPR/Cas9 system. Because the CRISPR/Cas9 system provides greater ease of use and targeting efficiency over other nuclease systems, we are now applying this technology in the monarch with the goals of 1) improving the efficiency of non-homologous end-joining-mediated targeting to facilitate the recovery of germline mutants, and 2) developing knock-in approaches to introduce reporter tags into loci of interest.
This work should provide a framework for genetic analyses in other lepidopterans and “non-model” insects.