L3/Lhx8 is a pivotal factor for cholinergic differentiation of murine embryonic stem cells.
L3/Lhx8 is a member of the LIM-homeobox gene family. Previously, we demonstrated that L3/Lhx8-null mice specifically lacked cholinergic neurons in the basal forebrain. In the present study, we conditionally suppressed L3/Lhx8 function during retinoic acid-induced neural differentiation of a murine embryonic stem (ES) cell line using an L3/Lhx8-targeted small interfering RNA (siRNA) produced by an H1.2 promoter-driven vector. Our culture conditions induced efficient differentiation of the ES cells into neurons and astrocytes, but far less efficient differentiation into oligodendrocytes. Suppression of L3/Lhx8 expression by siRNA led to a dramatic decrease in the number of cells positive for the cholinergic marker ChAT, and overexpression of L3/Lhx8 recovered this effect. However, no significant changes were observed in the number of Tuj1+ neurons and GABA+ cells. These results strongly suggest that L3/Lhx8 is a key factor in the cholinergic differentiation of murine ES cells and is involved in basal forebrain development.
Manabe
T
T
Department of 2nd Anatomy, Faculty of Medicine, Nara Medical University, 840 Shijyo-cho, Kasihara City, Nara 634-8521, Japan. manabe@cdb.riken.jp
Tatsumi
K
K
Inoue
M
M
Makinodan
M
M
Yamauchi
T
T
Makinodan
E
E
Yokoyama
S
S
Sakumura
R
R
Wanaka
A
A
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D060850:LIM-Homeodomain Proteins; D051379:Mice; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors; D014162:Transfection
D018398:Homeodomain Proteins; C112960:LIM homeobox protein 8; D060850:LIM-Homeodomain Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D002795:Choline O-Acetyltransferase