体軸上で、四肢の出る位置はどのようにして決まるのか？

 

 

 

Patterning mechanisms controlling vertebrate limb development. J. Capdevila, J. C. I. Belmonte · 2001　脊椎動物の四肢芽では、シグナル伝達経路間の複雑な相互作用により、四肢の位置決め、成長、パターン形成が制御されます。　https://www.annualreviews.org/content/journals/10.1146/annurev.cellbio.17.1.87　本文有料 総説論文

 

Analysis of Hox gene expression in the chick limb bud. C. Nelson, Bruce A. Morgan, Ann C. Burke+7 more · 1996年5月1日　肢芽における Hox 遺伝子の発現は、肢の近位遠位節 (上腕、下腕、手) の指定とパターン形成に関連して、最大 3 つの独立した段階で制御されます。https://journals.biologists.com/dev/article/122/5/1449/39007/Analysis-of-Hox-gene-expression-in-the-chick-limb　Expression of the Hoxa genes during normal chick limb development. Whole mount in situ hybridizations to wings (upper row) and legs (lower row) for the genes: Hoxa-9 (A), Hoxa-10 (B), Hoxa-11 (C) and Hoxa-13 (D). Approximate stages are: stage 19/20, stage 23, stage 25 and stage 28 (from left to right). All panels are dorsal views with anterior to the top and distal to the right. (A) Hoxa-9 is expressed uniformly in limb mesenchyme at the earliest stages of limb bud outgrowth. By stage 19, there is a region in the anterior/proximal region of the wing bud that does not express Hoxa-9. This region, which does not express Hoxa-9, persists and expands through subsequent stages of development. Expression throughout the rest of the bud is strong through stage 23 and then fades slowly throughout the limb bud. By stage 25, only low levels of transcript are detected. By stage 28 expression is no longer detected in the limb. In the leg bud, Hoxa-9 expression parallels that in the wing, but there is no region of non-expressing tissue in the anterior/proximal region. (B) By stage 19 Hoxa-10 is expressed in the wing bud, but it is largely excluded from the marginal mesenchyme. By stage 22 Hoxa-10 is expressed at moderate levels throughout the wing mesenchyme with the exception of a region in the anterior/proximal portion of the wing. In the leg bud Hoxa-10 is expressed uniformly by stage 19 and never appears to be excluded from the anterior/proximal region seen in the wing. As development proceeds, progressively lower levels of transcript are detected throughout both limbs. At late stages, Hoxa-10 expression is still detectable in the upper arm/leg and lower arm/leg, but is largely absent from the hand/foot. (C) At early stages a region of expression is detected in the medial region of the wing bud, where scattered punctate staining is also observed. In late stage 19, a separate domain of expression along the distal margin of the wing is also observed. At stage 22, Hoxa-11 expression is observed in the distal half of the wing bud across the A/P axis of the limb. By stage 25, expression is restricted to the presumptive lower arm and is largely absent from the upper arm and hand. This pattern of expression persists through stage 28. By stage 19 Hoxa-11 expression in the distal margin of the leg bud is strong and uniform. Expression is also detected in more proximal regions, but in a less uniform, punctate pattern with higher levels observed in the posterior two thirds of the limb. By stage 22, expression is strong and uniform through most of the leg bud, although Hoxa-11 is not expressed in the proximal anterior region. At later stages, expression persists in the lower leg but is greatly reduced or absent in the upper leg and foot. This pattern persists through stage 28. Like many of the Hox genes, Hoxa-11 expression becomes excluded from regions of condensing cartilage. (D) Hoxa-13 is first expressed in the hind limb at stage 19 in the posterior distal mesenchyme. Expression in the fore limb begins shortly thereafter at the posterior distal margin (data not shown). Later, Hoxa-13 is expressed at high levels in a crescent along the distal margin of both limbs. By stage 25 expression is strong throughout the hand/foot with no obvious bias in expression level along the anterior/posterior axis; no Hoxa-13 expression is detected in the proximal segments of the limb. High levels of expression persist in the hand/foot through at least stage 28.

分子生物学とは？生化学との違い

大雑把にいうと、分子生物学はDNAの科学、生化学はタンパク質の科学と捉えてよいものでしょうか？ChatGPTに訊いてみました。

分子生物学とは？

  • 分子生物学は、DNA、RNA、タンパク質の相互作用を中心に、生物の遺伝情報の伝達と機能を探る学問です。
  • 遺伝子の複製（DNAのコピー）、転写（DNAからRNAへの変換）、翻訳（RNAからタンパク質への変換）など、遺伝情報がどのようにして細胞内で利用されるかを研究します。
  • たとえば、遺伝子の発現調節、遺伝子変異、シグナル伝達などが分子生物学の対象となります。

生化学とは？

  • 生化学は、生物を構成する化学的な物質やその化学反応を研究する学問です。
  • タンパク質に関する研究は生化学の大きな部分を占めますが、それだけではなく、脂質、糖、核酸、代謝経路（例えばエネルギーの生成や利用）、酵素反応など、生物のあらゆる化学反応が対象です。
  • 酵素の機能や、細胞の代謝過程における化学反応の詳細なども生化学の主要な研究テーマです。

まとめ：

  • 分子生物学＝「DNAや遺伝子の科学」に近いが、実際にはRNAやタンパク質も含む遺伝情報の流れ全体を研究する。
  • 生化学＝「生体内の化学反応」や「タンパク質を含む生体分子の機能」を広く研究する。

したがって、「分子生物学がDNAの科学、生化学がタンパク質の科学」という理解は、分子生物学と生化学の一部を反映していますが、全体の範囲や対象を理解するにはもう少し幅広く捉える必要があります。

(ChatGPT 4o）

カエルとマウスの発生学から得られた知見の統合：wnt, BMP, nordal, オーガナイザー、神経誘導などについて

 

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage　https://www.biorxiv.org/content/10.1101/2020.07.16.167320v1.full

 

Developmental Cell Volume 1, Issue 5, November 2001, Pages 605-617 Journal home page for Developmental Cell Review Nodal Signaling in Early Vertebrate Embryos: Themes and Variations　https://www.sciencedirect.com/science/article/pii/S1534580701000764

TGF-b family signaling gradients during gastrulation. (A ...

TGF-β Family Signaling in Early Vertebrate Development June 2017Cold Spring Harbor Perspectives in Biology 10(6):a033274 DOI:10.1101/cshperspect.a033274　https://www.researchgate.net/figure/TGF-b-family-signaling-gradients-during-gastrulation-A-Embryonic-tissues-patterned-by_fig1_317494952

上肢や下肢が出る位置はどのようにして決まるのか？

上肢や下肢が出る位置はどのようにして決まるのでしょうか？体軸の位置はHOXコードで決まるのだとすれば、ある特定のHOX遺伝子産物が転写制御因子となって上肢や下肢の肢芽で発現するTbx5やTbx4の発現を直接制御するのでしょうか（エンハンサーかプロモーターに特異的に結合するなどして）？

下の総説によれば、HOX遺伝子が活性化や抑制に働くようです。

A Combination of Activation and Repression by a Colinear Hox Code Controls Forelimb-Restricted Expression of Tbx5 and Reveals Hox Protein Specificity Satoko Nishimoto,Carolina Minguillon,Sophie Wood,Malcolm P. O. Logan Published: March 20, 2014 https://doi.org/10.1371/journal.pgen.1004245

最近の総説論文を読むと, HOXコードで前肢の出る位置は決まるようですが、そう単純ではなさそうです。ただTbx5はFgf10遺伝子のプロモーターに直接結合して発現を誘導するそうです。下肢については、前肢ほどには研究がなされておらず、HOXコードで位置が規定されるのか、FGF10を発現させるものが何なのかについては報告がなさそうです。

  1. Current research on mechanisms of limb bud development, and challenges for the next decade Takayuki Suzuki　Genes & Genetic Systems/Volume 99 (2024)　https://www.jstage.jst.go.jp/article/ggs/99/0/99_23-00287/_html/-char/en　

 

  1. Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo　Development (2003) 130 (3): 623–633.　https://journals.biologists.com/dev/article/130/3/623/42069/Tbx5-is-essential-for-forelimb-bud-initiation
    1. mouse embryos lacking Tbx5 do not form forelimb buds
    2. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation
  2. Tbx5 is required for forelimb bud formation and continued outgrowth　Development . 2003 Jun;130(12):2741-51. doi: 10.1242/dev.00473.

Fig. 3. Antero-posterior limb polarity. (A) Major regulatory interactions involved in the specification of forelimb field antero-posterior polarity. Retinoic acid (RA) signalling is implicated in the defined anterior to posterior order of expression of Hox5-Hox9 paralogous group (PG) genes in presumptive forelimb regions of the lateral plate mesoderm (LPM). Hox5 PG proteins repress anterior Shh expression indirectly through activation of Plzf. Gata4 and Gata6 proteins transcriptionally inhibit Shh and attenuate Shh signal transduction by promoting the repressor form of Gli3. RA stimulates the posterior expression of Hand2, the product of which both represses Gli3 in the posterior part of the limb bud and stimulates Shh expression at the posterior margin. Gli3 also represses Hand2. Sall4 is expressed in the presumptive forelimb and its protein product contributes to the expression of Gli3. (B) Major regulatory interactions involved in the specification of hindlimb field antero-posterior polarity. Gata6 directly represses anterior expression of Shh. Sall4, Irx3 and Irx5 regulate Gli3 expression anteriorly. Isl1 indirectly promotes the posterior expression of Shh in the hindlimb by inducing Hand2, which represses Gli3 in the posterior part of the hindlimb. A, anterior; P, posterior.

https://journals.biologists.com/dev/article/147/17/dev177956/225797/Establishing-the-pattern-of-the-vertebrate-limb

notchは原始線条nodeのmotile ciliaをもつ細胞でnodal遺伝子発現を誘導するか

左右差を生み出す最初の仕組みは、マウスの場合は原始結節（2層の細胞層）の下側（中胚葉由来である脊索が脊索板になって内胚葉の層と一体化している）の細胞がもつ線毛の回転により左向きの水流が生じて、それを原始結節の両側に存在する動かない線毛を持つ細胞が感知して、左側に特異的な遺伝子発現を始めるというものでした。

動かない線毛を持つ細胞は左右両側にありますが、その線毛に存在するカルシウムチャンネルPkd2は正中線側に偏って存在しているため、左向きの水流で曲げられた線毛の正中線側の部分は、左側の細胞では引っ張られ、右側の細胞では表面が縮まることになります。張力によって開くカルシウムチャネルPkd2は、左側でのみ開くので、Ca濃度上昇に依存した細胞内の変化が、特異的な遺伝子発現につながるわけです。その細胞内変化とはどのようなものでしょうか。

notchは原始線条nodeのmotile ciliaをもつ細胞でnodal遺伝子発現を誘導するのでしょうか？

  1. Notch signaling regulates left–right asymmetry determination by inducing Nodal expression Luke T Krebs 1,4, Naomi Iwai 2,3,4, Shigenori Nonaka 2,3, Ian C Welsh 1, Yu Lan 1,5, Rulang Jiang 1,5, Yukio Saijoh 2, Timothy P O’Brien 1, Hiroshi Hamada 2,3,6, Thomas Gridley 1,7　Genes Dev. 2003 May 15;17(10):1207–1212. doi: 10.1101/gad.1084703 https://pmc.ncbi.nlm.nih.gov/articles/PMC196059/
  2. Notch activity acts as a sensor for extracellular calcium during vertebrate left–right determination Ángel Raya, Yasuhiko Kawakami, Concepción Rodríguez-Esteban, Marta Ibañes, Diego Rasskin-Gutman, Joaquín Rodríguez-León, Dirk Büscher, José A. Feijó & Juan Carlos Izpisúa Belmonte Nature volume 427, pages121–128 (2004) figure 1
  3. Notch activity induces Nodal expression and mediates the establishment of left–right asymmetry in vertebrate embryos　Genes Dev. 2003 May 15;17(10):1213–1218. doi: 10.1101/gad.1084403 https://pmc.ncbi.nlm.nih.gov/articles/PMC196060/
  4. Developmental Biology Asymmetric distribution of dynamic calcium signals in the node of mouse embryo during left–right axis formation Developmental Biology Volume 376, Issue 1, 1 April 2013, Pages 23-30  https://www.sciencedirect.com/science/article/pii/S0012160613000365?via%3Dihub

カエルのオーガナイザーその他のシグナル分子の遺伝子のマウス胚での発現

カエルの発生学で分子シグナルが詳細に調べられてきましたが、それらの分子がマウス胚でも同様の働きをもつのかどうかの情報の整理が大変です。

Goosecoid

  1. Goosecoid Regulates the Neural Inducing Strength of the Mouse Node　Developmental Biology Volume 216, Issue 1, 1 December 1999, Pages 276-281
  2. Gastrulation in the mouse: the role of the homeobox gene goosecoid　M Blum, SJ Gaunt, KWY Cho, H Steinbeisser, B Blumberg, D Bittner, EM De Robertis　Cell, 1992 本文有料
  3. TGIF1 and TGIF2 regulate Nodal signaling and are required for gastrulation. 　January 2010Development 137(2):249-59 DOI:10.1242/dev.040782　https://www.researchgate.net/figure/Regulation-of-goosecoid-expression-by-Tgifs-AEmbryos-of-the-indicated-ages-and_fig6_40812258　Regulation of goosecoid expression by Tgifs. (A)Embryos of the indicated ages and genotypes were analyzed for expression of goosecoid by in situ hybridization. The brackets indicate the extra-embryonic region. Arrowheads indicate expression in the extraembryonic region. (B)A goosecoid promoter reporter construct was used to test repression by co-expressed Tgif1 (+, 20 ng/well, ++, 50 ng/well) in transfected HepG2 cells. Cells were treated with TGFb, as indicated. Results are presented as means±s.d. of triplicate transfections, normalized to a Renilla luciferase control. Activity in the presence of Tgif1 was significantly different from the control (*P<0.05, **P<0.01), as determined by Student's t-test. (C)Expression of goosecoid in 8.0 dpc embryos of the indicated genotypes was analyzed by in situ hybridization. (D)Sections through the embryos in C. All in situ images are representative of at least three embryos. Scale bars: 250mm in A,C; 100mm in D. 
    (C)Expression of goosecoid　in 8.0 dpc embryos of the indicated genotypes　was analyzed by in situ hybridization.　(D)Sections through the embryos in C.

    Cがgoosecoidの発現。nodeに局在していることがわかります。

Chordin

下の図でaがchordinのin situ hybridazation （ｍRNAの局在）です。bcはnoggin遺伝子の発現。chordinは原始線条の前側で発現しはじめて原始結節に局在したと本文で説明されていますが、図には向きなどの記載がなくわかりにくいです。Chordin遺伝子をのノックアウトしても表現型は意外なくらいマイルド（図efg）なようです。

Figure 1

The organizer factors Chordin and Noggin are required for mouse forebrain development　Nature volume 403, pages658–661 (2000)　10 February 2000 　https://www.nature.com/articles/35001072

wntシグナリング分子

https://www.semanticscholar.org/paper/Wnt-signalling-in-mouse-gastrulation-and-anterior-Arkell-Fossat/1ebceb654e1ea878ac1bd663735be63e1a14a0fb

Canonical Wnt Signaling and Its Antagonist Regulate Anterior-Posterior Axis Polarization by Guiding Cell Migration in Mouse Visceral Endoderm Developmental Cell Volume 9, Issue 5, November 2005, Pages 639-650 https://www.sciencedirect.com/science/article/pii/S1534580705003734

体軸形成に関する最近の総説

 

最近の総説&原著論文

  1. In vitro modelling of anterior primitive streak patterning with human pluripotent stem cells identifies the path to notochord progenitors　Posted April 22, 2024.　bioRxiv　https://www.biorxiv.org/content/10.1101/2023.06.01.543323v3.full
  2. Recent advances in understanding cell types during human gastrulation　Semin Cell Dev Biol. 2022 May 21;131:35–43. doi: 10.1016/j.semcdb.2022.05.004 https://pmc.ncbi.nlm.nih.gov/articles/PMC7615356/
  3. The Organizer and Its Signaling in Embryonic Development　J Dev Biol. 2021 Nov 1;9(4):47. doi: 10.3390/jdb9040047 Figure 1　https://pmc.ncbi.nlm.nih.gov/articles/PMC8628936/　MDPI誌オープンアクセス論文
  4. Mesoderm induction and patterning: insights from neuromesodermal progenitors　Semin Cell Dev Biol. 2021 Nov 25;127:37–45. doi: 10.1016/j.semcdb.2021.11.010
  5.  A gene regulatory program controlling early Xenopus mesendoderm formation: network conservation and motifs　　Semin Cell Dev Biol. 2017 Mar 22;66:12–24. doi: 10.1016/j.semcdb.2017.03.003
  6. Vertebrate Axial Patterning: From Egg to Asymmetry　Adv Exp Med Biol. 2017;953:209–306. doi: 10.1007/978-3-319-46095-6_6 　https://pmc.ncbi.nlm.nih.gov/articles/PMC6550305/

心臓はなぜ左側にできるのか？左右軸の決定機構との関係

人間の体は外側から見ると一見、左右対称ですが体の中を見ると、心臓が左側にあり、肝臓は右側にあります。肺の枝の分岐パターンにも左右で違いがあります。これらの左右の違いは、いつ、どのように生じたのでしょうか？

 

Relationship between asymmetric nodal expression and the direction of embryonic turning　Published: 09 May 1996  Jérôme Collignon, Isabella Varlet & Elizabeth J. Robertson Nature volume 381, pages155–158 (1996)

マウスの胚における非対称な nodal 発現は、心臓のループ方向と胚の回転と相関しており、左右の体軸経路が脊椎動物で保存されていることを示しています。

ニュークープセンター Nieuwkoop Center ：アフリカツメガエルにおけるオーガナイザーの誘導メカニズム

ニュークープセンターは、オーガナイザーを誘導するセンターという概念です。カエルの胞胚の時期にアニマルキャップと植物極側とに分けて、くっつけて培養したおｔきにアニマルキャップ（外胚葉）から中胚葉が誘導されたことから生じた概念です。

したのレビュー論文は非常に網羅的で、かつ、歴史的な経緯や解釈が詳しくて勉強になります。

figure 1　figure 3

Molecular specification of germ layers in vertebrate embryos 14 December 2015 Cellular and Molecular Life Sciences  Volume 73, pages 923–947, (2016)　https://link.springer.com/article/10.1007/s00018-015-2092-y

  1. The organization center of the amphibian embryo: its origin, spatial organization, and morphogenetic action P D Nieuwkoop PMID: 4581327 DOI: 10.1016/b978-0-12-028610-2.50005-8　Adv Morphog . 1973:10:1-39. doi: 10.1016/b978-0-12-028610-2.50005-8.

二次軸を形成する背側化因子

β－カテニン

Fig. 1. (A) A secondary axis can be induced in developing Xenopus embryos by injection of RNA encoding β-catenin into a ventral cell of 4-cell stage embryos. Ventral cells are usually distinguished by their larger size and darker pigment compared to dorsal cells. For detailed methods see (Kuhl and Pandur, 2008a). (B) The duplicated axis is visible in neurula stage embryos within 2 days of injection. Embryos in these images have undergone in situ hybridisation for neuralβ-tubulin to illustrate the bilateral stripes of primary neurons and trigeminal　https://www.researchgate.net/publication/272524241_An_oncologist%27s_friend_How_Xenopus_contributes_to_Cancer_research/figures