Sea Lampreys Rewrite Our Understanding of Vertebrate Brain Evolution

If you were to encounter a sea lamprey while swimming, you might think that you had stumbled upon an eel. That is, until you spot its suction cup mouth, ringed with razor-sharp teeth.

Admittedly, this 500-million-year-old animal has not fared well in the looks department.

Native to the Northern and Western Atlantic Ocean, the sea lamprey is a jawless vertebrate, often nicknamed the “vampire fish”. It spends a considerable portion of its life latching on to other fish, rasping away tissue in order to feed on blood, all the while secreting an enzyme that prevents its prey’s blood from clotting. Pretty gruesome, right? 

The notion that we humans have anything in common with this parasitic predator might sound ludicrous, but over recent years, scientists have compiled a robust body of evidence demonstrating that the development of our hindbrain is very similar to that of the sea lamprey.

The hindbrain is a complex region of the vertebrate brain, made up of the upper part of the spinal cord, the cerebellum and the brain stem. It regulates functions that are vital to an organism’s survival, such as breathing, heart rate, digestion, sleep and motor control. As the hindbrain is considered to be the oldest part of the brain, its study can shed light on the evolution of vertebrates.

The sea lamprey is an interesting model in this context, given that it shares features with other vertebrates – a backbone and a skeleton – but lacks a jaw on its head. Scientists assumed the development of the sea lamprey's brain and head must be orchestrated by very different mechanisms to those that are at play in other vertebrates.

This assumption was recently shattered by researchers in the lab of Professor Robb Krumlauf at the Stowers Institute for Medical Research. They discovered that the Hox genes, which orchestrate the structuring and subdividing of jawed vertebrates’ hindbrains, also regulate this process in the sea lamprey.

Their most recent work, published in Nature Communications, extends our understanding of vertebrate evolution further still. Krumlauf and colleagues identified retinoic acid (RA) as a common molecular cue that initiates and directs the Hox genetic circuitry, responsible for correct hindbrain formation, across all vertebrates.

RA was known to initiate hindbrain development in complex vertebrates, but the fact that it also guides the development of a primitive, jawless vertebrate is a discovery that “makes us rethink some of the textbook explanations for how cues and signals are used to regulate development,” said Krumlauf. It implies that these molecular features were present in the common ancestor of all vertebrates today. 

Technology Networks recently had the pleasure of interviewing Dr. Alice Bedois, a former postdoc in the Krumlauf lab and lead author of the study, and Dr. Hugo Parker, a study co-author and senior scientist in the Krumlauf lab.

We wanted to gain insight into the backstory of this research project, how the Krumlauf lab team designed their methodology and the implications of a study that “rewrites the textbooks on evolution”.

Molly Campbell (MC): Can you explain why the sea lamprey is an interesting creature, and your motivation for studying it?

Alice Bedois (AB): Sea lampreys belong to the group of jawless vertebrates that split from jawed vertebrates (which include mammals) close to 500 million years ago.

It’s important to keep in mind that lampreys have kept evolving over that period of time, but they lack important features that jawed vertebrates have, such as a jaw. Using lamprey as a model and comparing it to what we know from other well-established vertebrate models, like mice, we can get an idea of what the ancestor to all vertebrates was like. In the context of our research, we were interested in the origins of hindbrain development, which is unique to vertebrates.

There are different types of lampreys that, together with hagfish, constitute the only remaining (non-extinct) jawless vertebrates. The sea lamprey is an attractive model organism and jawless vertebrate representative, because adults can produce thousands of embryos that develop slowly, allowing us to conduct gene-editing experiments in batch over the breeding season. This is an embryologist’s dream.

MC: Can you discuss the previous research in which the genes structuring and subdividing the sea lamprey hindbrain to those in jawed vertebrates were identified?

Hugo Parker (HP): In jawed vertebrate models, such as mice, decades of research have identified a complex network of genes that subdivide the hindbrain into segments, and then generate unique circuits of neurons within each segment. This process is fundamental for correct brain formation, as well as for the development of the head and neck.

A gene family called the Hox genes plays a crucial role in the formation and patterning of these segments. In lamprey, previous research had suggested that Hox genes were only partially associated with hindbrain segmentation, and that the lamprey hindbrain represented an intermediate step in vertebrate brain evolution with respect to Hox gene-dependent brain patterning. I sought to investigate this further by comprehensively characterizing the lamprey Hox genes and their roles in hindbrain patterning.

During that early work, I developed a technique to insert genetic elements into the genomes of lamprey embryos. These genetic elements contained regulatory sequences that drive stripes of green fluorescent protein (GFP) in the hindbrain when inserted into jawed vertebrates, such as zebrafish. Using this technique, I could test whether these regulatory sequences from jawed vertebrates could also generate stripes of fluorescent gene expression in lamprey.

This provided a direct readout on the similarities and differences between the gene regulatory networks of lamprey and jawed vertebrates. It led to a series of experimental results that really knocked our socks off: many of the regulatory sequences drove beautiful segmental expression of GFP in the hindbrain of lamprey embryos. This showed that many aspects of early hindbrain segmentation and patterning are in fact shared between lamprey and jawed vertebrates and were thus present in the common ancestor of all vertebrates that are around today.

MC: In this study, you identified a common molecular clue as being part of the gene circuitry guiding hindbrain patterning in sea lampreys. This cue is RA. Can you talk about what RA is, and what was already known about its role in gene circuitry?

AB: RA is a molecule derived from vitamin A that plays an important role in regulating gene expression. RA is provided very early on by the mother and plays a critical function in the early development of the embryo. In skin, it helps with cell turnover and promotes cellular growth.

It has long been known that RA plays a central role in the network regulating hindbrain development in jawed vertebrates, where it initiates and later coordinates a cascade of regulatory events that eventually lead to the formation of segments in the hindbrain. This is extremely important for later head development and the establishment of the neuron circuitry for coordinating autonomous functions, like breathing.

This complex process of hindbrain development doesn’t exist beyond vertebrates that do not have a hindbrain. However, RA is present in invertebrate chordates, where it regulates Hox genes in axial development, which means that this RA/Hox relationship is a very ancient feature shared by many animals.

It is thought that this ancient RA/Hox hierarchy was later integrated into a complex molecular process, which permitted the evolution of the hindbrain structure in jawed vertebrates. Twenty years ago, people thought that lampreys do not share this aspect of hindbrain development, and that their hindbrain is set up independently of the Hox genes and RA. In this study, we show that this is not the case, as RA is essential in initiating and maintaining hindbrain development in lamprey using a similar molecular network as other vertebrates.

MC: Can you discuss the key methods that you adopted in this study, and why?

HP: We implemented a multi-pronged strategy to investigate the role of RA in lamprey embryos, focusing on characterizing the machinery that synthesizes and degrades RA, where and when this machinery is activated, as well as observing what happens to the hindbrain when this machinery is perturbed.

Key to this was using new, state of the art techniques such as hybridization chain reaction (HCR) and CRISPR-mediated gene perturbation. HCR provides a fluorescent readout of gene activation at a cellular level and enabled us to characterize multiple genes simultaneously in the same embryo, which was crucial for characterizing segmental domains of gene activation.

We used both CRISPR-mediated gene knockout and drug treatments to perturb RA synthesis and degradation. The CRISPR approach is relatively new but is becoming more widely used and enabled us to target specific genes directly, rather than relying solely on drug treatments which can sometimes have other indirect effects.  

MC: What was the feeling in the lab when you discovered RA to be the molecular cue?

AB: I think we were all excited to see that our dear lampreys were not so primitive after all, and that important mechanisms evolved early in the evolution of vertebrates, before the split between jawed and jawless vertebrates, including the integration of the ancient RA/Hox regulatory hierarchy into a broader network underlying this unique process of hindbrain development.

We thought that it made a lot of sense, considering the ancestral role that RA plays in axial development in most animal groups, but it was important to demonstrate it. We were also very excited about some differences in this network in lamprey as compared to other vertebrate models, including in terms of the number of genes implicated in it, or where these are expressed in the hindbrain. In our view, the global conservations and the small particularities could explain how two vertebrate groups evolved and how original hindbrain development likely resembled the vertebrate ancestor.

HP: The biggest excitement for me was seeing Alice’s beautiful data from the HCR characterization of the RA-degrading genes, which showed their clear activation in specific hindbrain segments. This really indicated that RA activity is regulated in a segment-specific manner in the lamprey hindbrain. This was a smoking gun, and we spent a good while marveling at how clear and compelling these images are.

Another exciting moment was observing the effects of the CRISPR perturbations and drug treatments in lamprey embryos for the first time. Seeing whole dishes of truncated embryos that strongly resembled those obtained from equivalent drug treatments really brought home the powerful effect of this multi-pronged approach of RA perturbation.

MC: What are, in your opinion, the key implications of this work?

AB: I think this work really brings lamprey and jawless vertebrates closer to us jawed vertebrates. It is possible that the lamprey and its interesting genome could be more systematically considered and looked into in the future when exploring the origins of certain mechanisms.

MC: What are your next steps to advance this field of research?

AB: We are hoping that future research will help us better understand the origin of certain regulatory aspects of the hindbrain network, including how RA is able to regulate its own synthesis and degradation via the Aldh1a2 and Cyp26 families, and we know sea lamprey can help with that!

Dr. Alice Bedois and Dr. Hugo Parker were speaking with Molly Campbell, Senior Science Writer for Technology Networks.

About the interviewees

Dr. Alice Bedois completed her PhD in the laboratory of Dr. Robb Krumlauf at the Stowers Institute for Medical Research. She is now a scientific expert in genetics at Eurofins Biomnis.

Dr. Hugo Parker obtained his PhD in functional genomics from Queen Mary University of London. He joined the Stowers Institute for Medical Research in 2011 as a postdoctoral researcher, where he is currently a senior research associate.