Embryology of the Nervous System: Neurulation, the Neural Crest, and How Position Builds a Brain

01Closing the neural tube

The human neural tube closes from two separate sites and seals by day 28

Neurulation folds the neural plate into the neural tube, and in the human embryo the sequence is dated to the day. The neural groove and folds first appear at Carnegie stage 8, about 18 postovulatory days O'Rahilly 1994. Fusion of the folds begins at stage 10. Every date on that scale is read off staged human specimens, which matters because the rodent series usually borrowed for the human case closes on a different plan.

Human closure runs from two starting points. Two de novo sites of fusion appear in succession, alpha in the rhombencephalic region and beta in the prosencephalic region beside the chiasmatic plate O'Rahilly 2002. Fusion from alpha proceeds in both directions, rostrad and caudad, while fusion from beta proceeds caudad only. That geometry comes from 98 staged human embryos, 61 of them checked against precise graphic reconstructions.

Two neuropores are left open by that arrangement. The rostral neuropore closes within a few hours at stage 11, about day 24, and the caudal neuropore takes a day to close at stage 12, about day 26 O'Rahilly 1994. Final caudal closure sits at future somitic pair 31, the level of the second sacral vertebra, which is the same level from which the sacral parasympathetic outflow will later leave the cord. At stage 13, four weeks, the tube is closed along its whole length.

This hollow cylinder is the founding structure of the central nervous system, the single source from which the spinal cord, brainstem, and forebrain are all carved. A two dimensional sheet becomes a three dimensional tube. In that closure the embryo commits an entire lineage of cells to neural fate, and every nucleus and tract downstream inherits its address from the first act of invagination.

Folic acid works only inside the closure window

Neural tube defects were estimated at 260,100 affected birth outcomes worldwide in 2015, a prevalence of 18.6 per 10,000 live births, with about half of cases ending in elective termination or stillbirth Blencowe 2018. Folic acid is required while the tube is forming, which is finished by about 28 days after conception, before most women know they are pregnant. For a first pregnancy the periconceptional recommendation is 400 to 800 micrograms daily. Mandatory fortification reached 68 countries by 2022 and prevented 63,520 cases of folic-acid-preventable spina bifida and anencephaly, 23.7 percent of what fortification could prevent worldwide Wagh 2024.

Closure is modifiable by input, and the evidence is a randomized trial. The Medical Research Council Vitamin Study randomized 1,817 women who had already had an affected pregnancy MRC Vitamin Study 1991. Recruitment ran across 33 centers in seven countries. Four arms tested 4 mg of folic acid daily, a mixture of seven other vitamins, both together, and neither. Among 1,195 completed pregnancies there were 27 neural tube defects, 6 in the folic acid groups and 21 in the other two, a 72 percent protective effect with a relative risk of 0.28. The other seven vitamins produced no significant effect, at a relative risk of 0.80.

The two arms of that trial show what the Unified Model of Tone means by correspondence. Folate matches one process, the closure of a tube, inside one window of about ten days. Supplied after day 28 it changes nothing, because the structure it corresponds to is already sealed. Seven other vitamins supplied inside the window changed nothing either. What decided the outcome was the fit between the input and the process running at that moment, and no quantity of a different vitamin substituted for it.

02Findings

What the research shows

03Position sets neural tube fate

Cell fate in the neural tube is read off a concentration and a position

Two morphogens run in opposing gradients through the neural tube. Sonic Hedgehog is released from the notochord and floor plate, and Bone Morphogenetic Protein is produced in the roof plate. A cell's identity falls out of where it sits on those two gradients. The dorsoventral axis of the spinal cord is divided into eleven molecularly discrete progenitor domains, with the five ventral domains contributing to motor circuits and the six dorsal domains to sensory circuits Zannino 2015. A cell reads its position off the gradient rather than off any label it carries of its own.

One signal read at two concentrations produces two different cells. In neural plate explants, the threshold concentration of the amino-terminal cleavage product of Sonic Hedgehog needed to induce motor neurons is about five-fold lower than the threshold needed to induce floor plate Roelink 1995. Higher concentrations induce floor plate cells at the expense of motor neuron differentiation. More of the same signal builds a different structure.

The same notochord in a different position builds a different cord

Moving the inducing tissue changes the nervous system it induces. A notochord grafted 20 to 30 degrees from the sagittal plane in the chick embryo induces ventral genes and ventral structures, and the roof plate still differentiates normally Monsoro-Burq 1995. A mediodorsal graft placed on top of the roof plate induces no floor plate genes and no motoneuron differentiation at all. The grafted tissue is identical in both cases.

The Unified Model of Tone takes those two grafts as correspondence in its earliest measurable form. The angle between notochord and neural tissue carries the instruction for a floor plate. A five-fold rise in Sonic Hedgehog concentration yields a different cell rather than a larger crop of the same one, which is why a dose in this tissue reads as an address.

Induction here is positional, a dialogue between notochord and neural tissue rather than a fixed genetic script. That single fact is what makes the rest of development legible. A gradient can be read wrongly, and a signal delivered to the wrong address does nothing.

04Alar and basal plates

The dorsoventral split of the neural tube is graded rather than binary

The neural tube partitions into an alar plate dorsally and a basal plate ventrally. Neurons developing from the alar plate are predominantly sensory and receive the dorsal nerve roots arriving from the spinal ganglia. Neurons of the basal plate are predominantly motor and give rise to the ventral nerve roots. At the appropriate spinal levels those ventral roots also carry axons from developing autonomic neurons, so the basal plate becomes the embryonic seat of both somatic motor and visceral motor output. The sulcus limitans marks the boundary between the two.

That two-part picture is a simplification of a graded map. Up to eight alar and five basal longitudinal microzones are resolved in the hindbrain as molecularly and histogenetically distinct progenitor domains, which makes the classic four-column model of the hindbrain excessively simplistic Puelles 2019. The eleven dorsoventral domains of the spinal cord and the thirteen longitudinal microzones of the hindbrain are the same graded axis counted in two regions. Branchiomotor neurons are born beside the floor plate and then migrate dorsally into the alar mantle, so a motor cell can finish development living in sensory territory.

The split also has a limit along the axis. The sulcus limitans terminates in the supramamillary recess, so the alar and basal division is inappropriate for the human forebrain Müller 1997. Above that point the tube is organized by transverse segments instead of by a dorsoventral column pair, which is why forebrain anatomy is named in prosomeres rather than in plates.

This dorsoventral logic is the Bell and Magendie law written into tissue before there is a body to test it on. The separation of afferent and efferent exists as a developmental fact, a structural commitment made long before the first signal travels a root. The intermediolateral cell column that carries every sympathetic command out of the cord is basal plate territory, mapped in detail on the autonomic nervous system page.

Thirteen microzones in the hindbrain hold more organization than four columns can. On a graded axis, a state held at one level shows up in sensation and in visceral output together, because the domains reading that signal are neighbors on one gradient.

05Crest cells leave the fold

Neural crest cells carry the fold's address out into the periphery

Neural crest cells separate from the apex of each neural fold as the tube closes. They originate in the dorsal region of the developing neural tube, undergo an epithelial to mesenchymal transition, and migrate widely through the embryo Griswold 2012. Crest leaves in streams sorted by level. Cranial crest builds the facial skeleton and vagal crest the neurons of the gut. Trunk crest builds the dorsal root and sympathetic ganglia, and sacral crest the ganglia of the hindgut. One embryonic origin populates structures as distant as the gut wall and the face.

Quail-to-chick grafting puts crest fate in the environment. Early-migrating and late-migrating cranial crest populations have equivalent developmental potential in vivo, and the dorsal fate restriction of the late population comes from the earlier cells already occupying the route Baker 1997. Transplanted cells build whatever their new position builds. A crest cell's identity therefore turns on three things: its level of origin along the rostrocaudal axis, the signals it meets in transit, and the microenvironment it stops in Griswold 2012.

The nerve carries the cells that build the gland

The adrenal medulla reaches its place by an unexpected route. Chromaffin cells and sympathetic neurons do not share an immediate common ancestor in the form of a sympathoadrenal cell, the arrangement assumed for decades. In the mouse, chromaffin cells arise instead from Schwann cell precursors traveling along the preganglionic nerve Chan 2018. Autonomic wiring and endocrine output share a physical lineage, and the nerve that will later drive the gland delivered the cells that became it.

Sacral crest cells arrive four days too late to rescue the hindgut

Timing belongs to the address as much as position does. Crest adjacent to somites 3 to 6 supplies the enteric ganglia along the whole gut in the chick, and producing an aneural hindgut requires ablating that population before the 13-somite stage Burns 2000. Sacral crest cells do reach the aganglionic hindgut. They arrive in significant numbers only four days after the vagal-derived cells finish migrating, and the ganglia they form are small and infrequent.

The same dorsal cells that will read the world also build the ganglia that govern the viscera, so sensory input and autonomic output leave the fold in one migration. In this model, timing is part of the address, and a population that arrives four days late does not build a thinner version of the same ganglia. An input that arrives outside its window fails to organize the tissue.

06Segments and organizers

The developing brain is built as addressed compartments defined by their boundaries

The developing human brain is divided into numbered compartments before it has any structure worth the name. Six primary neuromeres are detectable at stage 9, and a maximum of sixteen secondary neuromeres at stage 14, counted across 215 staged human embryos with 85 graphic reconstructions Müller 1997. Late in week four the rostral tube flexes at the mesencephalon while the prosencephalon and rhombencephalon take shape around it, the lateral walls enlarging into the brain vesicles and the internal cavity becoming the ventricles.

The hindbrain carries eight of those segments, the rhombomeres r1 through r8, and the forebrain carries prosomeres. Each rhombomere is tabulated against its neural crest stream, its pharyngeal arch, and its cranial nerve exit Müller 1997. The trigeminal and facial roots emerge in register with that segmental order.

Rhombomere boundaries are physical partitions in cell lineage. Clonal analysis by intracellular marking in the chick hindbrain shows that a clone marked before boundary appearance spreads into the neighboring rhombomere Fraser 1990. A clone marked after boundary appearance expands freely inside its own rhombomere and stops at the border. The compartment becomes an address the cell cannot leave.

The isthmic organizer appears only where midbrain meets rhombomere 1

Fgf8-expressing tissue with the properties of the isthmic organizer appears when midbrain and rhombomere 1 are juxtaposed in the chick, and it does not appear when midbrain contacts any other rhombomere Irving 1999. The induction follows a direct planar interaction between the two tissues and involves a diffusible signal. The organizer exists at the interface where those two particular tissues meet.

The zona limitans patterns the diencephalon

The zona limitans intrathalamica is the Hedgehog source that patterns the diencephalon. Enhanced Hedgehog signaling in the zebrafish enlarges the prethalamic and thalamic gene expression domains, and loss of Hedgehog signaling abolishes them Scholpp 2006. Hedgehog expression in the zona limitans alone is sufficient for diencephalic differentiation in that animal, even when the basal plate is genetically ablated.

That organizer also carries a brake. Pax6 acts cell-autonomously to repress Shh in the cells around the zona limitans, limiting its size and its influence Caballero 2014. Losing the brake produces widespread diencephalic patterning defects in mice, and blocking Shh signaling in Pax6 null mutants reverses the major ones.

An organizer that never fires leaves the thalamus unbuilt, and one that spreads past its boundary breaks the same map. What the tissue defends is a width.

07Windows and arriving traffic

Every step in this sequence has a window, and an input outside it does not build a smaller structure

Nothing in embryology is a schedule of accumulating growth. Each structure has a window, and inside that window one particular signal organizes it. The rostral neuropore closes within a few hours at about day 24. Vagal crest bound for the gut has to be on its way before the 13-somite stage. What the embryo hands the newborn is a system whose organization is still being written by what arrives.

Painful handling lowers the preterm cortex's response to touch

Fifteen million infants are born preterm every year, and the degree of prematurity at birth determines how far cortical responses to light touch are attenuated by hospital discharge Maitre 2017. Inside that population the kind of handling separates the responses further. Supportive experience such as breastfeeding and skin-to-skin care goes with stronger cortical responses. Painful experience such as skin punctures and tube insertions goes with reduced responses to the same touch stimulus. Both associations hold after controlling for prematurity and analgesic exposure. What is read there is the cortical evoked response, and the autonomic numbers that the same handling moves are reported on the pediatrics research page.

The tube lays the map down and afferent traffic writes the circuits onto it. That traffic does not stop at birth, and the largest continuous share of it comes from the spine and its joints. What the cortex then does with the traffic, layer by layer, is set out on cortical organization.

The commitment made here is testable at the earliest step development offers. Folate closes a tube on day 20 and not on day 40. A notochord induces a floor plate only where the responding tissue is competent to answer it. Correspondence between an input and the process running at that moment is doing the work, and that dependence on timing and place confirms this reading of development. The general form of that commitment is stated on the tone page.

An input at the wrong strength inside the window builds the wrong cell rather than a weaker one. An input that arrives after the window does not build a smaller version of the structure. Sonic Hedgehog at a fifth of the floor plate concentration makes motor neurons, and sacral crest arriving four days late leaves a hindgut whose ganglia are small and infrequent. The embryo states that rule more plainly than any adult tissue can, because its structures are still being laid down while the inputs arrive.

08Tone

How this system expresses tone

Tone in the embryo is measured in days, in degrees of arc off the midline, and in concentrations of a single signal. Nothing here is holding a state yet; the tissue is deciding what state it will be able to hold.

Set point: fate in the neural tube is decided at concentration thresholds, with motor neurons induced at a fifth of the Sonic Hedgehog level that makes floor plate. Prediction: the tube commits sensory territory above the sulcus limitans and motor territory below it before a single root carries traffic, an arrangement built for inputs that do not yet exist. Coupling: the same crest that builds the dorsal root ganglia builds the autonomic ganglia, so sensing and visceral control leave the fold in one migration. Gain: raising Hedgehog signaling in the zebrafish enlarges the prethalamic and thalamic domains and removing it abolishes them, so the size of a region tracks the strength of one source. Oscillation: the embryo counts time in somites added on a fixed cycle, which is why the window for building the chick's enteric ganglia closes at the 13-somite stage rather than at an hour. Load: tube closure runs on folate supply, and the supply has to be in place before the pregnancy is known.

09Across the library

How this page relates to the rest of the library

10Frequently asked

Questions about this topic

11The sources

References

Sources: primary literature, linked inline.