Chapter 10: Sea Urchins and Tunicates: Deuterostome Invertebrates

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Welcome back to The Deep Dives, where we break down complex topics into the core knowledge you need.

Today, we are taking a, well, a really massive leap across one of the most fundamental divides in the entire animal kingdom.

We really are.

For the past while, we've been deep in the world of protostome development.

We're talking about insects, snails, worms, that whole group.

And the defining feature there, just as a quick refresher, is that the first embryonic opening, the blastopore, becomes the mouth.

Right.

But today, we're flipping that script entirely.

We are moving into the realm of the deuterostome.

So our mission for This Deep Dive is to really understand those foundational first steps of how a body plan gets made in two really key invertebrate deuterostome groups, the sea urchins and the tunicates.

You might also know tunicates as sea squirts.

And the name deuterostome itself is a huge clue.

It's Greek for second mouth.

Second mouth.

And that name, it perfectly describes the pattern that defines this entire lineage.

That includes us, by the way, all vertebrates.

During gastrulation, that first opening, the blastopore becomes the anus.

And the mouth forms later, secondarily.

Exactly.

At a completely different spot.

Now, what makes studying these two specific invertebrates so fascinating is this historical paradox they created for the first developmental biologists.

Oh, this is a great story.

For decades, they were the absolute poster children for two completely opposite ideas about how embryos decide what to become.

On one side, you had the sea urchin.

It was the classic example of conditional specification.

Or regulative development.

That means the early cells are, you know, flexible, they're pluripotent, and their fate is decided by signals from their neighbors.

Right.

It's all about context.

But then, on the flip side, you have the tunicate.

It was the textbook example of autonomous specification.

Also called mosaic development.

So here, the cell's fate seems to be locked in from the get -go.

Almost immediately.

It's all predetermined by inheriting these little

molecular instructions, cytoplasmic determinants that the mother puts in the egg.

So one is flexible, the other is fixed.

That was the old view.

But here is the real takeaway for our deep dive today.

The reality is so much more sophisticated.

They both actually use a complex, highly integrated blend of both autonomous inheritance and conditional signaling.

So the paradox isn't real.

It's not.

They just lean one way or the other.

We're going to spend a good chunk of time dissecting the sea urchin's journey first, and then we'll see how the tunicate pulls off a similar trick to build its body.

Early development in sea urchins.

The regulative master.

Okay, let's start with the sea urchin.

And it's hard to overstate just how important these little spiny creatures are to the history of developmental biology.

Absolutely.

They are foundational.

I mean, when you think about the biggest conceptual breakthroughs in the field,

the sea urchin is almost always right there at the center of the story.

You have to start with Hans Drich, right?

Back in the 1890s.

You do.

He performed this, well, this revolutionary experiment that just shattered the dominant belief at the time, which is all about mosaic development.

And what did he do?

He took a four cell stage, sea urchin embryo, and he physically separated one of those four cells, one blastomere.

And according to the thinking back then that one cell should have only been able to make what a quarter of a larva.

Exactly.

A sad little quarter fragment.

That was the prediction.

But that's not what happened.

What happened instead?

Instead, that single isolated cell started to regulate its own development.

It adjusted its internal programming and went on to form a whole perfectly proportioned pluteus larva, a bit smaller, sure, but complete.

Wow.

So that proved beyond a doubt that the cells at that stage weren't fixed.

Their fate was conditional.

It depended on their interactions on their neighbors, which were now gone.

And the sea urchin's contributions just kept coming.

I mean, think about the discovery of cyclins.

The proteins that regulate the cell cycle.

And basic discoveries about the roles of DNA and RNA, how gene expression is controlled by enhancers.

It's just a legacy organism for the field.

So to understand how all this works, we have to start at the beginning with cleavage.

Right.

Sea urchins undergo what's called radial holoblastic cleavage.

Holoblastic just means the cut goes all the way through the egg.

Which makes sense for an egg that doesn't have a ton of yolk getting in the way.

Exactly.

The first three divisions are incredibly orderly.

The first two are meridional.

So slicing from the top pole, the animal pole, down to the vegetal pole at the bottom.

Right.

And they're perpendicular to each other.

So you go from one cell to two, then two to four equal cells, like cutting an orange into quarters.

Then the third division is equatorial.

It slices horizontally,

separating the top animal half from the bottom vegetal half.

So now you have an eight cell embryo, four on top, four on the bottom.

And this is where the real drama kicks in.

The fourth cleavage.

Because here things get unequal.

Very unequal.

In the top tier, the animal half, those four cells divide equally, meridionaly again, to make eight cells called mesomeres.

Very orderly.

But down in the vegetal tier.

It's a whole different story.

Those four vegetal cells divide horizontally, but it's this incredibly skewed, unequal cleavage.

And that creates two different cell types.

Yes.

You get four large cells, the macromeres, and four tiny little cells right at the very bottom at the vegetal pole.

Those are the micromeres.

And the size difference is just staggering.

It is.

In some species, the macromeres keep something like 95 % of the cytoplasm, leaving the micromeres with just a tiny fraction.

But as we're about to see, those tiny micromeres are the molecular powerhouses that are going to direct the entire body plan.

It's a classic case of big things coming in small packages.

So as these divisions continue, the embryo reaches about the 120 cell stage, and it's no longer just a loose clump of cells.

Right.

It organizes itself into a hollow sphere, the blastula.

And that central cavity is called the blastocall.

How does it form that sphere?

The cells form a really tight epithelial sheet.

They connect to each other with tight junctions, which kind of zip them together.

They're also stuck to an outer layer, the highline layer, and there's fluid inside the blastocall.

And the whole thing expands, right?

Yeah, largely because water gets drawn into the blastocall by osmosis, inflating it like a balloon.

This is also where the cells start to get their polarity.

The outside surface becomes ciliated.

And once it's ciliated, the whole blastula starts to rotate inside its protective fertilization envelope.

This leads to the next big step,

hatching.

How does it get out?

The cells in the animal hemisphere, the top half, start to secrete a special hatching enzyme.

This enzyme literally digests the envelope from the inside, and the blastula breaks free.

And now it's a free swimming larva.

At the same time, the cells at the very bottom, the vegetal pool, they're getting ready for the next phase.

They start to thicken and flatten, forming what we call the vegetal plate.

This is where the action is going to happen next.

Okay, so we've got this 60 cell stage blastula.

It's organized.

It's swimming around.

Now we need to know where all these different cells are going to end up.

We need a fate map.

And researchers figure this out by injecting fluorescent dyes into individual cells and then tracking where their descendants go.

A key thing to remember is that at this stage,

most cells are specified.

They have their instructions, but they're not yet determined.

They're still pluripotent.

They can still change their minds if they get a different signal.

Exactly.

So let's walk through that map from top to bottom.

The whole animal half from those mesomeres that gives rise to the ectoderms.

So the larval skin and the neurons.

Right.

Then below that, you hit the VEG -1 tier.

These cells are pretty flexible.

They can become either ectoderm or endoderm.

Depends on the signals they get.

Then the VEG -2 tier.

These are a bit more committed.

They're destined to become the endoderm, which forms the gut, and also the non -skeletogenic mesencom.

Which is what exactly?

Think of them as the support crew.

They make things like pigment cells, immune cells, and some internal muscles.

They're also called the secondary mesencom.

But the real key to this whole map, as we said, is right at the very bottom.

The micromeres.

The four large micromeres, specifically.

They have one and only one destiny.

They become the skeletogenic mesencom, or primary mesencom.

Their only job is to build the larval skeleton.

That's it.

And those tiny small micromeres,

they're basically put on paws.

They contribute to the adult tissues much later, during metamorphosis.

And importantly, they contain the germline cells.

So they're holding onto the future generations while the big micromeres build the temporary larval body.

Precisely.

And this brings us right back to that core idea.

The two -step process that beautifully integrates autonomous and conditional specification.

Okay, lay it out for us.

How does this work?

Step one is autonomous.

The large micromeres are specified autonomously.

They inherit specific maternal determinants that were parked at the vegetal pole of the egg.

So because they get that specific molecular baggage, their fate is sealed from the inside.

It is.

This is why if you take those micromeres out at the 16 -cell stage and put them in a dish by themselves, they'll still go on to make skeletal spicules.

They don't need neighbors.

They just know what to do.

Incredible.

So that's step one.

What's step two?

Step two is the conditional part.

Once these micromeres are autonomously specified, they immediately become the organizing center.

The conductor of the orchestra.

They start sending out signals.

Paracrine injects different signals.

And those signals are what conditionally specify all their neighbors, telling the VEG -1 and VEG -2 cells what to become, pushing them toward that endometoderm fate.

And the classic proof for this is the horse stadius experiment.

It's just so elegant.

It really is.

So you take the animal cap, the top half of the embryo, which normally only ever makes ectoderm, just skin.

Right.

You isolate it.

Then you place a few of those powerful little micromeres underneath it.

And what happens is just mind -blowing.

The micromeres induce a complete change of fate.

They force those presumptive skin cells to become endoderm, to form a gut.

You end up with a recognizable tiny larva, where the gut is made from cells that should have been skin.

It's like a cellular reprogramming driven entirely by the micromere signals.

And you can take it a step further.

If you transplant micromeres to the animal pole of an otherwise normal embryo, they'll induce a whole second gastrulation event and a second body axis.

They are the autonomous drivers of the whole conditional system.

Gene regulatory networks, GRNs, the specification.

Okay, this is where we get into the really deep stuff.

How do these little micromeres know what to do?

How do they get this incredible signaling power?

To answer that, we have to move from the cellular level to the molecular level.

We have to talk about gene regulatory networks, or GRNs.

A concept really pioneered by people like Eric Davidson.

Right, and a GRN is, I mean, it's essentially a biological logic circuit.

It's not just one gene turning on another.

It's this huge interconnected web.

Where transcription factors bind to the control regions of other genes, the enhancers and promoters, and tell them when and where to turn on.

Exactly.

The network starts with a few initial inputs from the mother, from the egg, and then it basically self -assembles, activating more and more specific downstream genes.

So what are those first maternal inputs that kick the whole sea urchin network off?

It starts with two key players.

A protein called disheveled, and another one you've probably heard of, bedeticatinin.

Right, bedicatinin is involved in a lot of things.

It is.

Both are already present in the egg cytoplasm.

But disheveled is specifically anchored.

It's tethered right to the vegetal pole.

So when that unequal cleavage happens,

only the micromeres inherit the disheveled.

That's the key.

And the job of disheveled is to protect bedicatinin from being destroyed.

Normally, it's degraded really quickly.

So in the micromeres, disheveled is present.

It protects bedicatin, which then accumulates.

It accumulates, and then it can move into the nucleus, team up with another factor called TCF, and start turning on the entire genetic program for endoderm and mesoderm.

And this nuclear accumulation is absolutely critical.

There's really strong experimental proof for this.

Oh, absolutely.

If you treat an embryo with lithium chloride, which blocks the enzyme that normally degrades bedicatin, you get bedicatinin accumulation in every cell.

And the result is chaos.

Complete chaos.

The cells that should have been ectoderm, skin, they all turn into endoderm and mesoderm.

You get a ball of gut and skeleton with almost no skin.

And the reverse is also true.

If you block bedicatinin from getting into the nucleus?

You get the opposite.

No endoderm, no mesoderm at all.

You just get a hollow, ciliated ball of ectoderm.

So bedicatinin in the nucleus is the master switch for all vegetal fates.

OK, but that raises a question.

If edicatinin is active in both the micromeres and the VIG2 cells, how do the micromeres get their unique skeletal fate?

Ah, now that is where the true elegance of the GRN comes in.

It uses a brilliant piece of genetic logic called the double negative gate.

A double negative gate.

OK, break that down for us.

It sounds complicated.

The logic is actually very clean.

It starts with nuclear bedicatinin and another maternal factor called oddex.

Together, in the micromeres, they turn on a gene called PMAR1.

And PMAR1 is only turned on in the micromere.

Only there.

No, the PMAR1 protein is a repressor.

Its job is to turn off its target gene.

And its target is a gene called HESC.

Correct.

Now, HESC is also a repressor.

And here's the key.

HESC is normally active in almost every cell of the embryo, except for the micromeres, because that's where PMAR1 is active and is shutting it down.

I see.

So you have a repressor, PMAR1 repressing another repressor, HESC.

Exactly.

So what does HESC normally repress?

It represses the entire suite of genes needed to build a skeleton, all the key transcription factors like ALX1, TBR, ETS1, and also signaling genes like delta.

So let's trace the logic.

In the micromeres, PMAR1 is on N.

It represses the HESC repressor.

And when you repress a repressor, you get— The skeletogenic genes are unlocked.

They're free to be expressed.

But everywhere else outside the micromeres, PMAR1 is OOFF.

So HESC is on N.

And it's actively sitting on those skeletogenic genes, keeping them silenced.

It's a perfect, clean, binary on -off switch.

And it's restricted just to those four crucial cells.

Once that switch is flipped, the cell needs to make sure it stays on.

It has to stabilize that decision.

Right.

You can't have it flickering.

The network uses things like positive feedback loops to do this.

For example, Bledocatin and Otagex turn on a gene called WinA8.

And WinA8 is a signaling molecule.

It is.

But instead of signaling to its neighbors, the 1A protein is received by the very same micromeres that made it.

That's called autocrine regulation.

The cell is signaling to itself.

Precisely.

And this signal reinforces their own betacatin levels, amplifying the initial decision and locking the state on.

It's like turning up the volume on your own fate.

So that explains the micromeres' own destiny.

Now, how do they use this power to tell their neighbors what to do?

The conditional part.

They use two main signaling pathways.

The first is a paracrine signal for inducing the endoderm.

The micromeres start to secrete a TGF beta factor called Activen.

And I'm guessing the Activen gene is one of those downstream targets that gets unlocked by the double negative gate.

You got it.

So Activen diffuses out from the micromeres, and it's the critical signal that tells the surrounding VEGU cells to become endoderm, to become gut.

And the second pathway is more for close -range communication.

Right.

That's a juxtacrine or cell -to -cell signal.

This involves the protein delta, which is also turned on by the micromere GRN.

So the micromeres have delta protein on their surface.

And that delta protein interacts directly with notch receptors on the surface of the immediately adjacent VEG2 cells.

Physical contact is required.

And that delta notch signal is like a fork in the road for those VEG2 cells.

It is.

It turns on a transcription factor called GCM, and that pushes those cells to become the non -skeletogenic mesenchyme.

At the same time, it represses an endoderm -promoting factor called FoxA.

So the VEG2 cells that are physically touching the micromeres get the delta signal and become secondary mesenchyme.

And the VEG2 cells a little further away don't get the signal, so they keep their FoxA on and become the bulk of the endoderm.

It's an incredibly precise way to pattern different cell types right next to each other.

C, urchin gastrulation, and morphogenesis.

All right, so the molecular decisions have been made.

Fates are specified.

Now the embryo has to actually build something.

It has to move all these cells into the right place, which brings us to gastrulation.

This is where things get really dynamic.

The first and most dramatic movement is the ingression of those skeletogenic mesenchyme cells, the descendants of the large micromeres.

They're going to leave the outer epithelial layer and move into the blastocool.

They are.

This happens just after hatching, and it's a process called an epithelial to mesenchymal transition, or EMT.

EMT.

It's a huge deal in development.

It's basically a complete change in cellular identity.

It really is.

These cells go from being part of this tight cohesive sheet to becoming individual migratory free -roaming cells.

They lose their affinity for their neighbors and gain an affinity for the proteins on the inside of the blastocool wall.

And what's amazing is that the same GRN we just talked about, the one that specified their fate, also orchestrates the five distinct physical steps of this transition.

It starts with a shape change.

First, the whole vegetal plate thickens.

Then step two, apical constriction.

The micromeres constrict the part of the cell facing outwards, which makes them change shape into little bottles.

This helps initiate the inward movement.

But they can't just push their way through.

There's a barrier, the basement membrane.

So that's step three.

They remodel that barrier.

The cells secrete enzymes, proteases, that literally digest little holes in the basal lamina, clearing a path for themselves.

Once the path is clear, they have to detach from their old neighbors.

Step four, de -adhesion.

This is controlled by a transcription factor called snail.

Snail's job is to go in and degrade the ket -herin proteins that were holding the cells together.

This is the moment they truly become individual.

And finally, they have to be able to move.

Step five, motility.

The GRN turns on genes that allow the cells to extend these long, thin, exploratory fingers, called filopodia, and start actively crawling along the extracellular matrix on the inside of the blasticle.

And they're not just wandering around randomly in there.

They're on a mission.

A highly targeted mission.

They extend these incredibly long filopodia to feel their way along the blasticle wall, searching for specific chemical cues.

And what are those two?

There are two big ones.

First, a signal called VEGF, which is released from the two spots where the cells are supposed to gather.

It's like a chemical beacon sink assembled here.

And the second one?

The second is FGS, which is made in a belt around the equator of the embryo.

This acts more like a road guiding their migration path.

Once they arrive at their destination, they cluster together.

They do.

They fuse to form syncytial cables, which is like a network of cells that share a cytoplasm.

And then they begin the amazing process of biomineralization,

depositing calcium carbonate to form the skeletal rods of the larva.

So while the skeleton crew is busy at work, the rest of the vegetal plate has to move in to form the gut, the archenturron.

And this begins the main phase of gastrulation.

Stage one is the initial invagination.

The whole vegetal plate just kind of folds inward, forming a short tube.

The opening that's left on the outside is the blast pore.

And since this is a deuterostome, that will become the anus.

Correct.

And the cells leading this invasion are the non -skeletogenic mesenchyme cells.

They form a little cluster at the tip of the growing archenturron.

Behind them come the endoderm cells that will form the gut itself.

But this initial fold only gets it part of the way in.

The archenturron has to get much, much longer.

It has to triple in length.

And it does this first through a process called convergent extension.

Convergent extension.

This is a beautiful mechanical process.

It is.

The endoderm cells basically slide past each other and intercalate.

Imagine two lanes of traffic merging into one long lane.

The tissue gets narrower, but much longer.

But even that doesn't get the gut all the way to its final destination.

No, that only gets it about two -thirds of the way there.

The final phase is a literal mechanical pull.

Driven by those non -skeletogenic mesenchyme cells at the tip.

Exactly.

They extend their own filopodia, but this time they're searching for a very specific target on the inner wall of the animal hemisphere.

The future mouth region.

The future mouth region.

Once they make contact, they contract, they shorten, and they physically pull the rest of the archenturron up to meet the wall.

It's an active, guided process that completes the gut tube.

A perfect example of molecular signaling leading to large -scale physical morphogenesis.

And with that, we pivot from the sea urchin to our other key player, the tunicate.

Early development in tunicates, the autonomous link.

It's always so surprising to talk about tunicates.

I mean, you look at an adult sea squirt, this kind of sessile blob on a rock, and it's hard to believe that it's one of our closest invertebrate relatives.

It is, but the connection becomes so obvious when you look at the larva.

The free -swimming tunicate larva is why they are classified as chordates.

It has a notochord.

It has a notochord, a dorsal neural tube, pharyngeal arches,

all the hallmarks of our phylum.

It's the key evolutionary link.

Now, unlike the sea urchin's radial cleavage, tunicates have bilateral holoblastic cleavage.

And that first division is incredibly important.

It establishes the future right and left sides of the animal, right from the very first cut.

And the divisions that follow are often very asymmetrical.

Yes, and this is driven by a structure called the centrosome attracting body, or CAB.

It positions the mitotic spindle off center, so you get one big cell and one small cell, and it also acts like a magnet, pulling specific maternal mRNAs into that smaller posterior cell.

So this is the mechanism for parceling out those cytoplasmic determinants we talked about at the beginning.

This is exactly.

And in tunicates, you can actually see these determinants because the cytoplasm has different colors.

Describe that for us.

After fertilization, there's this huge reorganization of the cytoplasm.

And you end up with distinct regions.

There's a clear cytoplasm that becomes ectoderm, a slate gray region for endoderm, and most famously, a vibrant yellow crescent that forms on the future posterior side.

And that yellow crescent has a very specific fate.

A very specific fate.

It is destined to become all of the tail muscles of the larva.

The cells that inherit that yellow cytoplasm are the only ones that can make muscle.

It's the visual proof of autonomous specification, and scientists have figured out what the key molecule is in that yellow crescent.

They have.

It's an mRNA called MACHA1, and MACHA1 encodes a transcription factor.

And this is a classic case of a molecule being both necessary and sufficient, right?

It is.

If you get rid of MACHA1 mRNA, no muscle forms.

If you take MACHA1 and inject it into a cell that should have been skin, that cell will turn into muscle instead.

It's the master regulator.

So MACHA1 turns on the whole cascade of muscle building genes.

It does.

But it also has a second really critical job, which is repression.

MACHA1 turns on the snail gene,

and snail is a repressor.

What's it repressing?

It's repressing another key gene called brachyri.

And this is so important because brachyri is the master gene for specifying the notochord.

Ah, so MACHA1 makes sure that its muscle cells can't accidentally get confused and try to become notochord.

It locks in the muscle fate by shutting down the alternative.

Exactly.

It's about ensuring developmental integrity.

But it's also worth noting before we go on that tunicates and sea urchins still share some deep similarities.

Endoderm specification in tunicates, for example.

Let me guess.

It involves beta -catenin.

It involves the nuclear accumulation of beta -catenin.

It's a deeply conserved pathway.

Which is a great reminder that even in this poster child for autonomous specification, it's not the whole story.

The really important coordinate structures, the notochord, the brain, they require conditional signaling.

Absolutely.

So for the tunicate to form its notochord, it needs an inductive signal.

And where does that signal come from?

It comes from the endoderm cells, the ones specified by beta -catenin.

They start secreting FGFs, fibroblast growth factors.

And that FGF signal is received by neighboring cells, and it tells them to turn on...

The brachiary gene.

And the activation of brachiary is what specifies the notochord.

So this is where it all comes together.

We have the eponymous determinant, macho 1, and the conditional signal, FGF.

How do they integrate?

This is the most elegant part of the whole system.

The presence or absence of macho 1 completely changes how a cell interprets the FGF signal.

Okay, so let's walk through it.

Let's take the posterior cells first.

They have macho 1 inside them.

They receive the FGF signal.

What happens?

They become messenkind.

They do not become notochord.

And the reason is, macho 1 has already turned on snail, which is actively repressing the brachiary gene.

The FGF signal arrives, but the door to the notochord fate is already locked.

Okay.

Now, what about the anterior cells?

They don't have macho 1.

In those cells, when the FGF signal arrives, it leads directly to notochord fate.

The brachiary gene is free to be activated.

There's no snail protein there to repress it.

So the exact same external signal, FGF, results in two completely different outcomes depending on the cell's internal inherited history.

That's the integration.

Macho 1 acts as a filter.

It takes this widespread conditional signal and dictates whether that signal means notochord or messenkind.

It's the perfect resolution to that old paradox.

Outro.

And that really brings us to the end of our deep dive.

We've gone from the sea urchin, the master of regulated development, to the tunicate, the icon of mosaic development.

Only to find out that the real genius is how they both use an integrated blend of both strategies.

We saw in the sea urchin that the autonomous specification of the micromeres is just the first step.

They then become this signaling hub that conditionally patterns all their neighbors using these incredibly complex gene regulatory networks.

Featuring that amazing double negative gate with PMAR1 and HESI.

And that molecular plan allows them to then execute these dramatic physical movements like EMT and the convergent extension of the gut.

And then in tunicates, we saw that even though they lean heavily on parceling out maternal determinants like macho 1, they absolutely rely on conditional FGS signaling to build their most important chordate structure, the notochord.

And macho 1 itself is what controls how that conditional signal is even read by the cell.

Which brings us to the final thought we want to leave you with.

A thought that connects these organisms directly to us.

Way back in the 1860s, a biologist named Alexander Kowalski first saw the tunicate larva's notochord and realized its significance.

He saw it as the evolutionary bridge linking the invertebrates to the vertebrates.

And today we have the molecular proof of his observation.

The fact that the brachiary gene, that master regulator of notochord fate, is conserved and does the same job in a humble sea squirt as it does in you.

That's one of the most powerful pieces of evidence we have for our shared ancestry.

It really makes you wonder, doesn't it, what other seemingly simple organisms out there are holding the molecular keys to understanding the very foundations of our own complex development.

That's definitely something to think about.

Thank you for joining us for the Deep Dive.

We'll see you next time.