ポリコーム群遺伝子Pcgf1は腸管神経系のニューロンサブタイプ決定に寄与している

概要

Kobe University Repository : Kernel
PDF issue: 2024-05-02

Pcgf1 gene disruption reveals primary
involvement of epigenetic mechanism in neuronal
subtype specification in the enteric nervous
system

BAYU PRATAMA PUTRA
(Degree)
博士（医学）

(Date of Degree)
2023-09-25

(Resource Type)
doctoral thesis

(Report Number)
甲第8731号

(URL)
https://hdl.handle.net/20.500.14094/0100485915
※ 当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

（課程博士関係）

学 位 論 文 の 内 容 要 旨

Pcgf1 gene disruption reveals primary involvement of epigenetic
mechanism in neuronal subtype specification in the enteric nervous
system

ポリコーム群遺伝子 Pcgf1 は腸管神経系のニューロンサブタイプ決定に寄与している

神戸大学 大学院医学研究科・生理学・細胞生物学講座
神経分化・再生分野
（指導教員：榎本

秀樹

教授）

Bayu Pratama Putra

SUMMARY
Introduction

The enteric nervous system (ENS) regulates gut functions independently from the
central nervous system (CNS) by its highly autonomic neural circuit that integrates
diverse neuronal subtypes. Although each neuronal subtype has its differentiation
timeline, varying from as early as embryonic day (E) 8 to postnatal periods, the molecular
mechanism underlying the generation of neuronal diversity in the ENS remains largely
unknown. In the CNS, the fate of a given neuronal subtype is determined by actions of
specific transcription factors induced by the concentration gradient of BMPs (bone
morphogenetic proteins) and Shh (sonic hedgehog) in the neural tube. Transcriptional
control of neuronal subtype specification was also demonstrated in the ENS by several
mouse studies in which genetic disruption of transcription factors, Pbx3, Sox6, and Tbx3,
showed impaired differentiation of some enteric neuronal subtypes. However, the overall
mechanisms underlying neuronal subtype specification in the ENS have yet to be
elucidated.

Polycomb group proteins are the epigenetic modifiers that regulate cell
differentiation via chromatin compaction and modification, which are grouped into two
multiprotein complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). In
cell lineage determination, it is generally accepted that transcription factors activate or
silence gene expression, thereby determining the cell fate, and epigenetic mechanisms
successively stabilize the gene expression status. A recent study demonstrated that the
epigenetic mechanism could primarily regulate cell fate determination as shown by

disruption of the components of PRC1 which led to cell fate conversion in the
hematopoietic lineage.

PCGF1 (Polycomb group RING finger protein 1), a member of the PRC1, is
involved in the neural induction stage, stem cell renewal, and differentiation. During midgestation, Pcgf1 is highly expressed in the peripheral nervous system, including the dorsal
root ganglion, sympathetic and parasympathetic ganglia, and ENS. PCGF1-PRC1
complex is essential to shape Polycomb chromatin domains and initiate gene repression
during differentiation along with PRC2 recruitment.

In this study, we examined the biological function of PCGF1 in the development
and differentiation of the ENS by using a Pcgf1fl/fl; Phox2bCre/+ (Pcgf1 cKO), which is
Pcgf1 gene was selectively deleted in the autonomic-lineage of Phox2b+ cells. Phox2b is
expressed immediately after the specification of the autonomic lineage, including the
enteric, sympathetic, and parasympathetic ganglia in the developing peripheral nervous
system. We provide evidence on the possible epigenetic regulation of neuronal subtype
specification in the ENS.

Methods
Pcgf1 cKO was obtained by first generating Phox2b-Cre knockin mice
(Phox2bCre/+) using gene targeting followed by CRISPR/Cas9 genome editing. We
crossed Pcgf1-floxed mice with Phox2bCre/+ mice to obtain Pcgf1 cKO mice. To confirm
Pcgf1 ablation, we performed in-situ hybridization (ISH) in the embryonic mice (E13.5)
and BaseScope analysis in the myenteric plexus from the proximal colon of three weeks
old wild-type (WT) and Pcgf1 cKO mice.

We analyzed the mice phenotype by survival rate and body weight measurement
analysis of WT, Phox2bCre/+, and Pcgf1 cKO mice. In addition, we performed
gastrointestinal transit time and fecal water content analyses to examine gut motility
between Phox2bCre/+ and Pcgf1 cKO mice. We then performed immunohistochemistry
analysis on embryonic (E14.5), newborn (P0), and postnatal mice (three weeks old) to
investigate the basic formation of ENS, such as the gut colonization by ENS precursors
and total neuron numbers, along with enteric neuronal subtype differentiation by using
several subtype-specific antibodies (calretinin, NOS, calbindin, and somatostatin).

Results
To confirm whether the Pcgf1 gene was successfully disrupted in the autonomic
lineage of the Pcgf1 cKO mice, we conducted ISH analysis on E13.5 embryos using
riboprobes that hybridize the floxed genomic region of the Pcgf1 gene. This analysis
revealed the absence of Pcgf1 expression in the ENS and the sympathetic chain but not
in the dorsal root ganglion (DRG) in Pcgf1fl/fl; Phox2bCre/+ embryos, confirming the
disruption of the Pcgf1 gene in the autonomic nervous system. In the postnatal ENS,
Pcgf1 is almost ubiquitously expressed in enteric neurons. We also validated the absence
of Pcgf1 transcripts in the myenteric plexus of Pcgf1fl/fl; Phox2bCre/+ mice (3-week-old),
as shown by the BaseScope in-situ assay.
Pcgf1fl/fl; Phox2bCre/+ mice displayed small body size at the postnatal stage 3week-old. Therefore, we compared growth and mortality rates between Pcgf1fl/fl;
Phox2bCre/+, WT, and Phox2bCre/+ mice. Although both WT and Phox2bCre/+ had similar
survival rates, we observed slight growth retardation in Phox2bCre/+ mice compared to
WT at 3 (p = 0.035) and 8 weeks (p = < 0.0001). This observation led us to use Phox2bCre/+

as the control for the following experiments. Pcgf1fl/fl; Phox2bCre/+ mice displayed
significantly lower body weight than Phox2bCre/+ mice at 3 weeks (p = 0.002), although
they could finally catch up with Phox2bCre/+ mice’s growth at eight weeks. Only half of
Pcgf1fl/fl; Phox2bCre/+ mice (p = 0.0002) survived to adulthood. These data suggest that
the absence of PCGF1 impacts growth and survival rate.
Macroscopic examination of the gut showed no difference between 3-week-old
Phox2bCre/+ and Pcgf1fl/fl; Phox2bCre/+ mice, including gut length. Furthermore, we
evaluated the effect of Pcgf1 deletion on gut functions, such as motility and water
secretion. GITT was significantly longer in the Pcgf1fl/fl; Phox2bCre/+ than Phox2bCre/+
mice (p = 0.005). Slower transit was associated with lighter fecal weights (p = 0.0395)
and drier feces in Pcgf1fl/fl; Phox2bCre/+ mice (p = 0.0271). These observations indicate
that the absence of PCGF1 impairs gut motility at the early postnatal stage.
Impaired gut motility in Pcgf1fl/fl; Phox2bCre/+ mice suggested the presence of
anatomical deficits of the ENS. Therefore, we analyzed ENS development and
differentiation from embryonic to postnatal stages. The basic structure of the ENS is
formed by colonization of the entire gut by migrating ENS precursors, a process known
to occur from E9 to E14. Examination of the embryonic gut at E14.5 revealed that ENS
precursors fully colonized the colon in Phox2bCre/+ and Pcgf1fl/fl; Phox2bCre/+ mice.
Moreover, the numbers of ENS precursors, revealed by Phox2B staining, were
comparable in these mice. These data suggest that Pcgf1 deficiency does not influence
migration and proliferation of ENS precursors. In newborn mice (P0), the numbers of
myenteric neurons in the lower small intestine and colon were comparable between
Phox2bCre/+ and Pcgf1fl/fl; Phox2bCre/+ mice. These results suggest that PCGF1 deficiency
does not impact ENS development before birth.

At a postnatal stage (3-week-old), we examined the total number of neurons and
several neuronal subtypes in the myenteric and submucosal plexus of the Phox2bCre/+ and
Pcgf1fl/fl; Phox2bCre/+ mice. The staining of myenteric neurons with HuC/D, a panneuronal marker, showed no difference in all gut regions. Examination of calretinin- and
NOS1-expressing neurons, two major neuronal subtypes, also revealed no significant
difference between Pcgf1fl/fl; Phox2bCre/+and Phox2bCre/+ mice. However, we found that
the numbers of somatostatin-expressing (Sst+) neurons (descending/inhibitory neurons)
were significantly decreased in the ileum (p = 0.016) and proximal colon (p = 0.032)
of Pcgf1fl/fl; Phox2bCre/+ mice as compared to Phox2bCre/+ mice. In contrast, the numbers
of calbindin+ neurons (ascending/excitatory neurons) were increased (p = 0.008) in the
proximal colon of Pcgf1fl/fl; Phox2bCre/+ mice. These findings reveal the opposing effects
of PCGF1 in determining the numbers of Sst+ and calbindin+ neurons.
Examination of the total numbers of neurons in the submucosal plexus with panneuronal marker PGP9.5+ revealed no significant difference in all gut regions between
Pcgf1fl/fl; Phox2bCre/+ and Phox2bCre/+ mice. Although we did not observe the difference
in the calretinin+ neurons, we detected a significant decrease in the numbers of Sst +
neurons in the jejunum (p = 0.032), ileum (p = 0.032) and proximal colon (p = 0.04) in
Pcgf1fl/fl; Phox2bCre/+ mice. These analyses reveal that the Sst+ neurons are commonly
affected by Pcgf1 ablation in both the myenteric and submucosal plexus. Taken together,
the data suggest that PCGF1 plays a crucial role in neuronal subtype specification in the
ENS, especially Sst+ and calbindin+ neuron determinations, without affecting the total
neuronal numbers.

Conclusion
Although ENS precursor migration and enteric neurogenesis were largely
unaffected, neuronal differentiation was impaired in the Pcgf1-deficient mice, with the
number of Sst+ neurons were decreased in multiple gut regions. Notably, the decrease in
Sst+ neurons was associated with the corresponding increase in calbindin+ neurons in the
proximal colon. These findings suggest that neuronal subtype conversion may occur in
the absence of PCGF1 and that epigenetic mechanism is primarily involved in
specification of some enteric neuron subtypes.

神戸大学大学院医学（系）研究科（博士課程）
論文審査の結果の要旨
一

甲

第3320号

氏

名

受付番号

BAYU PRATAMA PUTRA

Pcgfl gene disruption reveals primary involvement of epigenetic
mechanism in neuronal subtype specification in the enteric
．．
論文題目
nervous system
Title of
Dissertation
ー
ヽ
ポリコ ー ム群遺伝子Pcgflは腸管神経系のニュ
ロンサブタイプ決
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審査委員
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