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Cephalopods, such as squid and octopuses, seem to be highly intelligent creatures. They have large brains and complex nervous systems that they use to control pigment cells in their skin for camouflage and mate attraction. Although there’s no way for us to know exactly what’s going on in a cephalopod’s mind, it’s easy to anthropomorphize them — many people who work with them find them charming and relatable.

Doryteuthis pealeii embryos fertilized and cultured in vitro, or outside of the organism. Iridescent chorion (embryonic coating) can be seen. Credit: Karen Crawford

Karen Crawford, 2018 Whitman Fellow from St. Mary’s College of Maryland, finds cephalopod embryos fascinating. These minuscule, delicate, jelly-coated embryos could be the key to understanding how cephalopods evolved such large brains. Crawford also thinks cephalopods are just — well — cute.

Embryonic development in squid 30 hours post-Dil dye injection, revealing the cleavage patterns of embryonic cells. Credit: Karen Crawford

“Pyjama squid, similarly to cuttlefish, almost appear to have unique personalities, although we can’t say for sure,” Crawford suggests. “When working with them, it seems they’ll like you or they won’t. If they don’t, they won’t approach you. Or they’ll flash their chromatophores (pigment cells) red as a warning.”

During the summers of 2016 and 2017, Crawford collaborated with several cephalopod researchers at С����Ƶ to study the Woods Hole squid, Doryteuthis pealeii, a local species whose nervous system has been studied at С����Ƶ for nearly a century. She established methods for injecting a dye called DiI into cells in their early embryos to track the cells’ journey throughout development. This summer, she’s continuing to generate what’s known as a fate map for Doryteuthis — a diagram that shows where each embryonic cell will end up in the developing organism.

Karen Crawford in her С����Ƶ laboratory. Credit: Kirsten Peramba

The fate map will help her identify the cells responsible for forming the brain and visual system of the cephalopod. This will offer insight into how cephalopods evolved such large brains, Crawford says.

“Having a fate map will help us understand how cephalopod embryos are special and unique,” says Crawford. “Once we have a fate map, we’ll better understand the pressures and constraints that make them different, and perhaps this will shed light on how they developed what appears to be such high intelligence.”

Crawford is also expanding her research to two new species, the striped pyjama squid and the Hawaiian bobtail squid. She plans to create fate maps for these species, for comparative purposes. She also aims to develop methods to edit these species’ genes while they’re embryos, in order to test the function of specific genes in the developing organism and what happens when they are mutated.

This summer, Crawford is injecting both striped pyjama squid and Hawaiian bobtail squid embryos with molecules that snip and edit the embryo’s DNA. By editing just one cell during an embryo’s two-cell stage, she can create a chimera: an animal with both edited and normal genes. One cell from this stage becomes the tissue in the left half of the squid and the other cell makes up the right half. For example, if she edits one cell to knock out the production of pigment cells, one half of the animal will be albino, while the other half will be normal.

Once Crawford establishes the most effective protocol for embryonic gene editing, she’ll try the technique in other cephalopod species. With this knowledge in hand, scientists will be able to create cephalopod lineages with specific traits. (When embryonic cells are edited, those changes can be passed down to future generations.) Her hope is that these studies will help scientists to unlock the evolutionary secrets behind cephalopod behavior.

“Evolutionarily, these organisms are from the Paleozoic era and before. I want to know what they’ve been doing in all of this evolutionary time,” says Crawford. “They’re extremely successful — you’re looking at the biggest and most sophisticated invertebrate brain on the planet. For example, they have a camera eye like ours, but it evolved separately.”

Striped pyjama squid embryo in culture. Credit: Karen Crawford

For many of us, the fact an animal so evolutionarily distant from humans has such a sophisticated brain is fascinating — and maybe a little frightening. Although we’re intrigued by their sophisticated and seemingly intelligent behaviors, we can’t yet understand the world from their perspective. There’s a lot about them we’d like to know, and with Crawford’s help, we’ll soon understand these squishy, tentacled creatures better.