Vetebrateembryo
5.1 The organiser is an experiment before it is a molecule #
5.1.1 Spemann and Mangold #
In 1924 Hilde Mangold, working with Hans Spemann, grafted the dorsal lip of the blastopore from one newt gastrula into the ventral side of another. The host built a second embryonic axis. Pigment differences between donor and host showed that the notochord of the second axis came mostly from the graft, whereas the neural tube came mostly from host cells that would otherwise have made ventral epidermis. The graft did not merely differentiate on its own. It induced host tissue to change fate, and it organised that tissue into a coherent axis, with dorsal mesoderm, neural tube, and anterior–posterior order. Spemann called the dorsal lip the organiser. The result is still the standard against which any “signalling centre” is judged: it must do more, when grafted, than produce its own fate map fate.
The organiser is not a fixed cell population with one programme. The dorsal lip that involutes early induces head structures. Dorsal lip that involutes later induces trunk and tail. A graft therefore has an age as well as an address. The molecular version of that change is the difference between blocking BMPs, which is enough to neuralise trunk ectoderm, and also blocking Wnt and Nodal, which is what protects and specifies the head. Those molecules come in the next topic. The experiment stands without them, and any molecular story that cannot explain a second axis has gone wrong.
5.1.2 The Nieuwkoop centre #
The organiser does not appear from nowhere. Pieter Nieuwkoop combined pieces of blastula that normally never meet. An animal cap cultured alone becomes epidermis. An animal cap placed on vegetal cells forms mesoderm at the junction, and the mesoderm comes from the animal-cap cells, which were not fated to make it. Dorsal vegetal cells induce dorsal mesoderm, the organiser itself, with notochord character. Ventral vegetal cells induce ventral mesoderm, such as blood and mesenchyme. The dorsal vegetal cells are the Nieuwkoop centre. They induce the organiser in the marginal zone above them. They do not become the organiser. Their own descendants are largely endoderm.
The centre is placed by cortical rotation. Dorsal determinants, including Dishevelled and Wnt11 mRNA, move along vegetal microtubules to the future dorsal side and locally inhibit the β-catenin destruction complex. β-catenin therefore accumulates in dorsal nuclei, on top of a maternal supply, before zygotic transcription of the organiser genes. Disrupting those microtubules ventralises the embryo: no dorsal axis forms. An imposed rotation can restore one. The grey crescent, where it is visible, is the anatomical sign of the same movement, and it is why Spemann’s constriction experiment in Chapter 1 cared which half received the crescent.
Vegetal identity itself depends on VegT, a T-box transcription factor whose mRNA is anchored at the vegetal pole. VegT cell-autonomously promotes endoderm genes, including sox17. It is also required for the mesoderm-inducing signals. Deplete VegT and both endoderm and mesoderm fail. The Nieuwkoop centre is the region where this vegetal programme overlaps high β-catenin. Neither input alone is the centre.
5.2 BMP ventralises, the organiser says no, and Wnt changes its job #
5.2.1 Mesoderm induction and the dorsal β-catenin bias #
The vegetally produced mesoderm inducers are TGF-β-family signals. The clearest endogenous ones in Xenopus are Nodal-related proteins (Xnrs). Vg1 mRNA is also localised vegetally. Processed Vg1 protein can induce mesoderm, and in the chick Vg1 at the posterior margin participates in starting the streak, but the Nodals are the signals to learn as the frog’s inducers. They act through the activin/Nodal receptor branch and phosphorylate Smad2. High Nodal signalling, which is dorsal because β-catenin cooperates with VegT to drive higher Xnr expression on that side, specifies organiser mesoderm and turns on goosecoid, chordin, noggin, and the other organiser genes. Lower Nodal specifies ventral and lateral mesoderm and turns on genes such as brachyury more broadly. Brachyury marks mesoderm; goosecoid marks the organiser. The concentration logic is the same one Chapter 6 will define as a morphogen, and it was shown directly by treating dissociated animal-cap cells with different doses of activin: low doses give ventral mesoderm markers, higher doses give organiser markers, and the responding cells read the dose themselves rather than waiting for a relay.
5.2.2 Antagonists, and the two phases of Wnt #
BMP4, with BMP2 and BMP7, ventralises mesoderm and tells ectoderm to become epidermis. The organiser’s defining molecular act is to secrete BMP antagonists: chordin, noggin, and follistatin. Chordin and noggin bind BMPs and stop them engaging their receptors. Follistatin binds activin very tightly and BMPs as well, and in this context it contributes to the same anti-BMP effect. Where antagonists win, on the dorsal side, ectoderm becomes neural and mesoderm becomes dorsal (notochord, somites). Where BMPs win, ectoderm becomes epidermis and mesoderm becomes ventral. Tolloid, a metalloprotease more active ventrally, cleaves chordin and releases BMP, which sharpens the gradient rather than letting chordin win everywhere.
This is the default model of neural induction, and the wording needs care. “Default” means “in the absence of BMP”, not “in the absence of every signal”. Evidence for the BMP half is strong. An intact animal cap autocrines BMP and becomes epidermis. The same cap dissociated, so that its BMPs are diluted, becomes neural. Adding noggin, chordin, or follistatin neuralises an intact cap; adding BMP4 returns it to epidermis. In the embryo, FGF signals also contribute. MAPK downstream of FGF phosphorylates the linker of Smad1 and thereby inhibits BMP signalling, and FGFs have additional effects on neural genes. A sentence that says only “neural tissue is what ectoderm does when it is left alone” is too crude. Neural tissue is what competent ectoderm does when BMP signalling is low, with FGF as part of how that state is reached.
Wnt signalling has two phases, and collapsing them is the other standard error. Early, maternal dorsal β-catenin, downstream of the cortical Wnt pathway components, is required to create the organiser. Block that early input and the embryo is ventralised: no organiser, no axis. Later, during gastrulation, zygotic Wnt signalling posteriorises and can ventralise. Head specification requires the opposite: antagonists of Wnt, BMP, and Nodal from anterior endomesoderm. Dickkopf and Frzb oppose Wnt; Cerberus opposes Wnt, BMP, and Nodal; Lefty opposes Nodal. BMP inhibition alone gives trunk neural tissue. BMP inhibition plus Wnt inhibition gives head. The early dorsal lip, which first involutes and ends up anterior, is the source that does both. The later lip maintains the trunk. “Wnt dorsalises” is true before gastrulation and false as a description of the anterior–posterior decision during gastrulation.
5.3 Left–right asymmetry #
5.3.1 Flow at the node #
The vertebrate body is bilaterally organised and then systematically broken. Heart, lungs, gut, and liver are not mirror images. The bias is not a new axis invented independently in each organ. It is initiated at a ciliated structure in the midline during gastrulation and somitogenesis, and then broadcast to the lateral plate.
In the mouse, cells of the node carry motile cilia that are tilted posteriorly. Their rotation drives fluid across the node toward the left. This nodal flow is the symmetry-breaking event. The equivalent ciliated structure is Kupffer’s vesicle in zebrafish and the gastrocoel roof plate in Xenopus. How the flow is read is still taught as two live models: immotile sensory cilia at the edge of the node, or transport of vesicles and signalling cargo with the current. Both can be true in part. What is not in doubt is the direction, leftward, and the molecular asymmetry that follows. Motile cilia depend on dynein arms and on the microtubule cytoskeleton in Cell Biology. People with primary ciliary dyskinesia, Kartagener syndrome when situs is reversed, have immotile cilia. Left–right laterality is randomised rather than reversed by a mirror programme, so about half have situs inversus and others have situs solitus or a mixed heterotaxy. Respiratory cilia and sperm flagella fail in the same syndrome, which is a clinical hint that one motor protein is shared.
The chick reaches the same left-sided cassette by an upstream route that is not identical to mouse nodal flow. These notes do not pretend the initiating geometry is universal. They do require the conserved readout.
5.3.2 Nodal, Lefty, and Pitx2 #
Downstream of flow, an inhibitor of Nodal at the node, Cerl2 (Dand5), becomes weaker on the left. Nodal signalling is therefore freed on the left side of the node first. Nodal then induces its own expression in the left lateral plate mesoderm, together with Lefty2 and the transcription factor Pitx2. Lefty proteins are TGF-β-family antagonists that diffuse further than Nodal and block it. Lefty2 limits how far the left-sided domain spreads. Lefty1 at the midline is a barrier that stops the signal crossing to the right. The pair is a reaction–diffusion cassette: an activator that turns itself on, and an inhibitor that travels further and turns it off. The right lateral plate does not express Nodal or Pitx2.
Pitx2 remains on the left after Nodal and Lefty have gone, and it is the effector that asymmetric morphogenesis reads. It directs the direction of heart looping, the unequal number of lung lobes, and the rotation of the gut. Losing the cassette randomises those organs. It does not create a third lung or a new axis. Left–right patterning chooses between mirror forms of a body that dorsal–ventral and anterior–posterior patterning have already outlined. Pitx1, a related gene, is not this signal. It will appear in Chapter 10 in the pelvis, and the names are neighbours only.
5.4 Somitogenesis #
5.4.1 Clock and wavefront #
Somites are paired blocks of paraxial mesoderm, added in anterior-to-posterior order from a presomitic mesenchyme. A mouse makes about 65 pairs; many snakes make several times that, and the number is species-specific. Cooke and Zeeman proposed in 1976 that a cellular clock, together with a wavefront of determination moving up the axis, could turn a continuous rod of mesoderm into a periodic row. The molecular clock was found when c-hairy1 expression in the chick presomitic mesoderm was seen to oscillate, with a period matching the time needed to make one somite.
The period is about 90 minutes in the chick, about 2 hours in the mouse, and about 30 minutes in the zebrafish, and it changes with temperature. The oscillator is a negative-feedback loop in the Notch pathway. Hes/Her genes repress their own transcription; the delays of transcription and translation set the period. Lunatic fringe, a glycosyltransferase that modulates Notch, oscillates with them. Notch signalling between neighbours keeps their clocks in phase. If Delta or Notch is lost, individual cells may still tick, but they tick out of synchrony, and somite boundaries become irregular or fail. Human spondylocostal dysostosis, with mutations in genes of this cassette including DLL3, LFNG, HES7, and MESP2, is the clinical version of a broken clock: the vertebral column is segmented badly.
The wavefront is a front of maturation moving posteriorly relative to the tissue, set by opposing gradients. FGF8 and Wnt are high in the tail bud. Fgf8 mRNA is transcribed there and decays once cells are left behind, so anterior presomitic cells, which left the tail bud earlier, have less FGF. Retinoic acid, synthesised by Raldh2 in the formed somites, opposes FGF and is degraded posteriorly by Cyp26. Where FGF and Wnt fall below a threshold, cells become competent to make a boundary. The next tick of the clock in that competent zone stabilises a stripe of Mesp2, and that stripe starts the boundary. Ephrin and Eph interactions between the future caudal half of one somite and the rostral half of the next actually cut the boundary in. Speed the clock without speeding the wavefront, and the somites come out smaller and more numerous. Slow the clock, and they come out larger and fewer. Species differences in vertebral number are, in substantial part, differences in how many cycles fit into the time the axis is elongating.
5.4.2 What a somite becomes #
A new somite is an epithelial ball. Its subsequent division of labour is induction again, on a smaller scale. Sonic hedgehog from the notochord and the floor plate ventralises the somite: high signalling drives sclerotome, marked by Pax1, which will make vertebrae and ribs. The dorsal somite remains epithelial as the dermomyotome, maintained by Wnt from the dorsal neural tube and the surface ectoderm, and produces myotome (skeletal muscle) and dermatome (dermis of the back). Muscle progenitors that will enter the limb express Pax3 and migrate out of the dermomyotome. They do not make the limb skeleton. That skeleton is lateral plate mesoderm, colonised by these muscle cells. Chapter 7 depends on that distinction.
Rostrocaudal identity inside each somite, fixed at the Mesp2 step, is what neural crest and motor axons later read. They pass through the anterior sclerotome only. A clock defect is therefore also a peripheral-nerve defect, not only a bony one.
Hox genes, expressed in nested domains along the axis, give groups of somites their regional identity: occipital, cervical, thoracic, lumbar, sacral. The anterior boundary of expression of a given Hox paralogue group lines up with a transition in vertebral type. Loss of all Hox10 paralogues, for example, transforms lumbar and sacral vertebrae toward a thoracic, rib-bearing identity. The segments were made by the clock. Their identities were written by Hox codes on top of that periodicity, which is the same division of labour as pair-rule genes versus Hox genes in the fly. The molecules are homologous. The visible segments, somites and insect segments, are not the same structure, a distinction Chapter 10 insists on.
5.5 Key terms, summary, and self-check #
Key terms. Organiser; Nieuwkoop centre; cortical rotation; VegT; Nodal-related signal; β-catenin; chordin; noggin; follistatin; default model; anterior visceral endoderm; nodal flow; Pitx2; Lefty; clock and wavefront; Mesp2; sclerotome; dermomyotome.
Summary. The dorsal lip induces and organises a second axis; the Nieuwkoop centre, where vegetal VegT overlaps dorsal β-catenin, induces the lip and is not the lip. High Nodal specifies dorsal mesoderm, BMPs specify ventral fates and epidermis, and chordin, noggin, and follistatin protect the dorsal side. Early Wnt signalling dorsalises; later Wnt signalling posteriorises, so the head needs Wnt antagonists as well as BMP antagonists. Leftward flow at the mouse node frees a Nodal–Pitx2 cassette on the left. Somites are cut from presomitic mesoderm where an oscillating Notch clock meets a retreating FGF/Wnt wavefront, and Hox genes then give those somites regional identity.
Self-check.
1. In the Spemann–Mangold graft, which tissue of the second axis is mostly host-derived, and why does that prove induction rather than self-differentiation?
2. What does the Nieuwkoop centre do, and which two maternal inputs overlap there?
3. Why does an intact animal cap become epidermis, while a dissociated one can become neural? What extra inhibition turns trunk neural tissue into head?
4. Why do immotile node cilia randomise situs instead of reversing every affected person?
5. In the clock-and-wavefront model, what happens to somite size if the clock period shortens and the wavefront retreats at the same speed?
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