The double inhibition of endogenously produced BMP and Wnt factors synergistically triggers dorsal telencephalic differentiation of mouse ES cells

Embryonic stem (ES) cells are becoming a popular model of in vitro neurogenesis, as they display intrinsic capability to generate neural progenitors that undergo the known steps of in vivo neural development. These include the acquisition of distinct regional fates, which depend on growth factors and signals that are present in the culture medium. The control of the intracellular signaling that is active at different steps of ES cell neuralization, even when cells are cultured in chemically defined medium, is complicated by the endogenous production of growth factors. However, this endogenous production has been poorly investigated so far. To address this point, we performed a high‐throughput analysis of the expression of morphogens during mouse ES cell neuralization in minimal medium. We found that during their neuralization, ES cells increased the expression of members of Wnt, Fibroblast Growth Factor (FGF), and BMP families. Conversely, the expression of Activin/Nodal and Shh ligands was low in early steps of neuralization. In this experimental condition, neural progenitors and neurons generated by ES cells expressed a gene expression profile that was consistent with a midbrain identity. We found that endogenous BMP and Wnt signaling, but not FGF signaling, synergistically affected ES cell neural patterning, by turning off a profile of dorsal/telencephalic gene expression. Double BMP and Wnt inhibition allowed neuralized ES cells to sequentially activate key genes of cortical differentiation. Our findings are consistent with a novel synergistic effect of Wnt and BMP endogenous signaling of ES cells in inhibiting a cortical differentiation program. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 66–79, 2015

[1]  L. Allen.  Stem cells. , 2003, The New England journal of medicine.

[2]  Marc Peschanski,et al.  Embryonic stem cells neural differentiation qualifies the role of Wnt/β‐Catenin signals in human telencephalic specification and regionalization , 2013, Stem cells.

[3]  John T. Dimos,et al.  The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells , 2006, Nature Neuroscience.

[4]  Alex Gutteridge,et al.  Directing Differentiation of Human Embryonic Stem Cells Toward Anterior Neural Ectoderm Using Small Molecules , 2012, Stem cells.

[5]  S. Mcconnell,et al.  The determination of projection neuron identity in the developing cerebral cortex , 2008, Current Opinion in Neurobiology.

[6]  M. Gulisano,et al.  Nested expression domains of four homeobox genes in developing rostral brain , 1992, Nature.

[7]  N. Allen,et al.  Neural differentiation of mouse embryonic stem cells in chemically defined medium , 2005, Brain Research Bulletin.

[8]  R. A. Smith,et al.  Neuronal maintenance and neurite extension of adult mouse neurones in non-neuronal cell-reduced cultures is dependent on substratum coating. , 1988, Journal of cell science.

[9]  P. Gruss,et al.  Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification , 1998, The EMBO journal.

[10]  Pierre Vanderhaeghen,et al.  An intrinsic mechanism of corticogenesis from embryonic stem cells , 2008, Nature.

[11]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Xuan,et al.  Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres , 1995, Neuron.

[13]  A. Brivanlou,et al.  SMAD7 Directly Converts Human Embryonic Stem Cells to Telencephalic Fate by a Default Mechanism , 2013, Stem cells.

[14]  Paola Arlotta,et al.  Neuronal Subtype-Specific Genes that Control Corticospinal Motor Neuron Development In Vivo , 2005, Neuron.

[15]  D. van der Kooy,et al.  Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences , 2006, The Journal of cell biology.

[16]  W. Harris,et al.  Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures , 2013, Open Biology.

[17]  Marius Wernig,et al.  In vitro differentiation of transplantable neural precursors from human embryonic stem cells , 2001, Nature Biotechnology.

[18]  K. Mizuseki,et al.  Directed differentiation of telencephalic precursors from embryonic stem cells , 2005, Nature Neuroscience.

[19]  H. Wichterle,et al.  Directed Differentiation of Embryonic Stem Cells into Motor Neurons , 2002, Cell.

[20]  J. Rubenstein,et al.  T-Brain-1: A homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex , 1995, Neuron.

[21]  A. Joyner,et al.  Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain. , 1988, Genes & development.

[22]  G. Duester,et al.  Retinoic Acid Functions as a Key GABAergic Differentiation Signal in the Basal Ganglia , 2011, PLoS Biology.

[23]  S. Chandran,et al.  Embryonic Stem Cell‐Derived Neural Progenitors Display Temporal Restriction to Neural Patterning , 2006, Stem cells.

[24]  Ali H. Brivanlou,et al.  Neural induction, the default model and embryonic stem cells , 2002, Nature Reviews Neuroscience.

[25]  Jan Terje Kvaløy,et al.  Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: the balance between accuracy and precision. , 2006, Analytical biochemistry.

[26]  G. Horgan,et al.  Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR , 2002 .

[27]  M. Gulisano,et al.  Two vertebrate homeobox genes related to the Drosophila empty spiracles gene are expressed in the embryonic cerebral cortex. , 1992, The EMBO journal.

[28]  Yoshiki Sasai,et al.  Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. , 2008, Cell stem cell.

[29]  Austin G Smith,et al.  Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture , 2003, Nature Biotechnology.

[30]  Tomomi Shimogori,et al.  Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex , 2004, Development.

[31]  N. Copeland,et al.  Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. , 1995, Development.

[32]  O. Lindvall,et al.  Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. , 2012, Cell reports.

[33]  S. Goderie,et al.  Timing of CNS Cell Generation A Programmed Sequence of Neuron and Glial Cell Production from Isolated Murine Cortical Stem Cells , 2000, Neuron.

[34]  Michael Brand,et al.  The Midbrain–hindbrain Boundary Organizer , 2022 .

[35]  Stephen W. Wilson,et al.  Early steps in the development of the forebrain. , 2004, Developmental cell.

[36]  M. Johnson,et al.  Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells , 2009, Development.

[37]  M. Ishida,et al.  Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons. , 1999, Development.

[38]  A. Messina,et al.  The positional identity of mouse ES cell-generated neurons is affected by BMP signaling , 2012, Cellular and Molecular Life Sciences.

[39]  Sally Temple,et al.  Cell lineage tree models of neurogenesis. , 2009, Journal of theoretical biology.

[40]  C. Stern,et al.  Evolution of vertebrate forebrain development: how many different mechanisms? , 2001, Journal of anatomy.

[41]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[42]  M. Tomishima,et al.  Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling , 2009, Nature Biotechnology.

[43]  B. Doble,et al.  The ground state of embryonic stem cell self-renewal , 2008, Nature.

[44]  D. Surmeier,et al.  Floor plate-derived dopamine neurons from hESCs efficiently engraft in animal models of PD , 2011, Nature.

[45]  Janet Rossant,et al.  Direct Neural Fate Specification from Embryonic Stem Cells A Primitive Mammalian Neural Stem Cell Stage Acquired through a Default Mechanism , 2001, Neuron.

[46]  J. Davis.  Bioinformatics and Computational Biology Solutions Using R and Bioconductor , 2007 .

[47]  L. Recht,et al.  Murine Embryonic Stem Cell-Derived Pyramidal Neurons Integrate into the Cerebral Cortex and Appropriately Project Axons to Subcortical Targets , 2010, The Journal of Neuroscience.

[48]  J. Nichols,et al.  Naive and primed pluripotent states. , 2009, Cell stem cell.