神経誘導の分子シグナリング、神経板の形成

シュペーマンとマンゴールドによる両生類の原口背唇部の移植実験：誘導の発見とその概念の確立

シュペーマンHans SpemannとマンゴールドHilde Mangoldによるイモリを用いた移植実験で、原口背唇部（げんこうはいしんぶ）オーガナイザーと呼ばれる領域を胚の他の場所（胞胚腔など）に移植すると、周囲の組織に「誘導」をかけて二次の体軸が発生することが示されました。

  1. https://journals.biologists.com/dev/article/135/20/3321/19420/Induction-into-the-Hall-of-Fame-tracing-the　Ｆｉｇ．１

The term ‘organization centre’ was first introduced by Hans Spemann (Spemann and Mangold, 1924)

the blastopore lip of the early gastrula of the newt Triturus taeniatus had the ability to cause the formation of a full axis when transplanted onto the opposite side of a similarly staged embryo of Triturus cristatus, a different unpigmented species.

the 1924 report could discern between the host and the graft by pigment differences, which revealed the important point that the ectopic tissue developed from the host tissue.

On the nature and function of organizers Alfonso Martinez Arias* and Ben Steventon* Development. 2018 Mar 1; 145(5): dev159525. PMCID: PMC5868996

移植された組織自体は、二次軸の体の主要な部分すなわち神経系や筋肉などにはなっておらず、脊索や体節の一部（つまり、もともと自分がなる運命だった組織）といった中胚葉性の組織になっていました。つまり、本来、神経系や筋肉などになる予定ではなかった部分が、移植片によって発生運命を変更されたというわけです。この論文報告は、ある組織が他の組織に働きかけて発生の運命を変える「誘導」という現象を初めてしめした、発生学において非常に意義のあるものでした。

Über Induktion von Embryonalanlagen durch implantation artfremder Organisatoren (PDF　１９２４年のドイツ語の論文)

この１９２４年の論文の実験でつくられた標本（ガラスプレパラート）が残っているみたいで、異所的につくられた２次胚の組織が高解像度で見ることができます。

Hilde Mangold: Original microscope slides and records of the gastrula organizer experiments Wolfgang Driever a b, Jochen Holzschuh a, Luise Sommer c, Roland Nitschke b d, Angela Naumann b d, Jenny Elmer c 1, Peter Giere c　Cells & Development 28 February 2024, 203909

他の動物種でも同様の実験が行われて同様の結果が得られたことから、「誘導」という概念が確立しました。

  • the organizer is responsible for neural induction
  • the organizer dorsalizes the mesoderm

Induction into the Hall of Fame: tracing the lineage of Spemann’s organizer Richard Harland Author and article information　15 OCTOBER 2008 Development (2008) 135 (20): 3321–3323.

ちなみに、発生学の実験において背側の組織、腹側の組織が形成されたといったときに実際に指しているのは具体的にいうと

  • dorsal： neural plate, notochord and somites
  • ventral：blood and gut

ちなみに中胚葉（脊索）→外胚葉に働きかけて神経系が誘導されたわけですが、そのオーガナイザー（中胚葉）自身もそもそも「誘導」によって生じています（ニュークープの実験）。

当然生じる次なる疑問は、誘導を実際に担っている分子的な実体は何か？ということで、この分子実体の探求が、分子生物学の隆盛後の発生学の主要な潮流になりました。

 

アフリカツメガエルの神経誘導

Spemann-Mangold organizer and mesoderm induction　Makoto Asashima, Yumeko Satou-Kobayashi　Cells & Development Available online 1 February 2024, 203903

上の図では中胚葉が外胚葉に働きかけて神経誘導を行うことが模式的に示されています。

  1. Introducing the Spemann-Mangold organizer: experiments and insights that generated a key concept in developmental biology Int. J. Dev. Biol. 45: 1-11 (2001) PDF

Spemann and Mangold () provided the initial insight showing that transplantation of dorsal lip mesoderm of the gastrulating amphibian embryo would induce an ectopic secondary axis that included a central nervous system (CNS). This led to the view that neural inducers emanate from dorsal mesoderm, a region also called Spemann’s organizer.

neural induction may start very early in development with signals mediated by the β-Catenin pathway. 

Neural Induction in the Absence of Mesoderm: β-Catenin Dependent Expression of Secreted BMP Antagonists at the Blastula Stage in Xenopus Oliver Wessely,1 Eric Agius,1,2 Michael Oelgeschläger, Edgar M. Pera, and E. M. De Robertis* Dev Biol. 2001 Jun 1; 234(1): 161–173. doi: 10.1006/dbio.2001.0258 PMCID: PMC3039525 NIHMSID: NIHMS43280 PMID: 11356027

外胚葉の一部がどのようなシグナルによって神経板になるのかについては、別記事にまとめました。

  1. 神経板の形成：神経誘導

頭部形成因子：Ｗｎｔアンタゴニスト

神経発生に関与するシグナル分子はなかなか多彩で、ＢＭＰシグナリングだけでなくＷｎｔシグナリングも重要な役割を担っています。

Wnt antagonists, such as Cerberus (also inhibiting BMP and Nodal), Frizzled-related protein B (Frzb), and Dickkopf (Dkk), promote anterior neural development (Bouwmeester et al., 1996; Piccolo et al., 1999; Wang et al., 1997; Glinka et al., 1998).

Spemann-Mangold organizer and mesoderm induction　Makoto Asashima, Yumeko Satou-Kobayashi　Cells & Development Available online 1 February 2024, 203903

https://www.sciencedirect.com/science/article/pii/S2667290124000044

  1. Head organizer: Cerberus and IGF cooperate in brain induction in Xenopus embryos agmur Azbazdar a, Edgar M. Pera b, Edward M. De Robertis a Cells & Development Available online 16 December 2023, 203897 Spemann later found that early dorsal blastopore lips induced heads and late organizers trunk-tail structures. Identifying region-specific organizer signals has been a driving force in the progress of animal biology. Head induction in the absence of trunk is designated archencephalic differentiation. Two specific head inducers, Cerberus and Insulin-like growth factors (IGFs), that induce archencephalic brain but not trunk-tail structures have been described previously.
  2. Neural and Head Induction by Insulin-like Growth Factor Signals Edgar M. Pera, Oliver Wessely, Su-Yu Li, E.M. De Robertis Developmental Cell Volume 1, Issue 5, November 2001, Pages 655-665 

尾部の神経誘導因子

Ｗｎｔシグナルのアンタゴニストが頭部を形成する活性があったことの裏返しで、Ｗｎｔシグナルは尾部の神経系の形成に関与しています。また、ＦＧＦやＲＡ（レチノイン酸）も尾部の神経系の形成に関与しています。

FGF, RA, and Wnt signaling are required for posteriorization of neural tissues (Cox and Hemmati-Brivanlou, 1995; Kengaku and Okamoto, 1995; Lamb and Harland, 1995; Durston et al., 1989; Sive et al., 1990; Blumberg et al., 1997; McGrew et al., 1995; Kiecker and Niehrs, 2001)

Spemann-Mangold organizer and mesoderm induction　Makoto Asashima, Yumeko Satou-Kobayashi　Cells & Development Available online 1 February 2024, 203903

https://www.sciencedirect.com/science/article/pii/S2667290124000044

上の論文の図では、神経誘導因子、頭部、尾部それぞれで働くシグナル経路がまとめられていてわかりやすいと思います。しかし、中胚葉誘導因子や神経誘導因子（シュペーマンのオーガナイザー分子）

神経板の分子マーカーSox2発現のメカニズム

  1. Sex-determining region Y-related HMG box 2 (SOX2)
  2. High-mobility group (HMG) box transcription factors

神経板が誘導される過程で働く分子シグナリングはかなり複雑のようです。下の論文はかなり詳細に分子シグナリングを報告して、その当時の既知の情報にもとづいた仮説（モデル）を提示しています。

  • the earliest definitive marker for the neural plate is the transcription factor Sox2.
  • N2, the earliest enhancer of Sox2
  • fibroblast growth factor 8 (FGF8)により誘導されるpre-neural genes ：Sox3, ERNI, , Churchill,Geminin （これらの遺伝子発現は、Sox2遺伝子発現に先行する）
  • FGF activity induces both ERNI [18,36] and Geminin (this study) in the epiblast.
  • Geminin binds to the chromoshadow-binding domain of Brm, displacing HP1α (Figure 4G).
  • the interaction of ERNI with Geminin recruits the transcriptional repressor HP1γ, thus continuing to prevent premature expression of Sox2 in the epiblast (Figure 7E).
  • ＦGF is required for both mesodermal [51–54] and neural induction [36,55,56]. 同じシグナルを受けて異なる分化をとげるメカニズムは、受けて側のタイミングや場所の違いによると考えられる
  • BERT is up-regulated within the neural plate, where it binds to both ERNI and Geminin and displaces ERNI-HP1γ complexes away from Brm, freeing the latter to activate N2 and thus Sox2 expression (Figure 9M).
  • We propose that BERT disrupts the interaction between Geminin and ERNI, displacing HP1γ from the N2 enhancer and thus allowing Geminin/Brahma(Brm) to induce Sox2 expression.（Ｆｉｇ．９Ｍ）
  • FGF signaling activates ERNI as well as Sox3 and Geminin expression in the epiblast.
  • BERTはＦＧＦで誘導されず、BMP antagonistsでも、 既知のどんな組み合わせのシグナル分子でも誘導されない。つまり、ＢＥＲＴが活性化される仕組みはまだ不明であり将来の課題である。

A Mechanism Regulating the Onset of Sox2 Expression in the Embryonic Neural Plate 2008年　https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0060002

上の論文では神経板の分子マーカー転写因子Soｘ２の発現制御が論じられていました。下の論文のアフリカツメガエルの胚のin situハイブリダイゼーションによる解析で、Sox2が神経板を見事に染めています。

Molecular Biology of the CellVol. 18, No. 6ArticlesFree Access The Activity of Pax3 and Zic1 Regulates Three Distinct Cell Fates at the Neural Plate Border This is the final version – click for previous version Chang-Soo Hong, and Jean-Pierre Saint-Jeannet Marianne Bronner-Fraser, Monitoring Editor Published Online:4 Apr 2007　https://doi.org/10.1091/mbc.e06-11-1047

分子シグナリング

  • the neural inducers, Noggin, Chordin, and Follistatin  emanate from Spemann’s organizer.3,4,5
  • Noggin, Chordin, and Follistatin directly bind to bone morphogenetic proteins, namely, BMP2/4/7, in the extracellular space and act as antagonists to block these BMPs from binding to the BMP receptor.6
  • the blockade of BMP signaling inhibits the phosphorylation of the carboxyl-terminal serine residues of the Smad1 protein, which is an intracellular mediator of the BMP signal, preventing the downstream genes of BMP signals from being activated.
  • the inhibition of the BMP signal induces the expression of a series of transcription factors, which in turn activate the downstream transcriptional network to further promote neural differentiation.
  • Fibroblast growth factors (FGFs) also have neural inducing activity.7,8,9
  • FGF promotes the phosphorylation of the intermediate linker domain of the Smad1 protein, instead of its carboxyl-terminal domain, and restricts the Smad1 activity.7
  • FGF directly induces the neural genes.8,9
  • the combination of BMP inhibitors and FGF is essential for directing naive cells toward the neural fate.
  • In mouse embryos, Chordin and Noggin homologues emanate from the node, or the anterior portion of the primitive streak, and t
  • Chordin/Noggin double mutants exhibit severe forebrain malformation at early embryonic stages,11,12 but the development of the posterior nervous system in the Chordin/Noggin double mutant mice is relatively normal （両生類のように神経誘導すべてが抑制されるのとは事情が異なる。
  • 哺乳類では、the anterior and posterior neural cells are already separated at the epiblast stage, and this differentiation progresses independently.13,14

 

  1. Reorganizing the Organizer 75 Years On M.Angela Nieto Cell VOLUME 98, ISSUE 4, P417-425, AUGUST 20, 1999 https://www.cell.com/fulltext/S0092-8674(00)81971-6
  2. The Organizer and Its Signaling in Embryonic Development Vijay Kumar,1 Soochul Park,2 Unjoo Lee,3,* and Jaebong Kim1, J Dev Biol. 2021 Dec; 9(4): 47. Published online 2021 Nov 1. PMCID: PMC8628936 
  3. Cell communication with the neural plate is required for induction of neural markersby BMP inhibition: evidence for homeogenetic induction and implications forXenopus animal cap and chick explant assays　Claudia Linkera,⁎,1, Irene De Almeidaa, Costis Papanayotoua, Matthew Stowera, Virginie Sabadob,Ehsan Ghorania, Andrea Streitb, Roberto Mayora, Claudio D. Stern　Developmental Biology 327 (2009) 478–486　　ResearchGate

BMＰとTGF-beta signaling

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. BMPs were originally identified from bone matrix using an ectopic bone formation assay.

https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/bone-morphogenetic-protein

About 60 TGF-β family members have been identified so far with two general branches: (i) BMP/growth and differentiation factor (GDF) and (ii) the TGF-β/activin/nodal branch/mullerian-inhibiting substance or anti-mullerian hormone.

TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation Rahman et al., Bone Res. 2015; 3: 15005. PMCID: PMC4472151

  1. The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4 Fainsod et al., Mechanisms of Development Volume 63, Issue 1, April 1997, Pages 39-50 Mechanisms of Development https://www.sciencedirect.com/science/article/pii/S0925477397006734
  • The transforming growth factor-beta (TGF-β) family of cytokines, including TGF-β, bone morphogenic proteins (BMPs), and activin/inhibin, plays crucial roles in embryonic development, adult tissue homeostasis and the pathogenesis of a variety of diseases.
  • The highly conserved core of the canonical TGF-β/BMP signaling is a simple linear cascade that involves the TGF-β/BMP ligands, two types of receptors (type I and II) and the signal transducers, Smads.
  • On activation, the receptor complex phosphorylates the carboxy-terminus of receptor-regulated Smad proteins (R-Smads), including Smad1, 5 and 8 for BMP signaling and Smad2 and 3 for TGF-β signaling.
  • Activated R-Smads interact with the common partner Smad, Smad4, and accumulate in the nucleus, where the Smad complex directly binds defined elements on the DNA and regulates target gene expression together with numerous other factors [–].

Signaling cross-talk between TGF-β/BMP and other pathways Guo and Wang  Cell Res. 2009 Jan; 19(1): 71–88.  PMC3606489

We have identified a new member of the transforming growth factor-beta (TGF-beta) superfamily, growth/differentiation factor-10 (GDF-10), which is highly related to bone morphogenetic protein-3 (BMP-3).

Growth/differentiation factor-10: a new member of the transforming growth factor-beta superfamily related to bone morphogenetic protein-3 N S Cunningham 1, N A Jenkins, D J Gilbert, N G Copeland, A H Reddi, S J Lee  Growth Factors . 1995;12(2):99-109. doi: 10.3109/08977199509028956.  https://pubmed.ncbi.nlm.nih.gov/8679252/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10232782/figure/F1/

  1. Bone morphogenetic protein signaling: the pathway and its regulation Takuya Akiyama, Laurel A Raftery, Kristi A Wharton Author Notes Genetics, Volume 226, Issue 2, February 2024, iyad200, https://doi.org/10.1093/genetics/iyad200
  2. BMP signaling during craniofacial development: new insights into pathological mechanisms leading to craniofacial anomalies Hiroki Ueharu and Yuji Mishina　Front Physiol. 2023; 14: 1170511. 2023 May 18.
  3. Calreticulin is a secreted BMP antagonist, expressed in Hensen’s node during neural induction Irene De Almeida a, Nidia M.M. Oliveira a, Rebecca A. Randall c, Caroline S. Hill c, John M. McCoy b 1, Claudio D. Stern a Developmental Biology Volume 421, Issue 2, 15 January 2017, Pages 161-170
  4. https://www.sciencedirect.com/topics/neuroscience/noggin