The dynamic right-to-left translocation of Cerl2 is involved in the regulation and termination of nodal activity in the mouse node

The determination of left-right body asymmetry in mouse embryos depends on the interplay of molecules In a highly sensitive structure, the node. Here, we show that the localization of Cerl2 protein does not correlate to its mRNA expression pattern, from 3-somite stage onwards. Instead, Cerl2 protein displays a nodal flow-dependent dynamic behavior that controls the activity of Nodal in the node, and the transmission of the laterality information to the left lateral plate mesoderm (LPM). Our results indicate that Cerl2 initially localizes and prevents the activation of Nodal genetic circuitry on the right side of the embryo, and later its right-to-left translocation shutdowns Nodal activity in the node. The consequent prolonged Nodal activity in the node by the absence of Cerl2 affects local Nodal expression and prolongs its expression in the LPM. Simultaneous genetic removal of both Nodal node inhibitors, Cerl2 and Lefty1, sustains even longer and bilateral his LPM expression.F.C.T.; IBB/CBME, LAinfo:eu-repo/semantics/publishedVersio

High-depth spatial transcriptome analysis by photo-isolation chemistry

光照射を用いた超高解像度な遺伝子解析技術の開発に成功 --組織内に潜むがん細胞の病理診断などに応用可能--. 京都大学プレスリリース. 2021-07-27.In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8, 000 genes were detected with 7 × 10⁴ unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions

Antagonism between Smad1 and Smad2 signaling determines the site of distal visceral endoderm formation in the mouse embryo

The anterior–posterior axis of the mouse embryo is established by formation of distal visceral endoderm (DVE) and its subsequent migration. The precise mechanism of DVE formation has remained unknown, however. Here we show that bone morphogenetic protein (BMP) signaling plays dual roles in DVE formation. BMP signaling is required at an early stage for differentiation of the primitive endoderm into the embryonic visceral endoderm (VE), whereas it inhibits DVE formation, restricting it to the distal region, at a later stage. A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal. DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal. An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling

Mechanism of the Establishment of Left-Right and Anteroposterior Axes in Mouse Embryo

私たちの体は外観は左右対称だが,臓器は顕著な左右非対称性を示す.例えば,胃や脾臓は左側に位置し,心臓や血管走行も左右非対称である.胚発生における左右の位置情報の乱れは,複雑心奇形につながることからも左右軸形成の重要性が理解される.このような左右非対称性の形成を制御する分子機序が明らかになってきたのは,1995年以降,左右非対称に発現する遺伝子が鶏,マウス,ツメガエルで報告されたことによる.私たちは,マウス胚で左右非対称に発現する遺伝子leftyを見出し,この遺伝子を糸口に左右軸形成の仕組みを明らかにして行った.本稿で詳述するように,左右軸形成ではNodal-Leftyの相互関係が大きな役割を果たすが,左右のみならず前後(頭尾)軸形成でもこの関係が軸の確立に必須であることが明らかになってきた(四つ足動物の頭尾は前後であるため,これを前後軸と記載する).この総説では,マウス胚における左右軸及び前後軸形成の機序を私たちの解析を中心に概説したい

The Role of Sulfated Glycosaminoglycans in Early Mouse Development

動物の胚発生はたった一個の受精卵から始まり，細胞分裂を繰り返しながら複雑かつ精巧な構造を造りあげていく．細胞自体が発生の主役を演じることは言うまでもないが，それらの間隙を埋める細胞外マトリクスという「場」がなければ，まともな発生は進行しない．細胞外マトリクスとは，細胞の外にあるタンパク質や糖，あるいは両者の結合体（糖タンパク質）などからなる，一群の分子のことを指す．当初はその物性から，機械的な強度や柔軟性を与える詰め物に過ぎないと考えられていた．しかし近年の研究により細胞の接着や増殖，さらにはシグナル伝達などに不可欠な存在であることがわかってきた．中でもヘパラン硫酸などの硫酸化グリコサミノグリカンは，分泌因子によるシグナル伝達に深く関わることが明らかになってきた1)2)．分泌性シグナル因子は，発生期の細胞が周囲や遠方の細胞に情報を伝達するための担い手であり，それらの交換によって細胞の分化や増殖が時空間的に制御される．硫酸化グリコサミノグリカンはそこにどのような役割を演じているのであろうか？ 本稿ではまず硫酸化グリコサミノグリカンのあらましから始め，分泌性シグナル因子との関わりを例示し，最後にマウスの初期発生における役割について我々の研究を中心に概説したい

Efficient establishment of EpiSC lines from single cells.

<p>(<b>A</b>) Establishment of EpiSCs from E6.5 mouse epiblasts cultured with the indicated combinations of Activin, Fgf2, and XAV939 (25 µM) for 3 days. The cells were observed by phase-contrast microscopy, stained for alkaline phosphatase (ALP) activity (blue), or subjected to immunochemical staining for Oct4 (brown). Scale bars, 200 µm. (<b>B</b>) Culture of dissociated epiblast cells on a feeder layer with Activin and Fgf2 as well as in the absence or presence of 25 µM XAV939 or 10 µM Y27632. Epiblast cells from one embryo were seeded in each well of a four-well plate. The cells were stained for alkaline phosphatase activity after culture for 5 days. (<b>C</b>) Enlarged view of each well in (B) showing EpiSC colonies. Scale bar, 200 µm. (<b>D</b>) Number of alkaline phosphatase–positive (black bar) and –negative (gray bar) colonies for cultures similar to those in (B). Data are means ± SEM for three independent cultures per condition.</p

EpiSCs cultured with XAV939 contribute to chimeric embryos.

<p>Mouse embryos at E6.5 were injected with EpiSCs that harbor a <i>lacZ</i> transgene (ROSA26-<i>lacZ</i>) and which had been cultured in the presence of 10 µM XAV939 for at least 10 passages and exposed to 10 µM Y27632 for 1 hours before dissociation into single cells. The embryos were cultured until the early somite stage and then subjected to X-gal staining (green). The embryo in (H) was also stained for alkaline phosphatase activity (purple). LacZ-positive descendants of EpiSCs were detected in the allantoic bud [asterisks in (A) and (C)], yolk sac [arrowhead in (A)], amnion [asterisk in (B)], hindgut endoderm [arrowheads in (C)], somitic mesoderm [arrowheads in (D)], surface ectoderm [arrowheads in (E)], neural plate [arrowheads in (F)], myocardium [arrowhead in (G)], and migrating PGCs [arrowheads in (H)]. Embryos are oriented with anterior to the left (A, B, and E), posterior to the top (C, D, and H), or anterior to the top (F and G). A distal view of somites (D), enlarged view of the headfold (E), dorsal view of the neural plate (F), and ventral view of the heart primordium (G) are shown. Scale bars, 100 µm.</p

β-Catenin is dispensable for the propagation of undifferentiated EpiSCs.

<p>(<b>A</b>) Two independent EpiSC lines (#3 and #4) derived from <i>β-catenin</i>^fl/fl epiblasts harboring the UBC<i>-CreER</i> transgene were incubated in the absence or presence of 100 nM tamoxifen (4OHT) for four days, after which <i>β-catenin</i> and <i>Cre</i> genotype was determined by PCR analysis. The positions of amplification products corresponding to the floxed (fl/fl) and null (Δ/Δ) alleles of <i>β-catenin</i> as well as to the <i>Cre</i> transgene are indicated. (<b>B</b>) Immunoblot analysis of β-catenin, E-cadherin, Oct4, and proliferating cell nuclear antigen (PCNA) in EpiSCs of line #3 in (A). (<b>C</b>) Quantitative RT-PCR analysis of the indicated transcripts in EpiSC lines as in (A). The amounts of <i>Nanog</i>, <i>Sox2</i>, <i>Oct4</i>, and <i>Foxd3</i> transcripts are shown relative to those in <i>β-catenin</i>^fl/fl cells of line #3. (<b>D</b>) Phase-contrast images and in situ hybridization analysis of the epiblast markers <i>Fgf5</i> and <i>Fgf8</i> in <i>β-catenin</i>^fl/fl or <i>β-catenin</i>^Δ/Δ EpiSC colonies of line #3. Scale bars, 200 µm. (<b>E</b>) In situ hybridization analysis of <i>T</i> and <i>Pax6</i> expression in <i>β-catenin</i>^fl/fl or <i>β-catenin</i>^Δ/Δ EpiSC colonies of line #3 cultured with or without 20 µM CHIR99021 for 6 h or 100 nM PD0325901 for 2 days. Scale bar, 200 µm. (<b>F</b>) Immunofluorescence analysis of E-cadherin, Oct4, Sox2, Nanog, and β-catenin in <i>β-catenin</i>^fl/fl or <i>β-catenin</i>^Δ/Δ EpiSC colonies of line #3. Scale bar, 200 µm.</p

Canonical Wnt/β-catenin signaling down-regulates the pluripotency of EpiSCs and perigastrulation embryos.

<p>(<b>A</b>) In situ hybridization analysis of EpiSCs cultured without Activin and Fgf2 as well as in the absence (control) or presence of 20 µM CHIR99021 for 6 hours. Probes are indicated in each pair of panels. Scale bar, 200 µm. (<b>B</b>) Quantitative reverse transcription and polymerase chain reaction (RT-PCR) analysis of EpiSCs cultured without Activin and Fgf2 as well as in the presence of 20 µM CHIR99021 for the indicated times. The amounts of <i>Nanog</i>, <i>Sox2</i>, <i>Oct4</i>, and <i>Foxd3</i> mRNAs are shown relative to those in untreated EpiSCs. (<b>C</b>) In situ hybridization analysis of E6.5 mouse embryos cultured in the absence (control) or presence of 40 µM CHIR99021 or 100 µM XAV939 for 6 hours. Lateral views of embryos are shown with anterior to the left. The PS region is marked with a line in the <i>T</i> panels. Asterisks in the <i>Sox2</i> panels indicate expression in the extraembryonic ectoderm. Scale bar, 100 µm.</p