Article
https://doi.org/10.1038/s41467-024-45911-x
Sea lamprey enlightens the origin of the
coupling of retinoic acid signaling to
vertebrate hindbrain segmentation
AliceM.H.Bedois
1
,HugoJ.Parker
1
,AndrewJ.Price
1
, Jason A. Morrison
1
,
Marianne E. Bronner
2
&RobbKrumlauf
1,3
Retinoic acid (RA) is involved in antero-
posterior patterning of the chordate
body axis and, in jawed vertebrates, h
as been shown to play a major role at
multiple levels of the gene regulatory network (GRN) regulating hindbrain
segmentation. Knowing when and how
RAbecamecoupledtothecorehind-
brain GRN is important for understandi
ng how ancient signaling pathways and
patterning genes can evolve and generate d
iversity. Hence, w
e investigated the
link between RA signaling and hindbrain segmentation in the sea lamprey
Petromyzon marinus
, an important jawless vertebrate model providing clues to
decipher ancestral vertebrate features. Combining genomics, gene expres-
sion, and functional analyses of major components involved in RA synthesis
(Aldh1as) and degradation (Cyp26s),
we demonstrate that RA signaling is
coupled to hindbrain segmentation in lamprey. Thus, the link between RA
signaling and hindbrain segmentatio
n is a pan vertebrate feature of the
hindbrain and likely evolved at the base of vertebrates.
The body plan of most deuterostomes develops using a similar set of
transcription factors (TFs) and signaling pathways (e.g., FGF, Wnt)
which are expressed in analogous axial domains of different devel-
oping embryos
1
–
5
. This suggests the presence of an ancient, conserved
core gene regulatory network (GRN) underlying axial patterning,
which integrates inputs from developmental TFs and signaling path-
ways. For example, in the evolution of chordates the coordinated
action of FGF and Wnt signaling
2
,
6
,
7
coupled with the Hox family of TFs
is essential for establishing and patterning the antero-posterior (A-P)
axis
5
,
8
–
10
.
In chordate embryos, nested domains of
Hox
expression play a
fundamental role in regulating p
atterning of the
nervous system
1
,
11
–
14
.
In addition to inputs from FGF and Wnt signaling, evidence from
cephalochordate and jawed vertebrate models indicate that retinoic
acid (RA) signaling plays a key role in coordinating the regulation of
Hox
gene expression along the A-P axis
5
,
7
,
15
–
23
.Regulatorystudieshave
also uncovered the presence of retinoic acid response elements
(RAREs) present in conserved positions in an amphioxus and mouse
Hox
cluster that contribute to nested domains of
Hox
expression
24
–
26
.
This suggests that a direct regulatory link between RA signaling and
Hox
expression may be an ancient feature of the core GRN coupled to
A-P patterning of the body axis in all chordates (Fig.
1
a)
26
.
In jawed vertebrates this RA/
Hox
regulatory hierarchy is coupled
to hindbrain segmentation, a vertebrate speci
fi
c feature of the nervous
system (Fig.
1
a). The hindbrain is a complex coordination center that
regulates vital functions and beha
viors and is the site of origin of a
subset of neural crest cells, whose derivatives form many craniofacial
structures
14
,
27
–
30
. Hence, processes that form and pattern the hindbrain
are believed to be important for the evolution and emergence of a
diversi
fi
ed and complex head in vertebrates
31
–
33
.
During early embryogenesis, the hindbrain is transiently orga-
nized into segments (rhombomeres), which lays down a ground plan
for regional patterning of neural differentiation, circuit formation and
head development
14
,
26
,
34
. Hindbrain segmentation is regulated by a
conserved GRN which can be visualized as a hierarchical series of
regulatory modules that govern sequential steps of the cellular and
Received: 5 July 2023
Accepted: 6 February 2024
Check for updates
1
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
2
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA 91125, USA.
3
Department of Anatomy and Cell Biology, Kansas University Medical Center, Kansas City, MO 66160, USA.
e-mail:
rek@stowers.org
Nature Communications
| (2024) 15:1538
1
1234567890():,;
1234567890():,;
developmental patterning process. The GRN is initiated by signaling
cues, including RA, which setup TFs that subdivide the region into
segments, followed by a patterning module, which imparts unique
properties to each segment (Fig.
1
b)
14
,
19
,
35
,
36
. In jawed vertebrates, RA
signaling is essential for the initiation and regulation of multiple
modules, and
Hox
genes subsequently play important functions in
specifying the identity of segments (Fig.
1
b). The importance of hind-
brain segmentation for craniofacial development of jawed vertebrates
raises the question of when during vertebrate evolution the ancient
RA/
Hox
hierarchy became coupled to the process of hindbrain seg-
mentation. Exploring the origin of the coupling of RA signaling to
hindbrain segmentation is also important for understanding how
ancient signaling pathways and patterning genes can evolve their
regulatory interactions to generate more complex GRNs that con-
tribute to morphological diversity.
Lampreys and hag
fi
sh belong to a group of jawless vertebrates
(cyclostomes) that diverged from other vertebrates ~500 million years
ago
37
. Thus, they have a unique position in the vertebrate tree as a
sister group to jawed vertebrates and constitute important models for
understanding the evolution of vertebrate traits
38
.Itwaspreviously
postulated that lamprey hindbrain segmentation is only partially
coupled to
Hox
expression, and that RA signaling in
fl
uences
Hox
-
dependent branchiomotor neuron speci
fi
cation but not hindbrain
segmentation itself
39
,
40
. This implies that roles for RA in hindbrain
segmentation may have arisen later in vertebrate evolution. This
fi
nding is consistent with the idea that there has been a gain in con-
nectivity between GRNs and signaling pathways in the evolution of
jawed vertebrates from their chordate ancestors, which may have
contributed to the formation of new cell types and morphological
novelties
41
. However, through gene expression and cross-species reg-
ulatory studies we have recently uncovered clear evidence that
Hox
genes and other TFs of the hindbrain GRN in jawed vertebrates are also
coupled to the process of segmentation in the sea lamprey (
Petromy-
zon marinus)
(Fig.
1
b)
42
,
43
. This conservation implies that hindbrain
segmentation is a pan-vertebrate trait. Paradoxically, RA signaling is
not thought to be involved in segmental processes in lamprey
39
,
40
,
while it plays major roles in regulating hindbrain segmentation in
jawed vertebrates. This disparity raises important questions about the
origin of the role of RA signaling in hindbrain segmentation and the
evolution of the vertebrate hindbrain GRN.
In light of our
fi
ndings regarding the presence of key reg-
ulatory components of the hindbrain GRN in lamprey
42
,
43
,we
wanted to re-examine whether or not RA is coupled to this GRN.
Thus, we investigated the link between RA signaling and hindbrain
segmentation using sea lamprey as a jawless vertebrate model,
comparingittothecurrentknowledgebuiltfromotherjawed
vertebrates. Combining genomic, gene expression, and functional
analyses of major components involved in the synthesis (Aldh1a
enzymes) and degradation (Cyp26 enzymes) of RA, we demon-
strate that RA signaling is coupl
ed to hindbrain segmentation in
lamprey. Our
fi
ndings reveal that the GRN for hindbrain segmen-
tation and the roles for RA signalin
g in its regulation were already
present in the vertebrate ancestor before the split between jawless
and jawed vertebrates.
hb
hb
hb
Cephalochordates
Jawed
Vertebrates
Cyclostomes
Ant.
Post.
Central
H
o
x
Is RA Signaling
coupled to
Hindbrain
Segmentation?
RA Signaling
coupled to
Hindbrain
Segmentation
Hindbrain
Segmentation
A
P
RA Signaling
and
Hox
coupled
to A-P
Patterning
a
Hindbrain
GRN
Cyp26a1
Aldh1a2
RA
Cyp26s
kreisler
krox20
Hox
PG1-4
RA Signaling
hb
23456 7
r1
mb
RA
Cyp26
?
Aldh1
?
RA
Cyp26
?
kreisler
krox20
Hox
PG1-4
hb
23456 7
r1
mb
Segmental
Subdivision
Hindbrain
GRN
RA Signaling
A-P
Patterning
of CNS
A
P
Aldh1a
Cyp26-2
RA
Cyp26-1,3
Hox
1 to 4
RA
CNS
RA Signaling?
b
?
Segmental
Identity
Segmental
Subdivision
Segmental
Identity
?
Fig. 1 | What is the origin of the coupling of RA signaling to vertebrate hind-
brain segmentation? a
An ancient RA/
Hox
hierarchy is involved in axial (A-P)
patterning in all chordates (e.g.,
Aldh1as
,
Cyp26s
,
Hox 1-4
);
b
Many of these ancestral
genes are wired into a complex and dynamic GRN underlying the process of
hindbrain segmentation in jawed vertebrates. Some aspects of the GRN have been
shown to be highly conserved in sea lamprey (e.g., segmental expression of
Hox
PG1-4
,
krox20
and
kreisler
) suggesting that hindbrain segmentation originated early
in vertebrate evolution. However, it remains unknown whether RA signaling plays a
role in the GRN for segmentation in sea lamprey. Here we use the sea lamprey as a
jawless vertebrate model to understand the origin of the coupling of RA to the
hindbrain GRN for segmentation in the evolution of vertebrates.
Article
https://doi.org/10.1038/s41467-024-45911-x
Nature Communications
| (2024) 15:1538
2
Results
Analysis of sea lamprey
Cyp26
and
Aldh1a
gene families
To investigate potential roles for RA signaling in hindbrain develop-
ment of the sea lamprey (
Petromyzon marinus, Pm
), we
fi
rst identi
fi
ed
members of gene families predicted to encode components of the
enzymatic machinery involved in synthesis (Aldh1a) and degradation
(Cyp26) of retinoids associated with neural tube development in jawed
vertebrates
26
.Weidenti
fi
ed three predicted
Cyp26-like
genes in the sea
lamprey germline genome assembly (KPetmar1)
44
,
45
,
Cyp26A1
,
C
yp26B1/C1a
,and
Cyp26B1/C1b
,andtwo
Aldh1a-like
genes,
Aldh1a1/
a2a
and
Aldh1a1/a2b
.Thelamprey
Aldh1a1/a2a
gene corresponds to
the previously identi
fi
ed
Aldh1a2
gene
46
. Within the cyclostome group,
we also searched the hag
fi
sh genome (Eburgeri3.2) and found two
Cyp26-like
genes and one
Aldh1a1
gene. The gene structures and
lengths of the predicted proteins of lamprey and hag
fi
sh
Cyp26
and
Aldh1a
genes show a high degree of similarity to their putative jawed
vertebrate counterpart genes (Supplementary Tables 1
–
4).
To explore properties of the enzymes encoded by the lamprey
Cyp26
and
Aldh1a
genes, we searched for speci
fi
c catalytic sites
essential for the activity of these protein families in other vertebrate
species. Protein alignments of the Cyp26 family show conserved
amino-acid (AA) sequences for the I/K Helices and the Heme domain in
lamprey and hag
fi
sh (Supplementary Fig. 1)
47
. Similarly, alignments
between Aldh1a proteins reveal that key catalytic domains of this
family - the Cysteine (Cys) and Glutamic Acid (Glu) residues - are
conserved in the lamprey Aldh1a1/a2 proteins (Supplementary Fig. 2)
48
.
Phylogenetic analyses of predicted protein sequences were per-
formed to examine their evolutionary relationship with jawed verte-
brate
Aldh1a
and
Cyp26
genes. All vertebrate
Cyp26A1
genes group
together as a clade, with high bootstrap support (81) (Fig.
2
a). We did
not retrieve a
Cyp26A1
homolog in the hag
fi
sh genome, which may
re
fl
ect an incomplete assembly or that
Cyp26A1
was lost in hag
fi
sh. The
vertebrate
Cyp26B1/C1
genes form a sister clade to the
Cyp26A1
clade,
with strong bootstrap support (99) (Fig.
2
a), indicating a separation
between
Cyp26A1
and
Cyp26B1/C1
genes prior to the divergence of
jawed and jawless vertebrates. Within the
Cyp26B1/C1
clade, there is
support for separate jawed vertebrate
Cyp26C1
and
Cyp26B1
clades.
While lamprey
Cyp26B1/C1a
and
Cyp26B1/C1b
appear to group with the
two hag
fi
sh
Cyp26C1-like
genes, we are unable to infer any 1:1 orthology
between cyclostomes and jawed vertebrates. In addition, hidden
paralogy between cyclostome and gnathostome Cyp26A1s cannot be
excluded based on the phylogenetic analysis. Thus, it is possible that
the cyclostome and gnathostome Cyp26A1s may not be direct
orthologues.
For the
Aldh1a
complement, the tree shows clear jawed vertebrate
clades for
Aldh1a1
and
Aldh1a2
. However, it does not resolve clear
relationships between the cyclostome and jawed vertebrate
Aldh1a
genes, since the cyclostome
Aldh1a
genes do not group with either
Aldh1a1
or
Aldh1a2
clades, nor with each other. In jawed vertebrates,
the lineage that led to
Aldh1a3
genesappearstohavedivergedearly
from the ancestor that led to the
Aldh1a1
/
Aldh1a2
clade
49
.Hence,we
examined whether the
Pm Aldh1a1/a2
genes could be related to
Aldh1a3
. However, including the
Aldh1a3
family in this analysis did not
help to resolve the relationship of the sea lamprey
Aldh1a1/a2a
and
Aldh1a1/a2b
with the vertebrate
Aldh1as
(Supplementary Fig. 3).
Synteny analysis of vertebrate
Cyp26
and
Aldh1a
genomic loci
To gain further insight into the relationship between the sea lamprey
and jawed vertebrate
Cyp26
and
Aldh1a
complements, we looked for
evidence of conserved local synteny across vertebrate lineages
(Fig.
2
b). Despite observing clear syntenic groups in the jawed verte-
brate
Aldh1a1
and
Aldh1a2
genomic loci, we did not
fi
nd any indication
of shared synteny with lamprey
Aldh1as
(Fig.
2
b).
In most jawed vertebrate lineages
Cyp26A1
is positioned directly
adjacent to a
Cyp26C1
gene (Fig.
2
b). In sea lamprey,
Pm Cyp26A1
and
Cyp26B1/C1a
are also located adjacent to each other on chromosome
11, and we uncovered evidence of syntenic relationships around the
Pm
Cyp26A1
-
Cyp26B1/C1a
locus (
Myof, Exo6
) shared with other verte-
brates. These similarities in the genomic organization suggest that
these genes arose by tandem duplication before the split between
jawed and jawless vertebrates, and that this arrangement was subse-
quently maintained in most vertebrate lineages. Our analysis of the
third
Cyp26
gene,
Cyp26B1
or lamprey
Cyp26B1/C1b
, also reveals evi-
dence for shared synteny (
Dysf
and
Exo6b
) between lamprey and jawed
vertebrates (Fig.
2
b).
In summary, the data from phylo
genetic and synteny analyses
support an early separation of vertebrate
Cyp26A1
and
Cyp26B1/C1
genes prior to the divergence of jawed and jawless vertebrate lineages.
Lamprey
Cyp26A1
appears to be a clear ortholog of jawed vertebrate
Cyp26A1
, but the evolutionary history of the
Cyp26B1/C1
group in ver-
tebrates is less clear. This suggests that an ancestral vertebrate had one
Cyp26A1
gene and at least one
Cyp26B1/C1-like
gene. In contrast, the
ancestry of the vertebrate
Aldh1a
genes is not as clear, with cyclostome
genes forming basal branches in the phylogeny and not falling within
either of the jawed vertebrate
Aldh1a1
and
Aldh1a2
clades.
Expression analysis of sea lamprey
Cyp26
and
Aldh1a
genes
We next investigated the expression patterns of
Cyp26
and
Aldh1a
genes in sea lamprey embryos. We analyzed existing RNAseq data sets
for a series of early developmental stages
50
,
51
to determine whether any
of these genes are expressed during periods relevant to hindbrain
segmentation (Fig.
3
a).
Cyp26A1
and
Aldh1a1/a2b
are expressed during
blastula and gastrulation stages (Tahara stages st7-st17)
52
but are
downregulated during neurulation and hindbrain segmentation (st17-
st24). In contrast,
Cyp26B1/C1a
and
Aldh1a1/a2a
are not expressed at
the earliest stages but are upregulated during gastrulation, neurula-
tion, and segmentation (Fig.
3
a).
Cyp26B1/C1b
is not expressed at any
of these developmental times.
Based on these data, we focused on
Cyp26A1, Cyp26B1/C1a
and
Aldh1a1/a2a
and characterized their spatio-temporal expression pat-
terns in the period between mid-gastrulation and late hindbrain seg-
mentation (st16-st23) by colorimetric in situ hybridization (cISH)
(Fig.
3
b, c).
Cyp26A1
expression is dynamic over these stages. Expres-
sion is detected in the anterior neural plate at the beginning of neur-
ulation (st16), persists between st17-st19 and then is progressively
down-regulated in the most anterior part of the developing neural tube
(Fig.
3
b). At st20 and later stages, low levels of
Cyp26A1
expression are
detected in lateral regions of the head, which appear to correspond to
the optic placode, and in the neural tube at the level of the caudal
hindbrain.
For
Cyp26B1/C1a
, between st16-st20, weak expression is
observed in small patches lateral to the developing neural tube and
at st20 a restricted domain of expression is visible within the hind-
brain primordium (Fig.
3
b). Between st21-st23, two non-adjacent
stripes are observed in the hindbrain and then form a series of stripes
of differing intensities that cover most of the hindbrain. This
dynamic pattern is reminiscent of the expression of
Cyp26B1
and
Cyp26C1
in speci
fi
c rhombomeres of zebra
fi
sh and mouse
embryos
36
,
53
,
54
, suggesting that
Cyp26B1/C1a
could also be coupled to
segmentation in lamprey.
A previous study characterized the expression of
Aldh1a1/a2a
in
dorsal interneurons of the lamprey spinal cord (st22-st25) and linked
this pattern with an ancient conserved intronic enhancer
46
. In earlier
stages, we
fi
nd that expression of
Aldh1a1/a2a
is
fi
rst visible at st16
around the blastopore. At st17, during the early stages of neurulation,
it is highly expressed in two posterior domains corresponding to the
presomitic mesoderm (PSM) and this expression is maintained as the
embryo undergoes neurulation (Fig.
3
c). At st19/20,
Aldh1a1/a2a
is
expressed in the PSM and newly developing somites adjacent to the
neural tube, as well as in the lateral plate mesoderm in the posterior
Article
https://doi.org/10.1038/s41467-024-45911-x
Nature Communications
| (2024) 15:1538
3