Anatomical Science International

, Volume 93, Issue 4, pp 414–421 | Cite as

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using EGFP transfection

  • Haiming Fan
  • Nobuyuki Sakamoto
  • Hirohiko Aoyama
Original Article


Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.


Chick Developmental fate In ovo electroporation Primitive streak Somite 



We thank Dr. Yoshiko Takahashi of Kyoto University for kindly providing pT2 K-CAGGS-EGFP and pCAGGS-T2TP. A part of this study was supported by Grants-in-Aid for Scientific Research from JSPS (20590171, 23590219, 26460254).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Bortier H, Vakaet LCA (1992) Fate mapping the neural plate and the intraembryonic mesoblast in the upper layer of the chicken blastoderm with xenografting and time-lapse videography. Development (suppl.) 116:93–97Google Scholar
  2. Brown JM, Storey KG (2000) A region of the vertebrate neural plate in which neighbouring cells can adopt neural or epidermal fates. Curr Biol 10:869–872CrossRefPubMedGoogle Scholar
  3. Catala M (2002) Genetic control of caudal development. Clin Genet 61:89–96CrossRefPubMedGoogle Scholar
  4. Catala M, Teillet M-A, De Robertis EM, Le Douarin NM (1996) A spinal cord fate map in the avian embryo: while regressing, Hensen’s node lays down the notochord and floor plate thus joining the spinal cord lateral walls. Development 122:2599–2610PubMedGoogle Scholar
  5. Chuai M, Weijer CJ (2009) Regulation of cell migration during chick gastrulation. Curr Opin Genet Dev 19:343–349CrossRefPubMedGoogle Scholar
  6. Garcia-Martinez V, Schoenwolf GC (1993) Primitive-streak origin of the cardiovascular system in avian embryos. Dev Biol 159:706–719CrossRefPubMedGoogle Scholar
  7. Gilbert SF, Barresi MJF (2016) Developmental biology, 11th edn. Sinauer Associates, SunderlandGoogle Scholar
  8. Griffith CM, Wiley MJ, Sanders EJ (1992) The vertebrate tail bud: three germ layers from one tissue. Anat Embryol (Berl) 185:101–113CrossRefGoogle Scholar
  9. Hamburger V, Hamilton H (1951) A series of normal stages in the development of the chick embryo. J Morph 88:49–92CrossRefPubMedGoogle Scholar
  10. Hatada Y, Stern CD (1994) A fate map of the epiblast of the early chick embryo. Development 120:2879–2889PubMedGoogle Scholar
  11. Kondoh H, Takemoto T (2012) Axial stem cells deriving both posterior neural and mesodermal tissues during gastrulation. Curr Opin Genet Dev 22:374–380CrossRefPubMedGoogle Scholar
  12. McGrew MJ, Dale JK, Fraboulet S, Pourquié O (1998) The lunatic Fringe gene is a target of the molecular clock linked to somite segmentation in avian embryos. Curr Biol 8:979–982CrossRefPubMedGoogle Scholar
  13. Momose T, Tanegawa A, Takeuchi J, Ogawa H, Umesono K, Yasuda K (1999) Efficient targeting of gene expression in chick embryos by microelectroporation. Dev Growth Differ 41:335–344CrossRefPubMedGoogle Scholar
  14. Nicolet G (1971) Avian gastrulation. Adv Morphogen 9:231–262CrossRefGoogle Scholar
  15. Psychoyos D, Stern CD (1996) Fates and migratory routes of primitive streak cells in the chick embryo. Development 122:1523–1534PubMedGoogle Scholar
  16. Sato Y, Kasai T, Nakagawa S, Tanabe K, Watanabe T, Kawakami K, Takahashi Y (2007) Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol 305:616–624CrossRefPubMedGoogle Scholar
  17. Sawada K, Aoyama H (1999) Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites. Int J Dev Biol 43:809–815PubMedGoogle Scholar
  18. Schoenwolf GC, Smith JL (1990) Mechanisms of neurulation: traditional viewpoint and recent advances. Development 109:243–270PubMedGoogle Scholar
  19. Schoenwolf GC, Garcia-Martinez V, Dias MS (1992) Mesoderm movement and fate during avian gastrulation and neurulation. Dev Dyn 193:235–248CrossRefPubMedGoogle Scholar
  20. Shimokita E, Takahashi Y (2011) Secondary neurulation: fate-mapping and gene manipulation of the neural tube in tail bud. Dev Growth Differ 53:401–410CrossRefPubMedGoogle Scholar
  21. Takemoto T, Uchikawa M, Yoshida M, Bell DM, Badge RL, Papaioannou VE, Kondoh H (2011) Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells. Nature 470:394–398CrossRefPubMedPubMedCentralGoogle Scholar
  22. Tenin G, Wright D, Ferjentsik Z, Bone R, McGrew MJ, Maroto M (2010) The chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites. MBC Dev Biol 10:24Google Scholar
  23. Wilson V, Olivera-Martinez I, Storey KG (2009) Stem cells, signals and vertebrate body axis extension. Development 136:1591–1604CrossRefPubMedGoogle Scholar
  24. Wright D, Ferjentsik Z, Chong SW, Qiu XH, Yun JJ, Malapert P, Pourquié O, Hateren NV, Wilson SA, Franco C, Gerhardt H, Dale JK, Maroto M (2009) Cyclic Nrarp mRNA expression is regulated by the somitic oscillator but nrarp protein levels do not oscillate. Dev Dyn 238:3043–3055CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Japanese Association of Anatomists 2018

Authors and Affiliations

  • Haiming Fan
    • 1
  • Nobuyuki Sakamoto
    • 1
  • Hirohiko Aoyama
    • 1
  1. 1.Department of Anatomy and Developmental Biology, Graduate School of Biomedical SciencesHiroshima UniversityHiroshimaJapan

Personalised recommendations