Core circadian clock genes control molecular and behavioral circatidal rhythms in Parhyale hawaiensis

This study demonstrates that while the four core circadian clock genes (BMAL1, Cry2, Per, and Clk) are collectively essential for driving both 24-hour circadian and 12.4-hour circatidal rhythms in the amphipod *Parhyale hawaiensis*, they utilize distinct transcriptional wiring mechanisms, including a unique BMAL1-independent repression of *Per* by *Clk* specifically in circatidal neurons.

The Gist

Imagine your body has an internal master clock that tells you when to sleep and when to wake up. This is the circadian clock, and it runs on a roughly 24-hour cycle, synced with the sun rising and setting.

But for creatures living in the ocean, like the tiny shrimp-like animal Parhyale hawaiensis (let's call it the "beach hopper"), there's a second, equally important clock: the circatidal clock. This one runs on a 12.4-hour cycle, synced with the rising and falling of the tides. When the tide is high, the beach hopper swims; when it's low, it hides.

For a long time, scientists were puzzled: How does an animal run two different clocks at the same time? Do they have two separate timepieces, or is it one giant clock that can stretch and shrink?

This paper is like a detective story where scientists took apart the beach hopper's internal machinery to see how it works. Here is the simple breakdown:

1. The "Gears" of the Clock

Think of the circadian clock as a complex machine made of four main gears that turn together: BMAL1, CLOCK, PER, and CRY.

• BMAL1 and CLOCK are the "gas pedals." They push the machine forward, telling genes to turn on.
• PER and CRY are the "brakes." They kick in to stop the machine, creating a cycle of on-and-off that creates the rhythm.

Scientists already knew that if you broke the BMAL1 gear, the beach hopper lost both its day/night rhythm and its tide rhythm. They suspected the other three gears (CLOCK, PER, and CRY) might also be shared between the two clocks.

2. The Experiment: Breaking the Gears

The researchers used a molecular "scissors" tool (CRISPR) to break each of the other three gears one by one in the beach hopper's DNA. They then watched what happened to the animal's behavior and its internal brain cells.

The Results were surprising:

• Breaking any single gear stopped everything. Whether they broke the CLOCK, PER, or CRY gear, the beach hopper lost its ability to track both the 24-hour day and the 12.4-hour tide.
• The Takeaway: The beach hopper doesn't have two separate clocks with different parts. Instead, it uses the exact same four gears to power two different rhythms. It's like using the same engine to drive a car on a highway (24 hours) and a boat in the ocean (12.4 hours).

3. The Twist: The Wiring is Different

If they use the same gears, why do the rhythms run at different speeds? The answer lies in how the gears are connected.

Imagine a factory assembly line.

• In the "Day/Night Factory" (Circadian Neurons): The CLOCK gear pushes the PER brake pedal. This is the standard setup: Gas pedal pushes, then the brake kicks in to stop it. This creates a 24-hour loop.
• In the "Tide Factory" (Circatidal Neurons): Here, the wiring is weird. The CLOCK gear actually pushes down on the PER brake pedal to stop it from working. It's like the gas pedal is also acting as a brake for a different part of the machine!

This "rewiring" is the secret sauce. Even though the parts are identical, the way they talk to each other is different in the two types of brain cells. This allows the same set of genes to create two different rhythms simultaneously.

4. Why This Matters

This discovery solves a biological mystery. It shows that evolution didn't need to invent a brand-new clock for the tides. Instead, it took the existing 24-hour clock, duplicated the brain cells that use it, and simply rewired the electrical connections between the parts.

The Analogy:
Think of it like a smartphone. You can run a "Work Mode" app and a "Gaming Mode" app on the exact same hardware (the same processor and screen). You don't need a second phone. You just change the software settings (the wiring) to make the phone behave differently depending on what you need it to do.

Summary

• The Problem: How do marine animals track both the day and the tides?
• The Discovery: They use the same four core "clock genes" for both rhythms.
• The Secret: The genes are wired differently in different brain cells. In the "tide cells," the CLOCK gene acts as a repressor (a brake) for PER, which is the opposite of what it does in the "day cells."
• The Big Picture: Nature is efficient. Instead of building a new clock from scratch, it just rewired the old one to handle a second schedule.

Technical Summary

1. Problem Statement

Marine organisms in the intertidal zone face two distinct environmental cycles: the 24-hour light/dark cycle (circadian) and the ~12.4-hour tidal cycle (circatidal). While the molecular mechanisms of circadian clocks are well-conserved across animals (involving a negative feedback loop of Clock, Bmal1, Period, and Cryptochrome), the mechanism driving circatidal rhythms remains elusive. Three main hypotheses exist:

1. Distinct clocks for each rhythm.
2. A single plastic clock capable of running at different periods.
3. Two circalunidian (~24.8h) clocks running in antiphase.

Previous studies in other species (e.g., Eurydice pulchra, Apteronemobius asahinai) suggested that core circadian genes might not be required for circatidal rhythms. However, recent work in the amphipod Parhyale hawaiensis showed that knocking out PhBmal1 abolished circatidal behavior, suggesting a mechanistic overlap. This study aims to determine if the other three core circadian genes (PhPer, PhClk, and PhCry2) are also essential for circatidal rhythms and to elucidate the transcriptional wiring differences between the two clock systems.

2. Methodology

The researchers utilized the marine amphipod P. hawaiensis and employed a multi-faceted approach:

• CRISPR-Cas9 Genome Editing: Generated knock-out (KO) lines for PhPer, PhClk, and PhCry2 (complementing existing PhBmal1 KOs).
  • PhCry2: Two lines with truncations in the photolyase domain.
  • PhPer: Three lines with truncations eliminating PAS and PAC domains.
  • PhClk: Two lines with truncations in the bHLH and PAS domains.
• Behavioral Assays:
  • Animals were entrained to Light/Dark (LD) cycles and tidal cycles (simulated by water level changes: 10.3h high tide / 2.1h low tide).
  • Activity was monitored under constant conditions (Constant Light [LL] or Constant Darkness [DD] for circadian; Constant Darkness [DD] + Constant High Tide for circatidal).
  • Swimming and roaming behaviors were recorded using Drosophila Activity Monitors (DAM).
  • Rhythmicity, period, and power were analyzed using autocorrelation and $\chi^2$ periodogram analysis.
• Molecular Analysis (HCR-FISH):
  • Hybridization Chain Reaction Fluorescent In Situ Hybridization (HCR-FISH) was used to quantify mRNA levels of PhPer and PhCry2 in specific neuronal populations.
  • Circadian Neurons: Medioposterior (MP) cells (entrain to LD).
  • Circatidal Neurons: Dorsal-Lateral (DL) cells (entrain to vibration/tides).
• Transcriptional Assays:
  • Luciferase reporter assays in HEK-293T cells to test the repressive capabilities of PhPER and PhCRY2 on the PhBMAL1/mCLK heterodimer.

3. Key Contributions

• Confirmation of Shared Core Machinery: Demonstrated that all four core circadian genes (PhBmal1, PhClk, PhPer, PhCry2) are strictly necessary for both circadian and circatidal behavioral rhythms in P. hawaiensis.
• Discovery of Cell-Type Specific Wiring: Revealed that while the same genes are used, the transcriptional logic (wiring) differs between circadian and circatidal neurons.
• Novel Repressive Function of CLK: Identified a non-canonical role for PhCLK in circatidal neurons, where it acts as a repressor of PhPer independently of PhBMAL1.
• Resolution of Phase Relationships: Confirmed that PhPer and PhCry2 oscillate in antiphase in circadian neurons but in phase in circatidal neurons.

4. Key Results

A. Behavioral Rhythms

• Circadian: Loss of PhCry2, PhPer, or PhClk severely disrupted free-running circadian rhythms (under LL and DD). Most homozygous mutants became arrhythmic, and those retaining weak rhythms showed abnormal periods.
• Circatidal:
  • PhCry2 and PhBmal1 KOs completely abolished circatidal behavioral rhythms.
  • PhPer and PhClk KOs resulted in a significant reduction in rhythmic animals, but a subset of mutants retained weak, entrainable circatidal rhythms (though molecular rhythms were lost). This suggests these genes are critical but perhaps slightly less redundant than Cry2 or Bmal1 for behavioral output.

B. Molecular Rhythms (HCR-FISH)

• Circadian Neurons (MP cells):
  • In WT, PhPer and PhCry2 showed robust 24h rhythms in antiphase.
  • In PhCry2 KO: PhCry2 mRNA was absent; PhPer levels were elevated and arrhythmic.
  • In PhPer KO: PhPer mRNA absent; PhCry2 levels elevated and arrhythmic.
  • In PhClk and PhBmal1 KOs: Both genes showed low, non-oscillating expression, confirming their role as activators in this context.
• Circatidal Neurons (DL cells):
  • In WT, PhPer and PhCry2 showed robust ~12.4h rhythms in phase.
  • In PhCry2 and PhBmal1 KOs: Both genes lost rhythmicity; PhCry2 was low, PhPer was high/arrhythmic.
  • Crucial Finding in PhClk KO: PhCry2 remained low (as expected for a loss of activator), but PhPer mRNA levels were constitutively high. This indicates that in circatidal neurons, PhCLK normally represses PhPer, a function opposite to its canonical role as an activator in circadian neurons.

C. Transcriptional Mechanisms

• Luciferase assays confirmed that PhCRY2 is the primary repressor of the PhBMAL1/mCLK complex, while PhPER plays a modulatory role.
• The data supports a model where the circadian and circatidal clocks use the same four core components but are wired differently:
  • Circadian: CLK/BMAL1 activate Per and Cry2; PER/CRY2 repress CLK/BMAL1.
  • Circatidal: CLK/BMAL1 activate Cry2, but CLK represses Per (potentially independently of BMAL1).

5. Significance

• Mechanistic Overlap vs. Distinct Clocks: The study supports the "separate clocks" hypothesis (Naylor model) where distinct neuronal populations (MP vs. DL) utilize the same core genes but with different transcriptional wiring to generate different periods (24h vs. 12.4h).
• Evolutionary Insight: It suggests that the evolution of circatidal rhythms did not require the invention of new genes but rather the rewiring of existing circadian components within specific neuronal circuits.
• Novel Gene Function: The discovery that PhCLK acts as a repressor of PhPer in circatidal neurons challenges the canonical view of CLK as a universal activator and highlights the plasticity of clock gene functions.
• Ecological Adaptation: This dual-clock system allows P. hawaiensis to integrate diurnal and tidal cues, enabling behavioral plasticity across diverse tidal environments (semi-diurnal, mixed, and diurnal).

In conclusion, the authors demonstrate that the circatidal clock in P. hawaiensis is not a separate molecular entity but a rewired version of the circadian clock, utilizing the same core genes in distinct neuronal populations with unique regulatory logic to achieve a ~12.4-hour period.