ABSTRACT
The transcription factor DLX5 belongs to a family of homeoproteins required for craniofacial morphogenesis and forebrain development. DLX5 is expressed during formation of several skeletal elements such as cartilage, teeth and bone, and its knockout causes severe craniofacial malformations with a delay in the ossification process. Bone marrow contains mesenchymal progenitor cells which may differentiate along multiple pathways, therefore representing an interesting in vitro and in vivo model to study the mesodermal lineage differentiation. Here we report the effect of DLX5 overexpression in ex vivo expanded human bone marrow stromal cells by retroviral infection on the osteogenic lineage differentiation. A reduced mineral deposition was observed in DLX5-transduced cells upon osteogenic induction in culture. When DLX5-transduced cells were implanted in immunodeficient mice, a 60% reduction in bone matrix deposition was observed, whereas the in vitro chondrogenic potential was unaffected. A quantitative gene expression study indicated that DLX5 overexpression does not affect the early osteogenic commitment of bone marrow stromal cells but prevents their terminal differentiation. This block may be mediated by the observed persistent expression of SOX2, a transcription factor known to inhibit osteogenic differentiation.
Subject(s)
Bone Marrow Cells/cytology , Homeodomain Proteins/biosynthesis , Osteocytes/cytology , Osteogenesis/physiology , Transcription Factors/biosynthesis , Adolescent , Adult , Animals , Bone Marrow Cells/metabolism , Cell Differentiation/physiology , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Gene Expression Profiling , Genetic Vectors/genetics , Homeodomain Proteins/genetics , Humans , Mesenchymal Stem Cells/cytology , Mice , Osteocytes/metabolism , Osteogenesis/genetics , Retroviridae/genetics , SOXB1 Transcription Factors/biosynthesis , SOXB1 Transcription Factors/genetics , Stromal Cells/cytology , Stromal Cells/metabolism , Transcription Factors/genetics , Transduction, Genetic , Young AdultABSTRACT
Emx2 plays a crucial role in the development of the diencephalon and dorsal telencephalon. Thus, Emx2-null mutants have abnormal cortical lamination and a reduction in size of the caudal and medial areas of the prosencephalon. Emx2 is expressed in neural precursors of the subventricular zone in vivo and in cultured neurospheres in vitro where it controls the size of the transit-amplifying population, affecting proliferation and clonal efficiency of neural stem cells. To identify the cellular processes mastered by Emx2, and possibly the molecular mechanisms by which the gene exerts its action, we compared the expression profile of cultured neurospheres derived from wild-type and Emx2-null mouse embryos. The differential expression of several genes was also confirmed by semiquantitative RT-PCR, real-time PCR and cytofluorimetric analysis in different preparations of neurospheres, and by in situ hybridization. The gene expression profile suggested a role for Emx2 in regulating the differentiation and migration properties of neural precursor cells. This involvement was confirmed in vitro, where the altered clonogenicity and impaired migration of Emx2-null cells were partially corrected by transduction of the Emx2 gene. Taken together, our results indicate that Emx2 is indeed involved in the transition between resident early progenitors (perhaps stem cells) and more mature precursors capable of migrating out of the ventricular zone, becoming postmitotic and differentiating into the appropriate cell type, and help explain the alterations observed in the brains of knock-out mice.
Subject(s)
Brain/cytology , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/metabolism , Neurons/physiology , Stem Cells/physiology , Animals , Blotting, Northern/methods , Cells, Cultured , Embryo, Mammalian , Enzyme Activation/physiology , Female , Immunohistochemistry/methods , In Situ Hybridization/methods , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microarray Analysis/methods , Pregnancy , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Transcription FactorsABSTRACT
Dlx genes play an important role in the control of the development of the central nervous system (CNS). Single or compound inactivation of Dlx1, Dlx2, or Dlx5 in the mouse causes defects of neuronal migration and differentiation. Dlx5, in particular, is essential for the correct development of the olfactory system. Targeted inactivation of Dlx1 and Dlx2 in the mouse results in abnormal neuronal differentiation in the embryonic subcortical forebrain and is associated to the loss of Dlx5 and Dlx6 expression. So far, however, it has been impossible to investigate the role of Dlx genes on late neurogenesis, as their inactivation leads to perinatal death. We have now generated cultures of neural stem cells (NSCs) derived from embryonic and newborn Dlx5-null mice, and we have compared their capacity to differentiate in vitro to that of equivalent cells derived from normal littermates. We show here that in the absence of Dlx5, NSCs derived from newborn animals have a severely reduced capacity to generate neurons. This is not the case for cells derived from E12.5 embryos. Forced expression of Dlx5 in cultures of newborn mutant NSCs fully restores their neuronogenic potential. Our data suggest that Dlx5 is essential for secondary (postnatal) neuronogenesis.
