Evolution of the New Head by gradual acquisition of neural crest regulatory circuits

Evolution of the New Head by gradual acquisition of neural crest

regulatory circuits

Megan L. Martik

Shashank Gandhi

Benjamin R. Uy

J. Andrew Gillis

2,3

Stephen A.

Green

Marcos Simoes-Costa

Marianne E. Bronner

1,*

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom

Marine Biological Laboratory, Woods Hole, MA, 02543, U.S.A.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

Summary:

The neural crest is a vertebrate innovation proposed to be a key component of the “New Head”

that imbued vertebrates with predatory behavior. To address how evolution of this cell type

impacted the vertebrate body plan, we examined the molecular circuits that control neural crest

development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene

expression analysis showed that the lamprey cranial neural crest lacks most components of an

amniote cranial-specific transcriptional circuit that confers the ability to form craniofacial cartilage

onto other neural crest populations

. Consistent with this, hierarchical clustering revealed that the

transcriptional profile of the lamprey cranial crest is more similar to the amniote trunk crest.

Intriguingly, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated

that the cranial-specific transcriptional circuit emerged via gradual addition of network

components to the neural crest of gnathostomes, which subsequently became restricted to the

cephalic region. Our results indicate that the ancestral neural crest at the base of vertebrates

possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by

progressive assembly of a novel axial-specific regulatory circuit, allowed for the elaboration of the

New Head during vertebrate evolution.

Gans and Northcutt’s “New Head” hypothesis proposed that emergence of the vertebrate

lineage was accompanied by advent of the neural crest (NC), an embryonic stem cell

population that arises within the forming central nervous system (CNS) in all vertebrates

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Corresponding author. Correspondence and request for materials should be addressed to mbronner@caltech.edu.

Author Contributions:

Project and analysis conception were designed by M.L.M., M.S.C., and M.E.B. Writing and interpretation was performed by M.L.M.,

S.G., B.R.U., J.A.G., S.A.G., M.S.C., M.E.B. Lamprey orthologue cloning and all

in situ

hybridization, imaging, and analysis was

performed by M.L.M. Bioinformatics and chicken RNAseq was performed by S.G. Phylogenetic analysis and lamprey embryo

acquisition was performed by S.A.G. Cloning of skate orthologues and skate embryo acquisition was performed by J.A.G. Lamprey

embryo dissections and library preparations were performed by B.R.U. and M.S.C.

The authors declare no competing financial interests.

HHS Public Access

Author manuscript

Nature

. Author manuscript; available in PMC 2020 April 23.

Published in final edited form as:

Nature

. 2019 October ; 574(7780): 675–678. doi:10.1038/s41586-019-1691-4.

Author Manuscript

These cells subsequently leave the CNS, migrate to diverse locations and differentiate into

many derivatives including peripheral ganglia and craniofacial skeleton

. As vertebrates

evolved, NC cells contributed to morphological novelties like jaws, that enabled expansion

of vertebrates.

A pan-vertebrate NC gene regulatory network (GRN), invoking sequential deployment of

signaling and transcriptional events, has been proposed to underlie formation of this unique

cell type. Primarily studied at cranial levels, the core of the NC GRN is largely conserved

across vertebrates, including the sea lamprey,

Petromyzon marinus,

a jawless (cyclostome)

vertebrate. However, differences exist in utilization of key transcription factors, like

Ets1

and

Twist,

which are deployed later in the lamprey GRN than in amniotes

, suggesting

regulatory differences between cyclostomes and gnathostomes. Furthermore, some NC

derivatives are novelties of gnathostomes, such as jaws at cranial levels, a vagal-derived

enteric nervous system, and sympathetic ganglia at trunk levels

. This raises the intriguing

possibility that network differences in axial regionalization of the neural crest may have

contributed to the presence of these gnathostome cell types.

In jawed vertebrates, the NC is subdivided along the body axis into cranial, vagal, and trunk

populations. In contrast, lamprey lack an intermediate vagal population, suggesting there are

two major subdivisions: cranial and trunk

. How axial identity in lamprey is controlled

molecularly remains unknown. Avian embryos possess a “cranial crest-specific” NC GRN

subcircuit with ability to drive differentiation of trunk NC into ectomesenchymal

derivatives

. In this kernel, transcription factors

Brn3c, Lhx5,

and

Dmbx1

are expressed at

the neural plate border and, in turn, activate expression of

Ets1

and

Sox8

in premigratory

cranial NC (Fig 1A). In contrast to their cranial-specific expression,

Tfap2b

and

Sox10

are

pan-NC genes expressed all along the body axis

Here, we assessed whether this cranial subcircuit is a general feature of vertebrates by

examining whether lamprey possess a homologous spatiotemporal regulatory state. Taking a

candidate approach, we analyzed expression of cranial circuit orthologues in lamprey

embryos at different developmental stages. In contrast to amniotes, our results show that

Brn3, Lhx5, Dmbx1,

and

Ets1

appear to be absent from lamprey premigratory or migratory

NC (Fig 1B). The lack of most cranial-specific regulatory factors suggests a high degree of

divergence between early regulatory states of lamprey and amniote NC. In contrast, lamprey

SoxE1

and

Tfap2

were robustly expressed in premigratory and migratory NC along the

entire body axis (Fig 1B, SupFig1A-D). No

SoxE

family member was restricted in

expression to the cranial NC as is Sox8 in amniotes. Of note, lamprey SoxE transcription

factors are homologous to gnathostome Sox8/9/10, and there is variation in SoxE paralog

usage across gnathostomes

–

. Consistent with the lack of restricted ‘cranial-specific’

expression, ectomesenchymal derivatives have been previously reported as present at trunk

levels in the lamprey dorsal fin

How then did this regulatory subcircuit evolve? Interestingly, genes from the cranial crest

subcircuit are present in the genome and expressed later in pharyngeal arches populated by

NC cells (Fig 1C,D, SupFig1E-L, SupFig6). An intriguing possibility is that these genes

were expressed only in late NC derivatives of early vertebrates, followed by gradual co-

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option of components of this regulatory program to earlier developmental stages in

gnathostomes. According to this scenario, genes involved in NC differentiation in early

vertebrates were co-opted to the specification program of gnathostomes at all axial levels.

With subsequent regulatory modifications, they became cranially restricted, possibly

endowing the cranial NC with novel morphogenetic features while the trunk NC lost ability

to make cranial-like derivatives

To explore this possibility, we examined candidate elements of the cranial NC subcircuit in

the little skate

Leucoraja erinacea,

a Chondricthyan gnathostome outgroup to the bony

fishes. Of the

SoxE

genes, expression of

Sox9

and

Sox10,

as well as

Tfap2b

and

Ets1,

were

present at all axial levels and not restricted to cranial crest (Fig 2A, SupFig2). Since

Ets1

appears in the little skate migratory NC as in other gnathostomes, we conclude that this early

node was a novelty acquired by the cranial NC GRN prior to divergence of cartilaginous and

bony fishes (Fig 2A,B, SupFig2). Later, after NC cells migrate to and populate the

pharyngeal arches,

SoxE, Tfap2b,

and

Ets1

were present within the arches (SupFig3). Trunk

NC in the little skate produce ectomesenchymal dermal denticles, “cranial-like” derivatives,

consistent with our observation that cranial subcircuit genes in the little skate are not

restricted to the head but can drive differentiation of skeletogenic derivatives in the trunk

In the fossil record, many stem-gnathostomes possessed extensive dermal armour, which has

been retained, albeit with the dental component reduced and modified, within the

gnathostome crown group (e.g. dermal denticles of chondrichthyans; dentinous scales of

Polypterus

and coelacanth). Thus, dental tissues in the post cranial dermal skeleton appears

to be ancestral for gnathostomes.

Interestingly, in the teleost

Danio rerio, lhx5

and

dmbx1

are present in the early cranial

circuit but absent from later pharyngeal arch derivatives (Fig 2C,D, SupFig4, SupFig5). In

addition,

sox8b, sox10, tfap2a,

and

ets1

are present in premigratory and migratory crest at

all axial levels, though

brn3c

is missing (Fig 2C, SupFig4). Rather than restricted to the

cranial NC, many of these factors also are present in the zebrafish trunk, raising the

possibility that resolution of axial level potential may have arisen within sarcopterygians.

Furthermore,

in situ

analysis of pharyngeal arch derivatives in both little skate and zebrafish

lend support to temporal shifts of cranial specific regulatory nodes from later NC derivatives

to an early specification program. With progressive loss of nodes from late derivatives and

addition to an earlier program, this suggests that regulatory modifications arose gradually

throughout gnathostome evolution (SupFig 1M).

Our candidate gene approach suggests that extensive changes occurred in the NC regulatory

state between jawless and jawed vertebrates. To investigate further, we conducted a

comparative transcriptome analysis of cranial and trunk NC subpopulations in lamprey and

chicken (Fig 3). Premigratory lamprey NC was obtained by micro-dissecting segments of

cranial and trunk dorsal neural tubes at stages T21 and T23.5. For chick, premigratory NC

populations were isolated using enhancers driving eGFP expression in cranial or trunk

neural crest populations for FACS at stages HH9+ and HH18, respectively. After cDNA

library preparation and sequencing, differential expression analysis revealed far fewer genes

(1233 genes in lamprey compared with 2794 in chicken) significantly enriched in lamprey

cranial versus trunk crest compared with chick cranial versus trunk (Fig 3A, 3B).

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To better understand how each library correlated to the others in an unbiased fashion, we

mapped each to a common reference transcriptome, created by aligning proteomes using

BLAT and compiling matching sequences as a consensus alignment between species for

Bowtie mapping. We next performed hierarchical clustering analysis of all known NC GRN

genes (Fig 3C). Consistent with our previous

in situ

hybridization analysis (Fig 1B),

Tfap2

was enriched at all axial levels in both chicken and lamprey (Fig 3C’);

Sox8

was enriched in

chicken cranial NC but was in both cranial and trunk lamprey populations (Fig 3C”).

Dmbx1

and

Ets1

were enriched in chick but not lamprey cranial datasets (Fig 3C”’).

Interestingly, we found that lamprey cranial populations correlated more closely to chicken

trunk than lamprey trunk libraries, suggesting that basal NC was “trunk-like” in its

regulatory program (Fig 3C). These results suggest that cyclostomes possess a simpler and

more trunk-like cranial crest GRN, with potentially important implications for evolution of

NC subpopulations (Fig 4A). Accordingly, we speculate that the ancestral neural crest may

have been relatively homogeneous and trunk-like. Throughout evolution of the vertebrate

lineage, we propose that key transcription factors were progressively co-opted into an early,

cranial-restricted circuit, whereas some features like skeletogenic potential were lost from

the trunk.

These differences in axial-specific genes contrast with the deep conservation of the pan-NC

program

. Transcription factors like

SoxEs, Tfap2,

and

may be the rudiment of a larger,

more complex cranial crest GRN that was expanded during early vertebrate evolution with

incorporation of novel players such as

Dmbx1, Brn3c, Ets1,

and

Lhx5.

Consistent with these

findings, the basal chordate Amphioxus lacks expression at the neural plate border of genes

Dmbx, Brn3, Ets,

as well as core NC genes like

SoxE, FoxD, Tfap2,

and

Id,

although

these genes are expressed in other tissues

–

. Our observations also show that some of

these “novel” genes are expressed at later stages of NC formation, consistent with the

possibility that elaboration of the GRN might have involved co-option of parts of

differentiation programs to earlier portions of the network perhaps by acquisition of new

regulatory elements responsible for their heterochronic shift

. Thus, the pan-NC program

was likely the ancestral molecular recipe to make NC, with the subsequent elaboration of

axial-specific regulatory programs conferring important differences in developmental

potential along the body axis. Given that many key NC derivatives are gnathostome

innovations, we hypothesize that gain of these derivatives may be due to gene regulatory

differences associated with axial-specific regulatory programs.

Taken together, our results suggest the following scenario to explain evolution of NC

subpopulations (Fig 4). We suggest that NC of early vertebrates was uniform and similar to

amniote trunk populations, and that the division of NC into cranial and trunk subpopulations

occurred early in vertebrate evolution (Fig 4A). Consistent with evolutionary expansion of

NC cells in the vertebrate lineage, our molecular analysis of the cranial NC reveals

surprising differences in lamprey compared with gnathostome counterparts (Fig 4B). Given

that the Hox code was already linked to segmentation of the CNS in basal vertebrates,

posteriorizing influences of Hox genes and other factors may be sufficient to account for the

subtle transcriptional differences observed between these two populations

. We cannot

rule out the possibility that cyclostomes lost NC subpopulations during the course of

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evolution. However, the relative scarcity of cranial-specific factors in the lamprey cranial

crest might suggest that the gnathostome cranial NC GRN has undergone extensive

elaboration from a regulatory standpoint. Thus, we propose that regionalization of the NC,

with both emergence of new subpopulations and expansion of the cranial crest GRN, played

a crucial part in vertebrate evolution as a key element for driving evolution and expansion of

gnathostomes.

What does this mean for the ‘New Head’ hypothesis? We posit that, the NC component of

the New Head, rather than arising

in toto

at the base of vertebrates, underwent continued

regulatory modifications, evolving gradually during the course of vertebrate evolution. Our

data suggest that early vertebrates possessed a relatively simple NC that initially arose as a

fairly uniform population along the body axis and lacked region-restricted regulatory

programming. During gnathostome evolution, the cranial NC appears to have gained

regulatory complexity that modulated differentiation capacity, gaining some individual cell

fates while restricting others. We propose that co-option of distinct genes into a cranial-

specific module enabled this progressive specialization of NC regulatory programs, leading

to unique axial populations and morphological novelties of the gnathostome body plan.

Methods:

Animal husbandry and embryo collection

Adult sea lamprey were obtained from the US Fish and Wildlife Service and Department of

the Interior. Embryos were cultured according to previously published protocols and staged

according to Tahara staging methods

. All lamprey embryology work was completed in

compliance with California Institute of Technology Institutional Animal Care and Use

Committee (IACUC) protocol 1436. Skate eggs were obtained by the Marine Biological

Laboratory (MBL) Marine Resource Center, and embryos were cultured as previously

described

. All skate embryology work was compliant with animal protocols approved by

the IACUC at the MBL. Adult zebrafish were maintained in the Beckman Institute Fish

Facility at Caltech, and all animal and embryo work was compliant under approved IACUC

protocol 1346. Fertilized chicken eggs were obtained from a local farm in Sylmar, CA. No

statistical methods were used to predetermine sample size for analyses. For in situ

hybridization, embryos were pooled from different breeding pairs (fish), brooding stocks

(skates), or embryo batches (lamprey) to ensure replication of results in multiple fixed

collections.

Cloning of lamprey, skate, and zebrafish orthologues

RNA was extracted from desired embryo stages using the RNAqueous-Micro Kit (Thermo

Fisher Scientific), and cDNA was synthesized using a SuperScript III Reverse Transcriptase

Kit (Invitrogen). The following gene specific primers used to amplify probe template

sequences (accession numbers in parentheses):

PmTfap2

(MN410935):

F: 5’-GCATCGCGACAGTTGTTTGCTG-3’; R: 5’-

GATGCTGTGGTGCCCTAATCC-3’

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PmSoxE2

(MN410934):

F: 5’-CGAGTCACGTGGATCTGCTGC-3’; R: 5’-

CCGTCCAGCACTTGACTCACG-3’

PmSoxE1

(MN410933):

F: 5’-CGGGCTGAGTCATTACTCGCATCG-3’; R: 5’-

CTCTCGTCGCTGTCGGAAGC-3’

PmEts1a

(SIMRbase: PMZ-0040201):

F: 5’-GGACCTTCAAGGAGTACATGAGC-3’; R:

5’-GAGAGCGGTACTCGTGGAAAGTC-3’

PmDmbx1

(Ensembl: ENSPMAG00000008114):

F: 5’-

GCGCATGAATACCGGCCGTCG-3’; R: 5’-TTGCTTTGATGCTGTTACAAGG-3’

PmLhx5

(MN410936):

F: 5’-CGTGCGTTCGTGACCCCATC-3’; R: 5’-

GAGGCCAGGTAGTCCTCCTTG-3’

PmBrn3

(SIMRbase: PMZ-0005302):

F: 5’-CGAGTCTCCTTAACGCGTTAGCTC-3’; R:

5’-GCTCTGGTGGGAGACAATATCCACG-3’

LeTfap2b

(MN410937):

F: 5’-TCCCACTTCCACAGAAGAAT-3’; R: 5’-

TCCTTGTCTCCAGTTTTGGTG-3’

LeSox10

(MN410938):

F: 5’-ACCCCCGTTCTGTGTGTCT-3’; R: 5’-

GGCAGGTACTGGTCGAACTC-3’

LeSox9

(MN410939):

F: 5’-CCCAGCCACTACAATGAGCAG-3’; R: 5’-

CCGTACGGCATCAGCAAATG-3’

LeSox8

(MN410944):

F: 5’-CAACTCCGCCCACCACTCC-3’; R: 5’-

TGGCCTAGTCAGGGTTGTGTAG-3’

LeEts1

(MN410940):

F: 5’-TTCAGCCTGAAGAACGTGGAC-3’; R: 5’-

GCAAGACTTGTCCGTCAGGAG-3’

LeDmbx1

(MN410941):

F: 5’-CAATCAACACGACAGGGACA-3’; R: 5’-

GTAAGCTGTCAAGCCCCAGA-3’

LeLhx5

(MN410942):

F: 5’-TCATCGACGAAAACAAATTTGTGTG-3’; R: 5’-

TGAATAACCCGCATGTTGAGGC-3’

LeBrn3c

(MN410943):

F: 5’-CTTCAAGCCGGACATCACCTAC-3’; R: 5’-

TAGATCCCTGCTTGTTCCTGC-3’

Drtfap2a

(NM_176859):

F: 5’-GTCACGGCATTGATACTGGACTC-3’; R: 5’-

TCATTGGCACACTGCTTTACTGAT-3’

Drsox10

(NM_131875):

F: 5’-GTGAAACACACTTCCCTGGGGATAC-3’; R: 5’-

GTGGAGACATGTGTGTATGGCGTC-3’

Drsox8b

(NM_001025465):

F: 5’-ATGAGCGAGGAGCGGGAAAAGTG-3’; R: 5’-

GGGTCTGGACAGAGTGGTGTAGAC-3’

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Drets1

(NM_001017558):

F: 5’-CAGACAGCGGATCTTGTTGAGGGA-3’; R: 5’-

CAGTCCAGCTGATGAAGGACTGG-3’

Drdmbx1a

(NM_152977):

F: 5’-CGTGCCAGTCCTACTATCAGTCTC-3’; R: 5’-

CTGCTGTGTAGTGCATGCAACC-3’

Drlhx5

(NM_131218):

F: 5’-CACGGACATGATATCCCATGCAGAC-3’; R: 5’-

CTAGCTCACTTCTGACCATCAGATGC-3’

Drbrn3c

(NM_131278):

F: 5’-ATGATGACCATGAACGGCAAGC-3’; R: 5’-

GTGCACTGCTGAATACTTCATCC-3’

Phylogenetic analysis of Dmbx proteins

Candidate Dmbx sequences were assembled as an ungapped Fasta file and imported into the

TCoffee server (

http://tcoffee.crg.cat

) and processed using default parameters in an Expresso

(

http://tcoffee.crg.cat/apps/tcoffee/do:expresso

) into a protein alignment

. The

TCoffee fasta alignment was imported into MegAlign Pro (DNAstar ver 15.0.0) and

ambiguous regions with poor alignment scores were removed, leaving only large, contiguous

regions of well-aligned sequence. This alignment of 218 amino acid resides was exported as

nexus format. The start of the

P. marinus

sequence is missing, and so residues were recoded

from gaps to indicate missing sequence. The file was modified to include a MrBayes block,

with aamodelpr=mixed, stopval=0.01, ngen=200000, and burninfrac=0.25. The file was

executed within MrBayes3.2.1, and resulting consensus tree visualized in FigTree v1.4.2 to

show posterior probabilities (as %) at corresponding branch labels. Image output files from

Megalign Pro and FigTree v1.4.2 were combined in Adobe Illustrator 2019 (Adobe Creative

Suite 2019) (Supplemental Figure 6). NCBI accession numbers or Ensembl identifiers for

Dmbx sequences used in phylogenetic analyses are as follows:

XP_003725762.1 (

S. purpuratus)

NP_001161526.1 (

S. kowalevskii)

AAT66431.1 (

B. floridae)

Ensembl ENSPMAG00000008114 (

P. marinus)

XP_020369662.1 (

R. typus)

AAI34895.1 (Dmbx1a,

D. rerio)

NP_001017625.1 (Dmbx1b,

D. rerio)

XP_017949066.1 (

X. tropicalis)

XP_001234036.2 (

G. gallus)

NP_671725.1 (

H. sapiens)

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In situ hybridization of lamprey, skate, and zebrafish embryos, Sectioning, Imaging, and

Biotapestry modeling

Whole mount

in situ

hybridization was was performed using previously published

protocols

. Cryosections of lamprey, skate, or zebrafish embryo

in situs

were

sectioned at 18 μm with a

Microm

HM550

cryostat. In situ

analysis of S25 skate embryos

sections was performed using paraffin sections as follows: After fixation, embryos were

embedded in paraffin and sections were prepared at 5 (skate) or 10μm (lamprey) thickness

on a Zeiss microtome. After paraffin removal with histosol, sections were hybridized with

1ng/μl anti-sense digoxygenin-labelled probes overnight at 70°C in a humidifying chamber.

After hybridization, sections were washed with 50% formamide/50% 1X SSCT buffer

followed by washes with MABT and a blocking step in 1% Roche blocking reagent.

Sections were then incubated overnight at room temperature with a 1:2000 dilution of anti-

DIG-Alkaline Phosphatase antibody (Roche). After several washes with MABT,

chromogenic color was developed using NBT/BCIP precipitation (Roche). Imaging was

performed on a Zeiss AxioImager.M2 equipped with an Apotome.2. Gene network models

were assembled using Biotapestry

Chicken embryo electroporation, dissociated, and cell sorting

Cranial and trunk neural crest cells were labeled using previously published neural crest

enhancers FoxD3-NC1.1 and FoxD3-NC2, respectively

. To isolate cranial neural crest

cells, stage Hamilton-Hamburger (HH) 4 embryos were bilaterally electroporated with

FoxD3-NC1.1>eGFP and cultured

ex ovo

until stage HH9+

. For each biological replicate,

at least 15 embryo heads were dissected in Ringers and washed thrice in chilled 1x PBS. For

trunk neural crest cells, stage HH10 embryos were bilaterally electroporated with FoxD3-

NC2>eGFP and cultured

in ovo

until stage HH18. Based on the expression of the reporter,

five embryo trunks spanning the length of five somites were dissected in Ringers and washed

thrice in chilled 1x PBS. The tissues were dissociated in Accumax (Innovative Cell

Technologies, Inc.) for 15 minutes at 37°C and GFP+ cells were collected using

Fluorescence Activated Cell Sorting.

Library preparation and sequencing

Chicken libraries were prepared using the SMART-Seq Ultra Low Input RNA Kit (Takara)

according to the manufacturer’s protocol. For lamprey embryos, tissue was dissected from

the cranial dorsal neural tubes of n=100 T21 and trunk neural tube of n=100 T23.5 embryos.

Total RNA was extracted using the RNAqueous kit (Ambion). RNA-Seq was performed at

the Millard and Muriel Jacobs Genetics and Genomics Laboratory (California Institute of

Technology, Pasadena, CA) at 50 million reads on 2 biological replicates for both the T21

cranial and T23.5 trunk neural tube samples. Sequencing libraries were built according to

Illumina Standard Protocols. SR50 sequencing was performed in a HiSeq Illumina machine.

Databases have been deposited to NCBI (BioProject # PRJNA497902)

Statistical analysis of lamprey and chicken axial population RNAseq

To identify orthologous genes between lamprey and chicken, the lamprey proteome obtained

from SIMRbase

was aligned to the chicken proteome using the BLAT alignment software

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available on the UCSC genome browser

. Briefly, every lamprey protein sequence was

locally queried against the chicken proteome, following which regions with the longest

alignment were matched to the respective chicken gene. Using this alignment-based

approach, proteins with an alignment percentage score between 52-100 (see Supplementary

table for exact scores for each orthologue) were identified as orthologues, and their

respective cDNA sequences were obtained from the chicken and lamprey databases. Chicken

cranial and trunk libraries were aligned to the chicken sequences, while the lamprey cranial

and trunk libraries were aligned to the lamprey sequences using Bowtie2

. Transcript

counts were calculated using HTSeq-Count and differential gene expression analysis was

performed using DESeq2

. Using chicken gene annotations as a reference, we added the

transcript counts for duplicated orthologues found in the lamprey genome to calculate an

“aggregated” transcript count for each gene. These aggregated transcript counts were then

normalized using the formula:

− min (

)

max(

) − min (

)

where

– Normalized transcript count

– Absolute transcript count

A subset of genes previously identified as being part of the neural crest gene regulatory

network

was then isolated from the count matrix and plotted as a heatmap to obtain the

gene expression matrix.

Data availability

All raw sequencing data for all RNAseq libraries (Figure 3) and merged reference

transcriptomes are available online (NCBI BioProject# PRJNA497902). Sequences of

in situ

probe templates for Figures 1B, 1C, 2A, and 2C are available through GenBank accession

codes found in the methods.

Code Availability

Code used to analyze sequencing datasets are available from the corresponding author upon

request.

Extended Data

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Extended Data 1. Heterochronic shifts of cranial specific gene regulatory nodes from later neural

crest derivatives to an early specification program happened gradually throughout gnathostome

evolution.

a-d.) Expression of lamprey orthologues of amniote cranial specific genes at T21 (cranial)

and T23 (trunk) in cross-section. e.) Pharyngeal neural crest derivative expression in Tahara

stage 26

Petromyzon marinus

frontal section (illustration based on Damas, et al (1944)

). f-

l.) Cranial circuit orthologues are expressed in pharyngeal arch derivatives, with the

exception of

Brn3

which is present in the neural crest-derived cranial sensory ganglia

lamprey frontal sections. m.) Gene expression matrix summarizing the heterochronic shift of

cranial crest specific circuit nodes. nc=neural crest, nt= neural tube, n=notochord,

end.=endoderm, ect.=ectoderm, mes.=mesoderm. Scale bars= 100μm. Cryosections of

situs

were reproducible on n≥5 embryos per time point for n≥2 experiments.

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Extended Data 2. Expression of cranial circuit genes in the neural crest of the little skate.

a.) Schematic of a stage 18

Leucoraja erinacea

embryo with the neural crest illustrated as

blue (cranial) and red (trunk). Placement of cross-sections depicted on the illustration for

figures b-f. nc=neural crest, scale bar= 50μm. Cryosections of

in situs

were reproducible on

n≥2 embryos for n≥2 experiments.

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Extended Data 3. Pharyngeal neural crest derivative expression of cranial circuit orthologues in

stage 25

Leucoraja erinacea

embryos.

a.) Dashed box on the illustration represents the region of the head for each embryo imaged

in figures b-i, and the purple dashed line depicts the location of the frontal section for figures

a’-i’. Pharyngeal neural crest derivative expression of cranial circuit orthologues is seen in

panels b-f’.

Dmbx1, Lhx5,

and

Brn3c

are absent in pharyngeal arch derivatives at stage 25

(g-i’). b-i, scale bar=500μm. b’-i’, scale bar= 100μm.

in situs

were reproducible on n≥2

embryos.

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Extended Data 4. Expression of cranial circuit genes in the neural crest of the zebrafish.

a.) Schematic of a 14ss

Danio rerio

embryo with the neural crest illustrated as blue (cranial)

and red (trunk). Placement of cross-sections depicted on the illustration for figures b-h.

nc=neural crest, n=notochord, scale bars= 50μm.

in situs

were reproducible on n≥10

embryos.

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Extended Data 5. Expression of cranial circuit orthologues in pharyngeal neural crest derivatives

of 3dpf

Danio rerio

embryos.

a.) Purple dashed line depicts the location of the frontal section for figures e’-h’. Expression

of cranial circuit orthologues in pharyngeal arches is seen in panels e-h’.

Dmbx1, Lhx5,

and

Brn3c

are absent from pharyngeal arch derivatives at 3dpf (b-d). Scale bar= 150μm.

in situs

were reproducible on n≥10 whole mount and cryosectioned embryos.

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Extended Data 6. P. marinus

Dmbx

is homologous to gnathostome

Dmbx

genes.

a.) Truncated alignment of Dmbx protein sequences. An alignment of full length Dmbx

protein sequences was assembled using TCoffee and contiguous regions tagged by the

program as poorly or moderately well-aligned were removed, leaving 218 well-aligned

residues. b.) Bayesian consensus phylogenetic tree, with posterior probabilities are shown at

corresponding nodes.

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Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgements:

We thank Joanne Tan-Cabugao and Eli Grossman for technical assistance. We would also like to thank David

Mayorga and Ryan Fraser for help with fish husbandry. We thank Brennah Martik for illustrating the adult animals

for our expression matrices and acknowledge the Caltech Millard and Muriel Jacobs Genetics and Genomics

Laboratory, in particular, Igor Antoshechkin for sequencing of our RNAseq libraries. We thank Rochelle Diamond,

Jamie Tijerina, and Patrick Cannon of the The Caltech Flow Cytometry Cell Sorting Facility for cell sorting

assistance.

Funding:

This work is supported by NIH grant R01NS086907, R01DE024157, and R35NS111564 to MEB. MLM is

supported by a Helen Hay Whitney Foundation postdoctoral fellowship. SG is supported by a graduate fellowship

from the American Heart Association (18PRE34050063).

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Figure 1. Lamprey cranial neural crest lacks most components of a chick “cranial crest circuit”.

a.) Biotapestry model of cranial specific gene regulatory circuit driving skeletal

differentiation in amniotes. b.) Expression of lamprey orthologues of amniote cranial

specific genes at T21 and T23. Blue arrows represent expression in the cranial neural crest

(CNC), and red arrows represent expression in the trunk neural crest (TNC). c.) Late

expression of cranial specific orthologues in the pharyngeal arch neural crest derivatives

(black arrow). d.) Biotapestry model of the lamprey circuit with the addition of late module

expression of markers in the pharyngeal arch neural crest derivatives. TGG, trigeminal

ganglia. Scale bars, 250μm. Reproducible on n≥5 embryos per time point for n≥10

experiments.

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Figure 2. Nodes of an early cranial-specific circuit were acquired in and restricted to the cranial

neural crest progressively throughout gnathostome evolution.

a.) Expression of cranial-specific orthologues in the Little Skate,

Leucoraja erinacea

at stage

(S) 17 and 18. Expression of orthologues in the cranial neural crest (CNC) are depicted with

a blue arrow and rostral trunk neural crest (TNC) with red arrows. b.) Biotapestry model of

the skate circuit with the addition of a novel node,

Ets1.

c.) Expression of cranial-specific

orthologues in the zebrafish,

Danio rerio

at 5-9 somite stage (ss) and 14ss. d.) Biotapestry

model of the zebrafish circuit with the addition of novel early nodes,

lhx5

and

dmbx1

FB/MB, forebrain/midbrain. St, stomodeum. Scale bars, 250μm. For skates,

in situs

were

reproducible on n≥2 embryos for n≥2 experiments. For zebrafish,

in situs

were reproducible

on n≥5 embryos per time point for n≥10 experiments.

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Figure 3. Tissue-specific RNA-seq comparisons between lamprey and chicken reveal ancestral

neural crest had a more trunk-like identity.

a.) Volcano plot showing lamprey differential enrichments of cranial (blue) and trunk (red)

genes by population RNAseq (100 embryos were dissected for each of n=2 biological

replicates) (adjusted p-value<0.05). b.) Volcano plot showing enrichment of genes in the

cranial (blue) versus the trunk (red) neural crest in chicken,

Gallus gallus

(≥15 heads and 5

trunks were dissected and prepared for FAC-sorting for each of n=3 biological replicates)

(adjusted p-value<0.05). c.) Hierarchical clustering analysis of all RNAseq libraries focused

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