Lineage domains and cytoskeletal cables organize a cellular square grid in a crustacean

Introduction

Most epithelia are packed in hexagons: this is the minimal energy configuration we would expect, assuming cells as contractile bubbles squeezed into a confluent sheet.

But then something peculiar must have happened in the embryos of Malacostracan crustaceans – shrimps, crabs and lobsters etc. – collectively the largest class in Crustacea. During their early development, the embryonic ectoderm forms a square grid of cells (Fig 1).

Fig 1. Emergence of the square grid during Parhyale hawaiensis development. Figure adapted from the preprint where it is available under a CC-BY 4.0 International license.

Both the square-packing and the numerous 4-way junctions are supposed to be energetically unfavourable, and it remains unknown how cells could establish and maintain this spectacular pattern.

Here, Beatrice L. Steinert and colleagues provided a detailed view of grid initiation and progression in the model crustacean Parhyale hawaiensis with long-term multiview lightsheet microscopy. Furthermore, using laser ablation and pharmacological perturbations, they probed the role of tensile actomyosin cables in grid formation, shedding light on the cellular mechanisms behind this extraordinary case of epithelium morphogenesis.

Key findings

1. Observation of the grid formation process

Researchers first obtained volumetric, long-term timelapse images of the grid formation process using multiview lightsheet microscopy, and annotated cell lineage, cell positions and mitotic activities.

Researchers found that the grid initiates at the boundary between anterior and posterior ectodermal lineages. This boundary, which researchers referred to as IL (initiation line), emerged immediately after gastrulation and gradually straightens into a line perpendicular to the anterior-posterior axis (fig 2). During this process, 4-way vertices and square-shaped cells gradually emerges on the IL.

Fig 2. Formation and straightening of the IL as a lineage boundary. Figure adapted from the preprint where it is available under a CC-BY 4.0 International license.

The second axis of the square grid is the ventral midline perpendicular to the IL. This midline is formed through mediolateral cell intercalation initiating at the IL and propagating both anteriorly and posteriorly, with cells acquiring square shapes after joining the midline (fig 3). In contrast to the IL, the midline is polyclonal and does not correspond to lineage boundary.

Fig 3. Formation of the midline through intercalation and subsequent cell shape changes. Figure adapted from the preprint where it is available under a CC-BY-NC 4.0 International license.

2. Simulation of IL formation and straightening

Researchers simulated the ectoderm on the CompuCell3D platform, asserting an increase in contact energy between the anterior and posterior lineages. The simulation reproduced the formation of the IL as a straight boundary between these lineages, showing that IL can be formed through a differential cell sorting process (fig 4).

Fig 4. Simulation results reproduces IL formation and straightening. Figure adapted from the preprint where it is available under a CC-BY 4.0 International license.

3. The role of tensile cytoskeletal cables in grid formation

Immunostaining showed that both the IL and the midline are enriched in f-actin and myosin. Laser ablation experiments further showed that these cytoskeletal cables in the IL and the actively intercalating part of the midline are under higher tension than other regions of the ectoderm.

Interestingly, the role of this tensile cytoskeleton seems to differ in the IL and the midline:

Laser ablation at the IL or slightly anterior to it did not disrupt 4-way vertices, and did not prevent the formation of the square grid. On the other hand, ablations lateral to the midline in the posterior would result in a local disruption of the square grid, while ablation on the midline would completely abolish the grid throughout the posterior ectoderm (fig 5). In line with this, drug inhibition of ROCK did not compromise IL formation and straightening, but significantly disrupted cells aligning into the midline
and subsequent grid formation.

Fig 5. Lateral or midline laser cuts disrupt the posterior grid without affecting IL. Figure adapted from the preprint where it is available under a CC-BY 4.0 International license.

4. The role of the grid in segment patterning

Previous works have shown that the segmentation gene Engrailed (En) is expressed in a stripe pattern corresponding to specific rows in the grid. Here, researchers found that laser ablation of lateral cells, which locally disrupted the grid as mentioned above, also decreased the number of En-expressing cells on the same side; ablation of midline cells, which disrupted the entire grid, resulted in a more significant decrease on both sides of the ectoderm (Fig 6).

Fig 6. Laser cuts disrupting the grid also reduce the number of En+ cells. Figure adapted from the preprint where it is available under a CC-BY 4.0 International license.

Why I highlight this preprint

I was at once intrigued by the unique square grid pattern studied here – it looks very improbable especially when thinking about the physics. I find the data presented by the authors very informative, in particular the cell-resolution timelapse and the outcomes of perturbing the actomyosin cytoskeleton. I very much look forward to future works uncovering more details behind this!

Questions for the authors

1. In the lateral and midline ablation experiments, while cutting one cable does not seem to alter the tension on other cables, the cut still has non-cell autonomous effects as it would abolish the whole grid. How could these be reconciled in your opinion?
2. Square cells may also be packed in a staggered grid (like a brick wall) without 4-way junctions. However, given the subsequent patterning on specific grid rows, it looks like the grid needs to stay aligned. How do you think cells could maintain the seemingly unstable 4-way junctions?

 

doi: https://doi.org/10.1242/prelights.41534

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