Involvement of Notch signaling in initiation of prechondrogenic condensation and nodule formation in limb bud micromass cultures

Ryoji Fujimaki, Yoshiaki Toyama, Nobumichi Hozumi, Ken Ichi Tezuka

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Notch signaling is an evolutionarily conserved mechanism that plays a critical role in the determination of multiple cellular differentiation pathways and morphogenesis during embryogenesis. The limb bud high-density culture is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) showed that Notch and its related genes were expressed in such cultures on day 1 and reached a peak between day 3 and day 5, when cell condensation and nodule formation were initiated. Immunohistochemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), to analyze the function of Notch signaling in the culture system. The blocking of Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. DAPT treatment also induced chondrogenic markers and bone morphogenetic protein (BMP)-related molecules, including type II collagen, Sox9, GDF5, and Id1. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.

Original languageEnglish
Pages (from-to)191-198
Number of pages8
JournalJournal of Bone and Mineral Metabolism
Volume24
Issue number3
DOIs
Publication statusPublished - 2006 May

Fingerprint

Limb Buds
Amyloid Precursor Protein Secretases
Bone Morphogenetic Proteins
Collagen Type II
Chondrocytes
Morphogenesis
Reverse Transcription
Embryonic Development
Cell Differentiation
Polymerase Chain Reaction
Genes
N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester

Keywords

  • Chondrogenesis
  • Mesenchymal cell condensation
  • Micromass culture
  • Nodule formation
  • Notch

ASJC Scopus subject areas

  • Endocrinology

Cite this

Involvement of Notch signaling in initiation of prechondrogenic condensation and nodule formation in limb bud micromass cultures. / Fujimaki, Ryoji; Toyama, Yoshiaki; Hozumi, Nobumichi; Tezuka, Ken Ichi.

In: Journal of Bone and Mineral Metabolism, Vol. 24, No. 3, 05.2006, p. 191-198.

Research output: Contribution to journalArticle

Fujimaki, Ryoji ; Toyama, Yoshiaki ; Hozumi, Nobumichi ; Tezuka, Ken Ichi. / Involvement of Notch signaling in initiation of prechondrogenic condensation and nodule formation in limb bud micromass cultures. In: Journal of Bone and Mineral Metabolism. 2006 ; Vol. 24, No. 3. pp. 191-198.
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