Ancient evolutionary origin of vertebrate enteric neurons from trunk-derived neural crest

Ancient evolutionary origin of vertebrate enteric neurons from

trunk-derived neural crest

Stephen A. Green

1,*

Benjamin R. Uy

1,*

, and

Marianne E. Bronner

1,§

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA

91125, USA

Abstract

The enteric nervous system (ENS) of jawed vertebrates arises primarily from vagal neural crest

cells that migrate to the foregut and subsequently colonize and innervate the entire gastrointestinal

tract. To gain insight into its evolutionary origin, we examined ENS development in the basal

jawless vertebrate, the sea lamprey. Surprisingly, we found no evidence for the existence of a

vagally-derived enteric neural crest population in the lamprey. Rather, DiI labeling showed that

late-migrating cells, originating from the trunk neural tube and associated with nerve fibers,

differentiated into neurons within the gut wall and typhlosole. We propose that these trunk-derived

neural crest cells are homologous to Schwann cell precursors (SCPs), recently shown in

mammalian embryos to populate post-embryonic parasympathetic ganglia

, including enteric

ganglia

. Our results suggest that neural crest-derived SCPs made an important contribution to the

ancient ENS of early jawless vertebrates, a role that was largely subsumed by vagal neural crest

cells in early gnathostomes.

The enteric nervous system (ENS) is comprised of thousands of interconnected ganglia

embedded within the wall of the gut

, making it the most complex portion of the peripheral

nervous system in amniotes. The ENS of jawed vertebrates innervates the entire

gastrointestinal tract to regulate muscle contraction, chemosensation, water balance, and gut

secretion

. Classical transplantation experiments have demonstrated that the neurons and

glia of the gut are largely derived from the “vagal” population of neural crest cells that arise

within the post-otic portion of the hindbrain

. They subsequently migrate to the foregut and

embark upon the longest migration of any embryonic cell type, from foregut to hindgut.

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Address for correspondence: mbronner@caltech.edu.

These authors contributed equally to the work.

Competing Interests Statement

: The authors declare no competing interests.

Author Contributions

Project was conceived by MB, and analyses were designed by SG and MB. Descriptive analyses of enteric neurons were performed by

SG. Cranial DiI labeling was performed by BU, MB, and SG. Trunk DiI labeling was performed by BU, SG, and MB. Surgeries were

performed, imaged, and analyzed by SG and BU. Schematics were drawn by SG. Manuscript was written by MB, SG, and BU.

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable

request.

HHS Public Access

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. Author manuscript; available in PMC 2017 September 20.

Published in final edited form as:

Nature

. 2017 April 06; 544(7648): 88–91. doi:10.1038/nature21679.

Author Manuscript

The sea lamprey,

Petromyzon marinus,

is a jawless (agnathan) vertebrate and an

experimentally tractable representative of the cyclostomes. As the sister group to jawed

vertebrates (gnathostomes), lamprey are an important model for identifying traits common

throughout vertebrates. Lamprey possess migrating neural crest cells that give rise to many

cell types, including melanocytes, cartilage, sensory neurons and glia, but lack other neural

crest-derived structures that are present in gnathostomes like jaws and sympathetic chain

ganglia

. Given that lamprey embryos lack sympathetic ganglia, we sought to examine

other components of the autonomic nervous system, with a focus on the ENS. Adult lamprey

have a simple ENS that includes ganglionated plexuses of serotonin (5-HT) producing cells,

as well as a smaller number of catecholamine-containing neurons

. However, the

developmental origin of these enteric neurons is unknown.

As a first step in analysis of lamprey ENS development, we examined the time course of

neuronal appearance along the developing embryonic gut by performing

in situ

hybridization

for

Phox2b

, which is expressed in enteric neurons in many jawed vertebrates

. Expression of

lamprey

Phox2b

is detectable along the gut from early stages (Tahara Stage 25 [T25]

), and

by stage T28 or embryonic day (E) 20,

Phox2b

expressing cells are associated with a

depression in the gut that will become the typhlosole, an hematopoietic tissue associated

with elements of the ENS (Fig. 1a–b). Using a 5-HT antibody, serotonin-immunoreactive

neurons were first observed within the gut wall at ~T28.5 (Fig. 1c–d) in the anterior trunk

region. With time, the numbers increased, and 5-HT

neurons were noted progressively

posteriorly, with particularly high cell numbers in the cloacal region, as reported

previously

. By T30 (E30), there are >100 neurons along the gut in association with the

typhlosole and vagus nerve (Fig. 1e; Extended Data Fig. 1). Interestingly, individual

serotonergic neuroblasts also were often associated with axon bundles emanating ventrally

from the dorsal root ganglia (Fig. 2f; Extended Data Fig. 1a). In addition to 5-HT

neurons,

we also noted 5-HT

columnar cells that may represent gut enterochromaffin cells

(Extended Data Fig. 1a–b).

We next sought to determine the embryological origin of the neurons in the gut by

performing lineage labeling with the lipophilic dye DiI

. In chicken, vagal neural crest cells

that contribute to the ENS arise from the hindbrain neural tube adjacent to somites 1–7

After exiting the neural tube, they migrate ventrally, invade the foregut, and undergo a

collective cell migration along the rostrocaudal extent of the gut. To test whether lamprey

possess a homologous cell population, we performed focal injections of DiI into the dorsal

portion of the caudal hindbrain (corresponding to the site of origin of vagal neural crest in

gnathostomes) of T20 (E6) embryos. Regardless of the exact injection site, dye labeled cells

spread within the hindbrain and emigrated from the neural tube as a stream directly above

the pharynx (Extended Data Fig. 2). From this site, they progressed ventrally, then turned

caudally to populate all of the branchial arches (Extended Data Fig. 2), similar to previously

reported migration patterns of cranial neural crest cells

. However, despite many focal

injections (n=46) into the caudal hindbrain homologous to “vagal”, we failed to find

evidence of DiI-labeled cells in the gut. We next looked for expression of the vagal neural

crest marker,

ret,

in lamprey, since it is required for vagal neural crest development in

gnathostomes

–

. To this end, we cloned a lamprey

ret

homolog and the ret co-receptor

GFR

and examined their expression patterns by

in situ

hybridization. The results show

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lamprey

ret

and

GFR

are expressed in many parts of the embryo, including the

typhlosole.

Ret

and

GFR

expression are first observed in a region anterior to the gut at

T26 (Extended Data Fig. 2i–j) in a manner reminiscent of

Phox2b

expression (Extended

Data Fig. 2k). However, we did not observe expression in neural crest cells migrating from

caudal hindbrain, raising the intriguing possibility that enteric neurons might arise from a

different cellular source.

In gnathostomes, migrating trunk neural crest cells fail to invade the immediately underlying

gut due to the presence of repulsive signals like Slit that block their entry

. Recently,

however, a secondary contribution to the mammalian ENS has been uncovered that comes

not from vagal neural crest but rather from trunk neural crest-derived Schwann cell

precursors (SCPs) associated with extrinsic nerves that contribute post-natally to

calretinin

containing neurons of the gut

. Using Dhh-cre mice to lineage trace immature Schwann

cells, Uesaka and colleagues found that ~15% of enteric neurons in mice were derived from

trunk SCPs. Intriguingly, this population persists in

ret

null mice

. To examine the

possibility that a homologous population may exist in lamprey, we asked whether cells

emerging from the trunk neural tube might contribute to neurons of the gut. To test this, we

performed focal DiI injections into the dorsal trunk neural tube as well as neural tube lumen

injections following its cavitation at T22-T23 (~E8), thus labeling the entire neural tube as

well as presumptive neural crest cells prior to their emigration. Both labeling techniques

yielded similar results. By T25 (E14), such injections label neural crest-derived cells in

dorsal root ganglia and mesenchyme cells of the fin

. Interestingly, at later stages, we

observed DiI-labeled cell bodies closely associated with nerve processes, recognized by

acetylated tubulin staining above the gut (n=47 embryos) (Fig. 2a–c). These individual DiI-

labeled cells were visible as early as T28 (E18–20) and persisted until the latest stages

examined, T30 (E30-E35), by which time the yolk is reduced, facilitating imaging. In the

trunk of lamprey, there are two nerve roots per trunk segment—a dorsal root that contains

fibers emanating from neural crest-derived dorsal root ganglia and a ventral root that

contains motor neuron nerve fibers. Interestingly, DiI labeled cells were associated with

these dorsal roots (Fig. 2c). Similarly, serotonergic neuroblasts were selectively associated

with and appeared to migrate only along these dorsal root bundles (Fig. 2d), consistent with

a neural crest origin.

To establish whether the DiI-labeled cells differentiated into enteric neurons, embryos were

sectioned and stained with antibodies to serotonin and acetylated tubulin as a mature

neuronal marker. The results, based on examination of transverse sections through 25

representative embryos at T30 (E30–35) in which we quantitated cells that were both

acetylated tubulin and serotonin-positive, are summarized in Table 1. We noted numerous

DiI-labeled serotonergic neurons (DiI/5HT/acetylated tubulin-positive cells) in the anterior

gut (Fig. 2d), esophagus (Fig. 2e), typhlosole, and adjacent tissues, as well as other DiI

neurons that were serotonin negative (Extended Data Fig. 1c). These results demonstrate that

neural crest cells originating from the trunk neural tube can contribute to enteric neuron

populations within the gut wall.

To determine if markers associated with neural crest-derived SCPs were present in the

lamprey trunk, we stained embryos with an antibody to GFAP, which labels glial and

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Schwann cells in amniotes. We observed prominent GFAP+ cells in lamprey embryos

including some that were DiI positive along the lateral line (Fig. 2g), consistent with the

possibility that neural crest cells give rise to the nonmyelinating Schwann cells of

lampreys

Finally, as an additional means of testing whether enteric neurons originate from within the

trunk neural tube, we performed neural tube ablation (n=17), a method classically used to

demonstrate the neural crest origin of enteric neurons

, at approximately T24. To

accommodate slight natural variation in the number of ENS neurons between embryos, we

expressed counts as the ratio of serotonergic cells in the zone adjacent to the ablation versus

an identically sized region spaced approximately one somite length to the anterior. In

comparison to control nonablated (n=6) or sham-ablated (n=4) embryos (Fig. 3a,c),

experimental embryos show a significant decrease in the ratio of ENS serotonergic cells

(Fig. 3b,d). We noted an ~12% decrease of serotonergic neurons in the ablated region

compared with sham-ablated embryos and an ~25% decrease relative to stage controls,

consistent with a model of ENS neuronal precursors migrating from trunk NC-derived cells.

This decrease was particularly surprising given the remarkable regenerative capacity of the

lamprey spinal cord

Recent evidence suggests that many neural crest derivatives in post-natal mammals,

including skin and peripheral ganglia, arise from SCPs that are closely associated with

extrinsic innervation to these structures. For example, SCPs along nerve processes

differentiate into melanocytes of the skin and parasympathetic ganglia

. Moreover, SCPs

that migrate along trunk spinal nerves contribute to a subpopulation of enteric neurons in

mice

. These studies prove the existence of neural crest-derived cells that contribute to the

peripheral nervous system and other derivatives at post-embryonic stages. Our results

suggest that these trunk neural crest-derived cell types may represent an ancient and

evolutionary conserved source of cells that contribute to the ENS. Moreover, our data

suggest that agnathans might lack a classical “vagal” neural crest, leading us to speculate

that a vagal neural crest population with ability to form enteric neurons arose in stem

gnathostomes (Fig. 3e). We cannot rule out contributions from other sources, but focus here

on the positive contribution of trunk neural crest-derived cells to enteric neurons. Although

we cannot formally exclude the possibility that the mechanisms for populating the ENS

arose independently in agnathans and gnathostomes, we favor the idea that the contribution

of SCPs to the ENS might represent a primitive (plesiomorphic) state retained from early

vertebrates, and perhaps common to all living vertebrates. With emergence of jawed

vertebrates, new traits, including jaws, sympathetic ganglia, and vagal neural crest-derived

enteric ganglia, appeared under the umbrella of embryonic neural crest derivatives.

Methods

Adult sea lamprey (

P. marinus)

were supplied by the U.S. Fish and Wildlife Service and

Dept. of the Interior, and cultured according to previous protocols

in compliance with

California Institute of Technology IACUC protocol #1436. Embryo batches with less than

70% survival were excluded from analyses, and all experimental embryos were randomly

chosen from appropriately staged cultures. Celltracker CM-DiI (Thermo Scientific) was

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resuspended as described

, and surgical ablations were performed using forceps and

tungsten needles. For surgeries, only embryos with ablations of 2–6 somite lengths, adjacent

to anterior intestine, and without damage to other tissues were included in analyses.

Serotonergic neurons associated with the typhlosole were counted in a 200–300 um region

adjacent to the ablation site, and an equivalently sized nonablated region beginning

approximately one myotome length anterior to the ablation. Measurements were taken in

sibling non-ablated embryos and sham-ablated embryos in which ectoderm and neural tissue

was cut, but not removed, at similar axial locations. Investigators were not blinded to group

allocations. Probes for

Phox2

GFR

, and

ret

were cloned from cDNA with primers for

Ensembl gene models ENSPMAG00000008433, ENSPMAT00000009324, and

ENSPMAT00000008763. In situ hybridizations and antibody stainings were performed

using previously described protocols

. Anti-acetylated tubulin (Sigma clone 6-11b-1,

#T7541; Mouse IgG2b) and anti-5-HT (Immunostar #20080, Rabbit IgG) were used at

1:500. Anti-GFAP (Dako, #Z0334; Rabbit IgG) was used at 1:400. Embryos were processed

for cryosectioning according to standard protocols, and were sectioned on a

Microm

HM550

cryostat. Microscopy was performed on a Zeiss AxioImager.M2 equipped with an Apotome.

2. Images were cropped, rotated, and resized using Adobe Photoshop CC and image panels

were constructed using Adobe Illustrator CC. Statistics were performed in Microsoft Excel.

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Extended Data

Extended Data Figure 1. 5HT immunoreactive cells in the gut, and DiI labelling

a–b

, 5-HT immunoreactivity in embryonic day (E) 25 (

) and E30 (

) embryos.

Serotonergic neurons (yellow arrowheads) are positioned within the typhlosole, near the

endodermal mucosa. A cell present in the columnar epithelium (white arrowhead) might

represent an endocrine (enterochromaffin) cell. Cells positioned dorsal to the typhlosole

might be neuroblasts (white arrow).

, DiI labeling results in labeled cells (yellow

arrowhead) originating from the neural tube, migrating to the gut and typhlosole. Red:DiI;

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Cyan:5HT; Blue:DAPI; Green:Acet. Tubulin.

, Serotonergic (Green: 5-HT; Red: Acet.

Tubulin) neurons in a T30 embryo, and

, Acetylated Tubulin staining alone shows the

position of the vagus nerve.

Extended Data Figure 2. DiI labeling of the caudal hindbrain population shows contributions to

the branchial arches

a–c

e–f

, Sample time lapse imaging of two separate DiI-labeled embryos around the

hindbrain level.

, Initial injection at E6-E6.5 (T20).

, Final DiI localization of embryo

in A, 10 days post injection (E16).

, Frontal cryosection through the branchial basket shows

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DiI along the branchial arches. Red: DiI, Green: Neurofilament-M, Cyan: Collagen type II.

, Final DiI localization of embryo in A 14 days post-injection (E20).

, Transverse section

through the lamprey branchial basket shows DiI within the DRG. Red: DiI; Green:

Neurofilament-M.

g–h

, Schematic depiction of individual injection sites for cranial (g) and

trunk (

) injections.

i–m

, Genes associated with gnathostome enteric neurons,

Ret

(

i, l

GFRalpha1

(

j, m

), and

Phox2b

(

) do not appear to be coexpressed at T26 (

i–k

) and T27 (

l–

), prior to enteric neuron differentiation in lamprey. DRG:dorsal root ganglia; Nt: neural

tube.

Acknowledgments

We thank Clare Baker, Michael Piacentino, and Laura Kerosuo for discussion as well as Megan Martik and Marcos

Simoes-Costa for their comments on this manuscript.

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Figure 1. Early formation of enteric neurons in the lamprey

P. marinus

Phox2b

expression in dorsolateral cells of a T28 embryo.

, Transverse cross-section of a

T28 embryo shows

Phox2b

expression in the depression of the typhlosole (black

arrowhead).

, 5-HT and acetylated tubulin immunoreactivity in a slightly older T28.5

embryo.

, 5-HT is detectable in neurons (white arrowheads) adjacent to the vagus nerve

(white arrow).

, Serotonergic neurons form small ganglia within the enteric plexus of a T30

embryo. NT: neural tube; Not: notochord. Scale bar indicates 50 um.

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Figure 2. DiI labeled cells in the neural tube contribute to enteric ganglia

Immunohistochemistry of T30 lamprey transverse cryosections.

, Uninjected control

highlighting neuronal projections (white arrowhead) and serotonergic neurons (white

arrows) around the gut. Green:Acetylated tubulin; Blue:DAPI; Cyan:5-HT.

b–d

, DiI injected

“tubefill” embryos. Red:DiI; Green:Acetylated Tubulin; Cyan:5HT; Blue:DAPI.

, DiI

within the neural tube, dorsal root ganglia and along axon bundles.

, A DiI-labeled enteric

neuron (acetylated tubulin

, 5-HT

) within the gut wall typhlosole (white arrowhead). Inset

shows DiI only.

, A DiI-labeled neuron (acetylated tubulin

, 5HT

) in the esophagus

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(yellow arrowhead). See cell counts in Table 1.

, 5-HT

cells along dorsal root (DR) nerve

processes but not ventral roots (VR).

, A DiI-labelled GFAP-positive cell associated with

the lateral line. Eso: esophagus; NT: neural tube; Not: notochord. Scale bar indicates 20 um.

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Figure 3. Neural tube ablation disrupts ENS development

a–c,

Whole-mount images of T30 lamprey embryos (Red:HuC/D and acetylated tubulin;

Cyan:5HT).

, Control nonablated embryo.

, Ablation of the neural tube at T24 results in a

decrease of serotonergic (5HT

) cells in the adjacent gut relative to an equivalently sized

anterior region.

, A sham-ablated embryo, in which epidermis and neural tube were cut but

not removed, does not show a reduction of cells.

, Ablated embryos show a significantly

lower ratio of serotonergic cells (# cells in surgical zone/# cells in anterior zone) than sham-

ablated controls (Ttest; P=0.0004) and non-ablated batch controls (Ttest, P=0.0176). Error

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bars indicate s.e.m.

, Schematic model of neural crest contributions to the ENS in mouse

compared with lamprey. Vagal neural crest (V; purple); Schwann cell precursors (SCP;

blue); Lamprey branchial crest (BC; purple). Scale bar indicates 100 um.

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Table 1

Number of DiI-labeled neurons (AcTub

) in 25 representative E30–35 embryos in which DiI had been injected

into the neural tube.

Location

DiI

, 5HT

DiI

, 5HT

−

Total DiI-labeled

Neurons

Esophagus

Intestine (Basal Typhlosole)

Intestine (Typhlosole mesenchyme)

Total

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