Biochemistry and Biophysics Reports 27 (2021) 101047

Contents lists available at ScienceDirect

Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Protocadherin-1 is expressed in the notochord of mouse embryo but isdispensable for its formation
Kanako Fukunaga a, b, c, Masafumi Tanji c, Nana Hanzawa c, Hiroki Kuroda b, d, Masafumi Inui c, e, *a

Systems Biology Program, Graduate School of Media and Governance, Keio University, Kanagawa, 252-0882, Japan
Institute for Advanced Biosciences, Keio University, Kanagawa, 252-0882, Japanc
Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, 214-8571, JapandFaculty of Environment and Information Studies, Keio University, Kanagawa, 252-0882, Japane
Department of Systems BioMedicine, National Institute for Child Health and Development, Tokyo, 157-8535, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
PCDH1
Notochord
Cell adhesion molecule

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and thesignaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal mostmesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form
an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step.
In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), isindispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, therole of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the
notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined thetemporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5,
prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that
PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expressionin endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separationfrom the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaf­
fected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These
results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, inmammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord,
but its function may differ among species.

1. Introduction
Notochord is an embryonic mesoderm-derived, transient, midline
structure common to all members of the phylum Chordata [1]. Two
important functions of the notochord during embryonic development
are serving as mechanical support for axis elongation and secreting
signaling cues to the surrounding tissues [1,2]. The vacuolization of thenotochord is required for the body axis elongation for zebrafish embryo[3], and notochord-derived signals are essential for patterning of tissues,
such as the neural tube [4,5], somite [6,7], vessels [8], and endoderm[9].
In vertebrates, the notochord arises from the axial mesodermal cells,

which constitute organizer or node [10]. Notochord precursors aremarked by the expression of several transcription factors, such as theforkhead domain genes Foxa1 and 2 [11], the T-box domain gene Bra­chyury/T/no tail [12,13], and the homeobox gene floating head(flh)/Noto [14,15]. Those cells rearrange their positions during gastru­
lation by mutual intercalation towards the midline, migrate anteriorly,
and then form an elongated stack of cells [16]. Notochord cells even­tually regress in later stages of development to become the nuclei pul­posi of the intervertebral discs [17]. During the cell rearrangement
process, notochord precursor cells are separated from surrounding tis­sue, such as paraxial mesoderm or endoderm [10], suggesting that celladhesion molecules may play an important role in this step.

* Corresponding author. Laboratory of Animal Regeneration Systemology, Department of Life Science, School of Agriculture, Meiji University, Kanagawa, 2148571, Japan.
E-mail address: inui_m@meiji.ac.jp (M. Inui).
https://doi.org/10.1016/j.bbrep.2021.101047
Received 2 February 2021; Received in revised form 25 May 2021; Accepted 6 June 2021
Available online 15 June 2021
2405-5808/© 2021 The Authors.
Published by Elsevier B.V. This is an(http://creativecommons.org/licenses/by-nc-nd/4.0/).

open

accessarticle

under

the

CC

BY-NC-ND

licenseK. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047

Indeed, in Xenopus laevis embryo, the cell adhesion molecule Axial
protocadherin (AXPC), an orthologue of mammalian Protocadherin-1(PCDH1), is expressed in notochord precursor cells and plays animportant role in the assembly of notochord cells [18,19]. In axpc
knockdown Xenopus embryos, the notochord is not properly formed, andthe elongation of the embryonic body axis is inhibited [19,20].
In the present study, we performed expression as well as functional
analyses under the hypothesis that PCDH1 has a conserved role inmammalian notochord formation.

2.5. Animals
Pcdh1 knockout mice were generated using the double nicking
approach of the CRISPR-Cas system, as previously described [22,23]. A
guide RNA pair was designed with the web based design tool ofgenomeengeneering.org and no possible off-target site was predicted(please note that the website is no longer available at the moment of thesubmission of this manuscript). D10ACas9 mRNA and guide RNAs tar­geting pcdh1 exon 2 were microinjected into mouse zygotes obtained bymating superovulated BDF1 females and BDF1 males. The pcdh1Δ11
allele was selected from the obtained G0 mice as a founder of a
PCDH1-deficient mouse line. The pcdh1Δ11/Δ11 mice used in this study
were backcrossed at least three generations to C57BL/6. Mice or em­
bryos were genotyped by a PCR analysis using genotyping primers(shown in Supplemental Table 1) and restriction enzyme treatment for
genomic DNA extracted from the tail tips of pups or yolk sac of embryos.
Three-to eight-week-old mice were measured for their body weight, andeight-week-old mice were measured for their body length. The mice
were housed under controlled environmental conditions with free accessto water and food. Light was provided between 07:00 and 19:00.
The protocols for animal experiments were approved by the Animal
Care and Use Committee of the National Research Institute for Child
Health and Development (Permission Number: A2004-003-C09) andMeiji University (Permission Numbers: MUIACUC-20-113).

2. Materials and Methods
2.1. Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from embryonic day (E) 7.5, 8.5, 9.5, and11.5 whole embryos of ICR strain using ISOGEN-LS (NIPONGENE,
Tokyo, Japan), according to the manufacturer’s protocol. Total RNA wasreverse-transcribed with oligod(T) primers and SuperScript II Reverse
Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Quanti­tative PCR (qPCR) were performed using Power SYBR® Green MasterMix (Thermo Fisher Scientific). Primer sequences are shown in Sup­
plemental Table 1.
2.2. Whole-mount in situ hybridization (WISH)

2.6. Protein extraction from adult mice

Probes to detect pcdh1 (NM_029357.3), sonic hedgehog (shh)(NM_009170.3), and platelet/endothelial cell adhesion molecule-1 (pecam1) (NM_001032378) mRNAs were designed. Three probes with nonoverlapping sequences and about 1 kb length were designed for pcdh1
mRNA (Fig. S2). Two probes for pecam-1 and one probe for shh were also
designed. Multiple probes were used as mixtures. Each probe sequence
was cloned into TOPO TA vector (Thermo Fisher Scientific).
Digoxygenin-labeled RNA probes were transcribed with T3 or T7 RNApolymerase. WISH was performed on E9.5, 10.5, and 11.5 ICR embryos,
E9.5 and 10.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos, as previously
described (Shimizu et al., 2009). Primer sequences are shown in Sup­
plemental Table 1.

Small piece of liver tissues was isolated from adult male pcdh1+/+ or
pcdh1Δ11/Δ11 mice. The flash-frozen liver tissues were crushed into a
powder using homogenizer Shakeman-3 (bio medical science, Tokyo,
Japan). Lysis buffer (50 mM HEPES [pH 7.8], 200 mM NaCl, 5 mM
EDTA, 1% NP40, 5% Glycerol), 1 μl of 1 M DTT, and 20 μl 50x Protease
Inhibitor Cocktail (Roche, Basel, Switzerland) were added to the tissue
powder. The solution was homogenized, and then the supernatant wasmeasured for the protein concentration using a Bio-rad DC protein assay
kit (#500–0111, Bio-Rad, Hercules, CA, USA) for equal loading.
2.7. Western blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed using a 4%–12% precast acrylamide gel (Thermo
Fisher Scientific). Extracts with equal protein amounts were loaded into
each well. Proteins were transferred to an Immobilon-P (Merck Milli­pore, Burlington, MA, USA) using a Trans-Blot Sd Semi-Dry Transfer Cell
(Bio-rad). Membranes were blocked with Blocking One (Nacalai Tesque,
Kyoto, USA). The following primary antibodies were used: anti-PCDH1(#H00005097-M01, 1:500; Abnova) and anti-GAPDH (#MAB375,
1:1000; Merk Millipore). Horseradish peroxidase conjugated anti-mouse
IgG (1:2000; Sigma-Aldrich) was used as a secondary antibody. Signals
were detected using Amersham ECL Western blotting Detection Re­
agents (GE Healthcare, Chicago, IL, USA) or SuperSignal West Femto
Maximum Sensitivity Substrate (Thermo Fisher Scientific).

2.3. Hematoxylin-eosin (HE) staining
E8.5 or E10.5 ICR embryos were fixed with 4% paraformaldehyde(PFA) for 2 h, cryoprotected by infiltration of 30% sucrose in phosphatebuffered saline (PBS) overnight and infiltration of 30% sucrose in PBS
and OCT (1:1) for 1 h, and embedded in OCT embedding compound.
Transverse sections (20 μm) were obtained using a cryostat. E9.5
pcdh1+/+ and pcdh1Δ11/Δ11 embryos were dissected, fixed with 4% PFA,
dehydrated, cleared, and embedded into paraffin wax. Transverse sec­
tions (14 μm) were obtained using a microtome, de-waxed, hydrated,and stained with hematoxylin (Wako, Osaka, Japan) and eosin (Wako).
Stained sections were dehydrated and mounted with Mount-Quick(OHMICH, Gunma, Japan).

3. Results

2.4. Immunofluorescence staining

3.1. Pcdh1 mRNA expression in mouse embryos

Immunofluorescence staining was performed as previously described[21]. In brief, cryosections were made as described as above, washedwith PBS, and blocked by PBS with 10% fetal bovine serum for 1 h.
Primary antibodies were diluted in PBS with 1% fetal bovine serum andincubated at 4 ◦ C overnight. Primary antibodies were anti-PCDH1(#H00005097-M01, 1:250; abnova, Taipei City, Taiwan), anti-CD31(#ab28364, 1:250; abcam, Cambridge, UK), and anti-Laminin(#L9393, 1:500; Sigma-Aldrich, St. Louis, MO, USA). Slides were cov­
erslipped with Fluoroshield Mounting Medium (ImmunoBioScience
Corp., Mukilteo, WA, USA).

First, to explore the temporal expression of pcdh1, we performed RTPCR on E7.5, E8.5, 9.5, and 10.5 embryos. Pcdh1 has several isoforms(see Fig. S1A), and we designed a primer pair in exons 2 and 3 shared by
all of the isoforms (Fig. S1B). As a result, we detected pcdh1 mRNA in all
the stages examined (Fig. 1A). Previously, pcdh1 mRNA was reported to
be expressed from E9.5 [24], but in our set-up, the mRNA was detected
as early as E7.5, prior to the stage when notochord cells detach fromother tissue at the surface of the embryo [10]. We also detected theexpression of pcdh1 mRNA in these stages with an independent primer
2

K. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047

Fig. 1. The pcdh1 mRNA expression in mouse embryos. (A) An RT-PCR analysis showing the expression of pcdh1 mRNA in mouse embryo. β-actin served as aloading control. RT, reverse transcription. (B, C) Whole-mount in situ hybridization using sense and antisense probes of pcdh1 with E9.5 embryos. (D–O) Wholemount in situ hybridization using antisense probes of pcdh1, shh, and pecam-1 with E9.5, 10.5 and 11.5 embryos. Black arrowhead: intersomitic vessel. White
arrowhead: notochord. Scale bar: 0.5 mm. The experiments were performed at least twice, and representative results are shown.

set (Fig. S1B), indicating that the mRNA was expressed at stage of
notochord formation (Fig. S2).
Next, to examine the spatial expression of pcdh1 mRNA in mouseembryos, we adopted the WISH approach. We designed three sense/
antisense probes that covered most of the pcdh1 mRNA sequence (iso­form NM_029357.3, Fig. S1B) and used them as a mixed probe (see theMaterials and Methods for details). First, to confirm the specificity of our
pcdh1 probes, WISH was performed with sense and antisense probes inE9.5 embryos. Tissue-specific signals were detected with the mix of thethree antisense probes (Fig. 1B) but not with the mix of the three sense
probes (Fig. 1C). We concluded that our mixed antisense probes detected
pcdh1 mRNA specifically and designated the mix of the three antisense
probes as “pcdh1 antisense” and that of the three sense probes as “pcdh1
sense”.
With the specificity of the probes confirmed, we examined the spatial
expression pattern of pcdh1 mRNA using embryos at various stages
3

K. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047(E9.5, 10.5, and 11.5). As we were focusing on the expression in noto­
chord, shh, a notochord marker gene, was included as a control. Since
the pcdh1 expression in blood vessels had been previously reported,
pecam-1, an endothelial cell marker gene, was also included. As shown in
Fig. 1D–G, pcdh1 expression was seen as lines on both sides of the dorsal
midline (neural tube) as well as in the intersomitic region in all stages
examined (Fig. 1D–G, black arrowheads). At E9.5 and 10.5, a mesh-likepattern of signals was detected at the surface of limb buds or branchialarches (Fig. 1D and E). These expression patterns coincided with those of
pecam-1 (Fig. 1L–O), indicating that the pcdh1 mRNA was expressed inblood vessels at these stages. Although the expression of shh in noto­
chord and the floor plate was clearly detected in E9.5–11.5 embryos(Fig. 1H–K, white arrowheads), pcdh1 mRNA was not detected in
notochord in these WISH experiments (Fig. 1D–G).

3.2. PCDH1 protein expression in mouse embryos
We also examined the expression of PCDH1 protein with immuno­
fluorescence using E10.5 embryo sections. PECAM-1 was used as anendothelial cell marker. As a result, PCDH1 expression was detected inblood vessels, such as the peri-neural vascular plexus (Fig. 2B), whichcoincided with the PECAM-1 expression (Fig. 2C), confirming its
expression in endothelial cells. In addition to the expression in the bloodvessels, we detected a PCDH1 signal at the midline of the embryo, where
PECAM-1 was not detected (Fig. 2B and C arrowheads). This signal
corresponds with the position of the notochord structure whencompared with the HE staining (Fig. 2A). These results indicate that
PCDH1 is expressed in both blood vessels and notochord in mouse em­bryos. As the RT-PCR analysis showed the pcdh1 mRNA expression at
E8.5, when the notochord formation occurs, next we examined if thePCDH1 protein is expressed in the notochord tissue. As the result, weFig. 2. The PCDH1 protein expression in mouse embryos. (A) HE staining of transverse sections of E10.5 embryo. (B) Immunofluorescence staining for PCDH1
with transverse sections of E10.5 embryo. (C) Immunofluorescence staining for PECAM-1 with transverse sections of E10.5 embryo. DAPI staining (blue) shows
nuclear positions. Note that the sections of A-C are consecutively taken from the same sample. White arrowhead: PCDH1-positive and PECAM-1-negative structure.
Diagrams illustrating the positions of notochord and vasculature are shown in Figure S3. (D) HE staining of transverse sections of E8.5 embryos. (E) Immunoflu­
orescence staining for PCDH1 with transverse sections of E8.5 embryos. DAPI staining (blue) shows nuclear positions. Note that the sections of D and E areconsecutively taken from the same sample. no: notochord. nt: neural tube. pnvp: peri-neural vessel plexus. En: endodermal layer. Scale bar: 20 μm. Three embryos
were used for each experiment. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)4

K. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047

3.3. Generation of pcdh1 knockout mice using the CRISPR-Cas9 systemhave detected PCDH1 in a part of forming notochord cells as well assurrounding endoderm layer (Fig. 2D and E). The reason why wedetected PCDH1 protein but not pcdh1 mRNA is probably due to the sizeand location of the mammalian notochord. The notochord in mouseembryos is a very thin tissue embedded in the middle of the embryo
body, so the detection of its gene expression is more difficult with wholemount analysis than with sections.

To reveal the function of PCDH1 in notochord, we generated pcdh1
gene knockout mice using the CRISPR-Cas9 system. We selected the
double-nicking approach, which uses D10ACas9 and two gRNAs [23], tominimize the off-target effect. We designed a gRNA pair (target 1 andtarget 2) (Fig. 3A) in exon2 of the pcdh1 gene, which is common to all ofFig. 3. Generation of pcdh1 knockout mice using the CRISPR-Cas9 system. (A) A schematic drawing showing the position and sequences of gRNAs targetingexon2 of pcdh1 gene. Exons (black boxes) and introns (black lines) indicate the structure of one of seven transcription variants of pcdh1 (NM_029357.3). Targeting
sequences and PAM sequences of gRNAs are indicated with underline and a gray background, respectively. (B) Genomic sequences around the targeting site of thewild-type (WT) and Δ11 alleles. Targeting sequences of gRNAs are indicated with underline. Dashes: deleted bases. (C) Gross appearance of pcdh1+/+ and pcdh1Δ11/
Δ11
female mice at 8 weeks old. (D) Western blotting to detect PCDH1 protein in liver tissue of pcdh1+/+ and pcdh1Δ11/Δ11 adult mice. GAPDH serves as a loading
control. (E) Body weight of pcdh1+/+ and pcdh1Δ11/Δ11 mice at 3–8 weeks old. At least three mice were measured for each genotype. Data represent the mean ± SE. A
t-test was used for the statistical analyses (*p < 0.05, **p < 0.01, and ***p < 0.001). (F) Body length of pcdh1+/+ and pcdh1Δ11/Δ11 adult male and female mice at 8
weeks old. At least three mice were measured for each genotype. Box plots show the median and 25-75th percentiles (middle line and box, respectively). Whiskers
extend to the maximum and minimum data points. A t-test was used for the statistical analyses (*p < 0.05). N.S., not significant
5

K. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047

the reported isoforms (Fig. S1A). We performed zygote microinjection
and obtained a pup with an allele with an 11-base deletion 138 bases
downstream from the start codon of the pcdh1 gene, which we hereafter
call the pcdh1Δ11 allele (Fig. 3B). We successfully established a mouseline from this founder mouse.
The mice with homozygote pcdh1Δ11 allele (pcdh1Δ11/Δ11) are viable,born at a Mendelian ratio, and fertile (Fig. 3C). We performed Western
blotting to confirm whether or not PCDH1 protein was depleted in
pcdh1Δ11/Δ11 mice. Total protein was extracted from the liver tissues of
pcdh1+/+ and pcdh1Δ11/Δ11 adult mice. The Western blotting analysis
using an anti-PCDH1 antibody detected 2 bands around 150 kDa in the
pcdh1+/+ (Fig. 3D) that were absent in pcdh1Δ11/Δ11 mice (Fig. 3D). Since
the Δ11 mutation is in exon2 which is common in all of the reported
isoforms, all PCDH1 proteins are expected to be depleted in pcdh1Δ11/Δ11
embryos and mice. We also confirmed that the PCDH1 protein was notdetected by immunofluorescent in the notochord and blood vessels of
pcdh1Δ11/Δ11 embryo (Fig. 4G and H). These results indicated that we
had successfully generated a pcdh1 knockout mouse line.
We measured the body weight of pcdh1+/+ and pcdh1Δ11/Δ11 mice atthree to eight weeks old. As a result, the body weight of pcdh1Δ11/Δ11
female mice was significantly lower at all ages examined than wild typecontrol (Fig. 3E), while no significant difference was observed in malemice (Fig. 3E). The body length of pcdh1Δ11/Δ11 of female mice, but notmales, was also significantly lower at eight weeks old than wild typecontrol (Fig. 3F).
3.4. Phenotypic analyses of PCDH1-deficient mice
Having generated PCDH1-deficient mice, we examined its notochordformation. The gross appearance of pcdh1Δ11/Δ11 embryo was indistin­
guishable from wild-type embryos at E9.5, indicating that the axis
elongation process had not been affected by the loss of PCDH1 (Fig. 4A
and B). A cross section of embryos at the level of the heart showed thatthe notochord had formed normally in pcdh1Δ11/Δ11 embryos (Fig. 4C
and D). WISH analysis with shh probe revealed that the notochord
structure along embryonic anterior-posterior axis was indistinguishable
between pcdh1Δ11/Δ11 and wild-type embryos (Fig. 4E and F). Immu­
nofluorescence of Laminin showed that there was no difference inbasement membrane formation of notochord of pcdh1Δ11/Δ11 and wildtype embryos (Fig. 4I and J). These results indicate that PCDH1 isdispensable for the induction of notochord and its segregation fromsurrounding tissue. The expression level of key transcription factors for
notochord such as T or Foxa1 was similar in pcdh1Δ11/Δ11 and wild-type
embryos (Fig. 4K).
Next, to examine the notochord function as a signaling center, weobserved the surrounding tissue, such as the neural tube, dorsal aorta,
and foregut of E10.5 pcdh1Δ11/Δ11 embryos and the spinal code orvertebrae of E13.5 pcdh1Δ11/Δ11 embryos, and found that those tissues
had formed similarly to their formation in pcdh1+/+ embryos(Fig. 5A–H). These results suggested that PCDH1 was dispensable for
notochord to function as a signal source for the surrounding tissues.
To investigate the effect of pcdh1 knockout on blood vessels, we
compared the vessel patterns and morphology between pcdh1+/+ andpcdh1Δ11/Δ11 embryos. We performed WISH using pecam-1 probes at
E10.5 and found that the major vascular patterns, including intersomitic
vessels, did not differ markedly between pcdh1+/+ and pcdh1Δ11/
Δ11
embryos (Figs. S4A and B). In addition, we performed immunofluo­
rescence staining for PECAM-1 in E13.5 pcdh1+/+ and pcdh1Δ11/Δ11
embryos. The expression of PECAM-1 in endothelial cells of pcdh1Δ11/Δ11
embryo was similar to that of pcdh1+/+ embryos (Figs. S4C and D).
These results indicate that PCDH1 is dispensable for blood vessel for­mation and its embryonic patterning.

Fig. 4. Notochord of pcdh1 knockout mice. (A, B) Gross appearance of E9.5
pcdh1+/+ and pcdh1Δ11/Δ11 embryos. Scale bars: 0.5 mm. (C, D) HE staining of
the cross-sections of E9.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. Scale bars: 20
μm. no: notochord. nt: neural tube. (E, F) In situ hybridization of E9.5 pcdh1+/+
and pcdh1Δ11/Δ11 embryos with shh probe. (G–J) Immunofluorescence of crosssections of E10.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. (K) qPCR analysis of
E11.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. A t-test was used for the statisticalanalyses (*p < 0.05). Four embryos were used for each genotype in eachexperiment. N.S., not significant

4. Discussion
In this study, we revealed that pcdh1 mRNA was expressed from E7.5,
6

K. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047

Fig. 5. Tissues surrounding notochord of pcdh1 knockout mice. (A–H) HE staining of the cross-sections of pcdh1+/+ and pcdh1Δ11/Δ11 embryos. (A–D) E10.5
embryos. Scale bars: 50 μm. (E–H) E13.5 embryos. Scale bars: 200 μm. cv: cardinal vein. da: dorsal aorta. es: esophagus. fg; foregut. lu: lung. no: notochord. nt: neuraltube. sc: spinal code. vb: vertebra. Three embryos were used for each genotype in each experiment.

prior to the stage when notochord cells detach from other tissue, and
PCDH1 protein was expressed in the notochord of mouse embryo fromthe stage in when notochord cells detach from other tissue. The pcdh1
knockout mice were viable and fertile with a reduced body length andweight in females. The reason for the female-specific reduction of bodysize is unclear at the moment, and a further study will be required toclarify the underlying mechanism. In pcdh1Δ11/Δ11 embryos, structureand gene expression of notochord and major vascular patterns werenormal, indicating that PCDH1 is dispensable for notochord and bloodvessel formation.

4.2. The role of the pcdh1 gene in mammalian development
As the deletion in pcdh1Δ11/Δ11 mice is located in exon 2, which isshared among all seven isoforms of pcdh1, the proteins of all isoforms are
expected to be depleted in pcdh1Δ11/Δ11 mice. In fact, we detected two
protein bands in our Western blotting analysis, both of which weredepleted in pcdh1Δ11/Δ11 mice (Fig. 3D). In Syrian hamsters, pcdh1
knockout results in animals that are viable, fertile, and with no majororgan abnormalities [26]. The knockout mouse phenotype in this study
is consistent with this result.
The notochord of PCDH1-deficient mice was formed in the normal
position. These phenotypes show clear differences compared to thephenotype of AXPC (PCDH1 orthologue)- knockdown Xenopus embryos,
where the notochord is not properly formed and axis elongation is
inhibited [19]. Two reasons may be responsible for this discrepancy: thestructural differences in amphibian and mammalian notochord, or afunctional redundant gene that might exist in the mammalian genome.
In amniotes, the notochord consists of a thin, acellular sheath covering
inner vacuolated cells [27,28]. In contrast, in lower vertebrates, thenotochord is a multilayered structure in which large vacuolated cells are
surrounded by an epithelial sheath, which is further encapsulated in
extracellular matrix layers [3,29]. The diameter of the notochord inlower vertebrates is much larger than that of mammalians, suggesting alarger contribution to mechanical support in axis formation. Alterna­
tively, PCDH1 may work redundantly with other adhesion molecules
expressed in notochord, such as P-cadherin [30]. PCDH11, which forms
a heterodimer with PCDH1, may also play some role with PCDH1 [31].
CDH1 and CDH2 are also expressed in notochord and endoderm layer,
respectively, during the notochord formation [32]. It would be inter­esting to analyze mice with multiple knockout of PCDH1 and thesecandidate genes in a future study. Since mammalian notochord has alimited cell number, and no immortalized cell line is available, thewholistic view of the adhesion molecules expressed in the notochord

4.1. Expression pattern of mammalian pcdh1
The mouse pcdh1 gene is located on chromosome 18. Seven isoforms,
including Refseq (NM_029357.3), are registered as mouse pcdh1 mRNAin the NCBI Gene data base (Fig. S1A). In a previous study, Redies et al.
detected a relatively low expression of short and long isoforms of pcdh1
from E9.5 using isoform-specific primers [24]. In this study, we detectedsimilar levels of pcdh1 mRNA in E7.5 to E10.5. The difference between
our results and the previous report might be due to the primer pairs weused, which detect all seven isoforms (Fig. S1B). By immunofluores­
cence, we found that PCDH1 protein in notochord, endoderm layer andblood vessel (Fig. 2). While the expression of PCDH1 (AXPC) in noto­
chord is conserved in amphibians and mammals, its expression domain
is broader in mouse embryos ([19] and this study). The pcdh1 gene has
isoforms with different transcription start sites (Fig. S1A). These iso­forms may have different transcription regulatory region, which may
account for the differences in the pcdh1 expression between amphibians
and mammals. Broad expression domain of pcdh1, including notochord,endoderm, and endothelial cells, is confirmed also in the published dataof single cell transcriptome analysis of E8.25 embryo [25].

7K. Fukunaga et al.

Biochemistry and Biophysics Reports 27 (2021) 101047

remains unclear. However, newly developed technologies, such as atranscriptomics analysis [33] or induction of notochord-like cells frompluripotent stem cells [34], may provide a comprehensive understand­ing of notochord adhesion molecules.
In conclusion, we identified the conserved expression of PCDH1 inmammalian notochord. Phenotypes of our pcdh1Δ11/Δ11 mice suggested
that the role of PCDH1 might differ among vertebrate species. It will beinteresting to examine the role of PCDH1 orthologues in other vertebrateclades as well as in Cephalochordata or Urochordata in a future study.

[10] S. Balmer, S. Nowotschin, A.K. Hadjantonakis, Notochord morphogenesis in mice:current understanding and open questions, Dev. Dynam. 245 (2016) 547–557.
[11] H. Sasaki, B.L.M. Hogan, Differential expression of multiple fork head related genes
during gastrulation and axial pattern formation in the mouse embryo,
Development 118 (1993) 47–59.
[12] P. Chesley, Development of the short-tailed mutant in the house mouse, J. Exp.
Zool. 70 (1935) 429–459.
[13] B.G. Herrmann, S. Labeit, A. Poustka, T.R. King, H. Lehrach, Cloning of the T gene
required in mesoderm formation in the mouse, Nature 343 (1990) 617–622.
[14] H. Ben Abdelkhalek, A. Beckers, K. Schuster-Gossler, M.N. Pavlova, H. Burkhardt,
H. Lickert, J. Rossant, R. Reinhardt, L.C. Schalkwyk, I. Müller, et al., The mousehomeobox gene Not is required for caudal notochord development and affected by
the truncate mutation, Genes Dev. 18 (2004) 1725–1736.
[15] G. von Dassow, J.E. Schmidt, D. Kimelman, Induction of the Xenopus organizer:
expression and regulation of Xnot, a novel FGF and activin-regulated homeo box
gene, Genes Dev. 7 (1993) 355–366.
[16] D.L. Stemple, Structure and function of the notochord: an essential organ for
chordate development, Development 132 (2005) 2503–2512.
[17] Y. Nibu, D.S. José-Edwards, A. Di Gregorio, From notochord formation tohereditary chordoma: the many roles of Brachyury, BioMed Res. Int. 2013 (2013),
826435.
[18] H. Kuroda, H. Sakumoto, K. Kinoshita, M. Asashima, Changes in the adhesive
properties of dissociated and reaggregated Xenopus laevis embryo cells, Dev.
Growth Differ. 41 (1999) 283–291.
[19] H. Kuroda, M. Inui, K. Sugimoto, T. Hayata, M. Asashima, Axial protocadherin is amediator of prenotochord cell sorting in Xenopus, Dev. Biol. 244 (2002) 267–277.
[20] M.D. Yoder, B.M. Gumbiner, Axial protocadherin (AXPC) regulates cell fate duringnotochordal morphogenesis, Dev. Dynam. 240 (2011) 2495–2504.
[21] M. Inui, M. Tamano, T. Kato, S. Takada, CRISPR/Cas9-mediated simultaneous
knockout of Dmrt1 and Dmrt3 does not recapitulate the 46,XY gonadal dysgenesis
observed in 9p24.3 deletion patients, Biochem. Biophys. Reports 9 (2017)
238–244.
[22] M. Inui, M. Miyado, M. Igarashi, M. Tamano, A. Kubo, S. Yamashita, H. Asahara,
M. Fukami, S. Takada, Rapid generation of mouse models with defined point
mutations by the CRISPR/Cas9 system, Sci. Rep. 4 (2014) 5396.
[23] F.A. Ran, P.D. Hsu, C.-Y. Lin, J.S. Gootenberg, S. Konermann, A.E. Trevino, D.
A. Scott, A. Inoue, S. Matoba, Y. Zhang, et al., Double nicking by RNA-guided
CRISPR Cas9 for enhanced genome editing specificity, Cell 154 (2013) 1380–1389.
[24] C. Redies, J. Heyder, T. Kohoutek, K. Staes, F. Van Roy, Expression of
protocadherin-1 (Pcdh1) during mouse development, Dev. Dynam. 237 (2008)
2496–2505.
[25] X. Ibarra-Soria, W. Jawaid, B. Pijuan-Sala, V. Ladopoulos, A. Scialdone, D.J. Jörg,
R.C.V. Tyser, F.J. Calero-Nieto, C. Mulas, J. Nichols, et al., Defining murineorganogenesis at single-cell resolution reveals a role for the leukotriene pathway inregulating blood progenitor formation, Nat. Cell Biol. 20 (2018) 127–134.
[26] R.K. Jangra, A.S. Herbert, R. Li, L.T. Jae, L.M. Kleinfelter, M.M. Slough, S.L. Barker,
P. Guardado-Calvo, G. Román-Sosa, M.E. Dieterle, et al., Protocadherin-1 isessential for cell entry by New World hantaviruses, Nature 563 (2018) 559–563.
[27] K.-S. Choi, M.J. Cohn, B.D. Harfe, Identification of nucleus pulposus precursor cells
and notochordal remnants in the mouse: implications for disk degeneration andchordoma formation, Dev. Dynam. 237 (2008) 3953–3958.
[28] L. Ward, A.S.W. Pang, S.E. Evans, C.D. Stern, The role of the notochord in amniote
vertebral column segmentation, Dev. Biol. 439 (2018) 3–18.
[29] K. Ellis, B.D. Hoffman, M. Bagnat, The vacuole within: how cellular organization
dictates notochord function, BioArchitecture 3 (2013) 64–68.
[30] A. Nose, M. Takeichi, A novel cadherin cell adhesion molecule: its expression
patterns associated with implantation and organogenesis of mouse embryos, J. Cell
Biol. 103 (1986) 2649–2658.
[31] O.J. Harrison, J. Brasch, P.S. Katsamba, G. Ahlsen, A.J. Noble, H. Dan, R.
V. Sampogna, C.S. Potter, B. Carragher, B. Honig, et al., Family-wide structural andbiophysical analysis of binding interactions among non-clustered δ-protocadherins,
Cell Rep. 30 (2020) 2655–2671.
[32] S.R. Fausett, L.J. Brunet, J. Klingensmith, BMP antagonism by Noggin is required
in presumptive notochord cells for mammalian foregut morphogenesis, Dev. Biol.
391 (2014) 111–124.
[33] S.H. Peck, K.K. McKee, J.W. Tobias, N.R. Malhotra, B.D. Harfe, L.J. Smith, Wholetranscriptome analysis of notochord-derived cells during embryonic formation of
the nucleus pulposus, Sci. Rep. 7 (2017) 10692–10695.
[34] Y. Zhang, Z. Zhang, P. Chen, C.Y. Ma, C. Li, T.Y.K. Au, V. Tam, Y. Peng, R. Wu, K.M.
C. Cheung, et al., Directed differentiation of notochord-like and nucleus pulposuslike cells using human pluripotent stem cells, Cell Rep. 30 (2020) 2791–2806.

Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the Yamagishi Student Project Support
Program, the TTCK RAship from Yamagata prefectural government andthe City of Tsuruoka, and Taikichiro Mori Memorial Research Fund
(Graduate Student Researcher Development Grant) for K.F.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbrep.2021.101047.
Author contributions
H. K. and M. I. planned the study, and K. F., M. T. and N. H. per­formed the experiments. K. F., H. K., and M. I. planned and wrote themanuscript.
References[1] D. Corallo, V. Trapani, P. Bonaldo, The notochord: structure and functions, Cell.
Mol. Life Sci. 72 (2015) 2989–3008.
[2] D.S. Adams, R. Keller, M.A. Koehl, The mechanics of notochord elongation,straightening and stiffening in the embryo of Xenopus laevis, Development 110(1990) 115–130.
[3] K. Ellis, J. Bagwell, M. Bagnat, Notochord vacuoles are lysosome-related organelles
that function in Axis and spine morphogenesis, J. Cell Biol. 200 (2013) 667–679.
[4] K.F. Liem, T.M. Jessell, J. Briscoe, D. Acampora, M. Gulisano, A. Simone,
H. Amthor, D. Connolly, K. Patel, B. Brand-Saberi, et al., Regulation of the neuralpatterning activity of sonic hedgehog by secreted BMP inhibitors expressed bynotochord and somites, Development 127 (2000) 4855–4866.
[5] M. Placzek, The role of the notochord and floor plate in inductive interactions,Curr. Opin. Genet. Dev. 5 (1995) 499–506.
[6] C.-M. Fan, M. Tessier-Lavigne, Patterning of mammalian somites by surface
ectoderm and notochord: evidence for sclerotome induction by a hedgehog
homolog, Cell 79 (1994) 1175–1186.
[7] O. Pourquié, M. Coltey, M.A. Teillet, C. Ordahl, N.M. Le Douarin, Control of
dorsoventral patterning of somitic derivatives by notochord and floor plate, Proc.
Natl. Acad. Sci. U.S.A. 90 (1993) 5242–5246.
[8] B. Fouquet, B.M. Weinstein, F.C. Serluca, M.C. Fishman, Vessel patterning in theembryo of the zebrafish: guidance by notochord, Dev. Biol. 183 (1997) 37–48.
[9] O. Cleaver, P.A. Krieg, Notochord patterning of the endoderm, Dev. Biol. 234(2001) 1–12.

8