NeuroD2/6 regulate expression balance of transcription factors controlling neurocortical cytoarchitecture

Abstract

BACKGROUND: Genes of the NeuroD family, including NeuroD1, NeuroD2, and NeuroD6, control neuronal survival, differentiation, maturation, and neurite specification in the nervous system. Deletion of NeuroD1 in the mouse brain results in complete loss of dentate gyrus because of neuronal apoptosis. NeuroD2 is required for neuron survival in the cerebellum and integration of thalamo-cortical connections into neocortex and formation of somatosensory whisker barrel cortex. In NeuroD2/6 double deficient (DKO) mice, callosal axon projections are defective due to abnormal EfnA4 signaling. In order to investigate the NeuroD2/6 controlled molecular cascade, we explored the expression of key transcription factors that control various aspects of cortical development in brains of NeuroD2 and NeuroD6 deficient mutants.

AIM: To investigate possible changes in differentiation programs downstream of NeuroD2/6 transcription factors.

METHODS: Embryos with NeuroD2/6 double deficiency were used in the experiments, and pregnant mice carrying E13.5 embryos were operated for in utero electroporation. We performed in situ hybridization at various stages of embryonic development to study the expression pattern of target genes. Analyzing the activity of a gene promoter, genomic DNA fragments containing NeuroD2/6 motifs were cloned into pMCS-Gaussia Luc vector for luciferase assays. Charts were made with GraphPad Prism software and data were presented as mean ± standard error.

RESULTS: Our findings showed that NeuroD1 expression is ectopically upregulated in postmitotic neurons of NeuroD2/6 DKO neocortex and hippocampus. We detected changes in expression of key transcription factors, Cux1, Tbr1, Lhx2, and Id2. Additionally, Cux1 was shown to be direct target of NeuroD2/6. Moreover, Olig2⁺ progenitors were increased in NeuroD2/6 DKO neocortex and expression of NeuroD2/6 and Olig2 was mutually exclusive. Thus, NeuroD2/6 regulates the expression of transcription factors in the developing brain.

CONCLUSION: Our findings indicate that cumulative action of NeuroD2 and NeuroD6 is required to initiate and maintain the expression of transcription factors Cux1, Tbr1, Lhx2, and Id2. Additionally, both genes are required to prevent premature differentiation of Olig2 positive glial precursors.

Keywords

NeuroD / transcription factors / ectopic expression / cerebral cortex

References

[1] Lodato S, Arlotta P. Generating neuronal diversity in the mammalian cerebral cortex. Annu Rev Cell Dev Biol. 2015;31:699–720. doi: 10.1146/annurev-cellbio-100814-125353

[2] Lodato S., Arlotta P. Generating neuronal diversity in the mammalian cerebral cortex // Annu Rev Cell Dev Biol. 2015. Vol. 31. P. 699–720. doi: 10.1146/annurev-cellbio-100814-125353

[3] Yan K. NeuroD family transcription factors regulate corpus callosum formation and cell differentiation during cerebral cortical development [dissertation]. Berlin; 2016. Available from: https://refubium.fu-berlin.de/bitstream/handle/fub188/3671/Diss_Kuo.Yan.pdf?sequence=1&isAllowed=y

[4] Yan K. NeuroD family transcription factors regulate corpus callosum formation and cell differentiation during cerebral cortical development : dissertation. Berlin, 2016. Режим доступа: https://refubium.fu-berlin.de/bitstream/handle/fub188/3671/Diss_Kuo.Yan.pdf?sequence=1&isAllowed=y

[5] Ferguson BR, Gao WJ. PV interneurons: critical regulators of e/i balance for prefrontal cortex-dependent behavior and psychiatric disorders. Front Neural Circuits. 2018;12:37. doi: 10.3389/fncir.2018.00037

[6] Ferguson B.R., Gao W.J. PV interneurons: critical regulators of E/I balance for prefrontal cortex-dependent behavior and psychiatric disorders // Front Neural Circuits. 2018. Vol. 12. P. 37. doi: 10.3389/fncir.2018.00037

[7] Fishell G, Hanashima C. Pyramidal neurons grow up and change their mind. Neuron. 2008;57(3):333–338. doi: 10.1016/j.neuron.2008.01.018

[8] Fishell G., Hanashima C. Pyramidal neurons grow up and change their mind // Neuron. 2008. Vol. 57, N 3. P. 333–338. doi: 10.1016/j.neuron.2008.01.018

[9] Longo A, Guanga GP, Rose RB. Crystal structure of E47-NeuroD1/beta2 bHLH domain-DNA complex: heterodimer selectivity and DNA recognition. Biochemistry. 2008;47(1):218–229. doi: 10.1021/bi701527r

[10] Longo A., Guanga G.P., Rose R.B. Crystal structure of E47-NeuroD1/beta2 bHLH domain-DNA complex: heterodimer selectivity and DNA recognition // Biochemistry. 2008. Vol. 47, N 1. P. 218–229. doi: 10.1021/bi701527r

[11] Tutukova S, Tarabykin V, Hernandez-Miranda LR. The role of neurod genes in brain development, function, and disease. Front Mol Neurosci. 2021;14:662774. doi: 10.3389/fnmol.2021.662774

[12] Tutukova S., Tarabykin V., Hernandez-Miranda L.R. The role of neurod genes in brain development, function, and disease // Front Mol Neurosci. 2021. Vol. 14. P. 662774. doi: 10.3389/fnmol.2021.662774

[13] Liu M, Pleasure SJ, Collins AE, et al. Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy. Proc Natl Acad Sci U S A. 2000;97(2):865–870. Corrected and republished from: Proc Natl Acad Sci U S A. 2000;97(10):5679. doi: 10.1073/pnas.97.2.865

[14] Liu M., Pleasure S.J., Collins A.E., et al. Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy // Proc Natl Acad Sci U S A. 2000. Vol. 97, N 2. P. 865–870. Corrected and republished from: Proc Natl Acad Sci U S A. 2000. Vol. 97, N 10. P. 5679. doi: 10.1073/pnas.97.2.865

[15] Ince-Dunn G, Hall BJ, Hu SC, et al. Regulation of thalamocortical patterning and synaptic maturation by NeuroD2. Neuron. 2006;49(5):683–695. doi: 10.1016/j.neuron.2006.01.031

[16] Ince-Dunn G., Hall B.J., Hu S.C., et al. Regulation of thalamocortical patterning and synaptic maturation by NeuroD2 // Neuron. 2006. Vol. 49, N 5. P. 683–695.

[17] Bormuth I, Yan K, Yonemasu T, et al. Neuronal basic helix-loop-helix proteins Neurod2/6 regulate cortical commissure formation before midline interactions. J Neurosci. 2013;33(2):641–651. doi: 10.1523/JNEUROSCI.0899-12.2013

[18] Bormuth I., Yan K., Yonemasu T., et al. Neuronal basic helix-loop-helix proteins Neurod2/6 regulate cortical commissure formation before midline interactions // Neurosci. 2013. Vol. 33, N 2. P. 641–651. doi: 10.1523/JNEUROSCI.0899-12.2013

[19] Yan K, Bormuth I, Bormuth O, et al. TrkB-dependent EphrinA reverse signaling regulates callosal axon fasciculate growth downstream of Neurod2/6. Cereb Cortex. 2023;33(5):1752–1767. doi: 10.1093/cercor/bhac170

[20] Yan K., Bormuth I., Bormuth O., et al. TrkB-dependent EphrinA reverse signaling regulates callosal axon fasciculate growth downstream of Neurod2/6 // Cereb Cortex. 2023. Vol. 33, N 5. P. 1752–1767. doi: 10.1093/cercor/bhac170

[21] Srivatsa S, Parthasarathy S, Molnár Z, Tarabykin V. Sip1 downstream Effector ninein controls neocortical axonal growth, ipsilateral branching, and microtubule growth and stability. Neuron. 2015;85(5):998–1012. doi: 10.1016/j.neuron.2015.01.018

[22] Srivatsa S., Parthasarathy S., Molnár Z., Tarabykin V. Sip1 downstream Effector ninein controls neocortical axonal growth, ipsilateral branching, and microtubule growth and stability // Neuron. 2015. Vol. 85, N 5. P. 998–1012. doi: 10.1016/j.neuron.2015.01.018

[23] Davis MW, Jorgensen EM. ApE, A Plasmid editor: a freely available DNA manipulation and visualization program. Front Bioinform. 2022;2:818619. doi: 10.3389/fbinf.2022.818619

[24] Davis M.W., Jorgensen E.M. ApE, a plasmid editor: a freely available DNA manipulation and visualization program // Front Bioinform. 2022. Vol. 2. P. 818619. doi: 10.3389/fbinf.2022.818619

[25] Iavarone A, Garg P, Lasorella A, et al. The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein. Genes Dev. 1994;8(11):1270–1284. doi: 10.1101/gad.8.11.1270

[26] Iavarone A., Garg P., Lasorella A., et al. The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein // Genes Dev. 1994. Vol. 8, N 11. P. 1270–1284. doi: 10.1101/gad.8.11.1270

[27] Bedogni F, Hodge RD, Elsen GE, et al. Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex. Proc Natl Acad Sci U S A. 2010;107(29):13129–13134. doi: 10.1073/pnas.1002285107

[28] Bedogni F., Hodge R.D., Elsen G.E., et al. Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex // Proc Natl Acad Sci U S A. 2010. Vol. 107, N 29. P. 13129–13134. doi: 10.1073/pnas.1002285107

[29] Ono K, Takebayashi H, Ikenaka K. Olig2 transcription factor in the developing and injured forebrain; cell lineage and glial development. Mol Cells. 2009;27(4):397–401. doi: 10.1007/s10059-009-0067-2

[30] Ono K., Takebayashi H., Ikenaka K. Olig2 transcription factor in the developing and injured forebrain; cell lineage and glial development // Mol Cells. 2009. Vol. 27, N 4. P. 397–401. doi: 10.1007/s10059-009-0067-2

[31] Marshall CA, Novitch BG, Goldman JE. Olig2 directs astrocyte and oligodendrocyte formation in postnatal subventricular zone cells. J Neurosci. 2005;25(32):7289–7298. doi: 10.1523/JNEUROSCI.1924-05.2005

[32] Marshall C.A., Novitch B.G., Goldman J.E. Olig2 directs astrocyte and oligodendrocyte formation in postnatal subventricular zone cells // J Neurosci. 2005. Vol. 25, N 32. P. 7289–7298. doi: 10.1523/JNEUROSCI.1924-05.2005

[33] Fode C, Ma Q, Casarosa S, et al. A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. Genes Dev. 2000;14(1):67–80.

[34] Fode C., Ma Q., Casarosa S., et al. A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons // Genes Dev. 2000. Vol. 14, N 1. P. 67–80.

[35] Mattar P, Langevin LM, Markham K, et al. Basic helix-loop-helix transcription factors cooperate to specify a cortical projection neuron identity. Mol Cell Biol. 2008;28(5):1456–1469. doi: 10.1128/MCB.01510-07

[36] Mattar P., Langevin L.M., Markham K., et al. Basic helix-loop-helix transcription factors cooperate to specify a cortical projection neuron identity // Mol Cell Biol. 2008. Vol. 28, N 5. P. 1456–1469. doi: 10.1128/MCB.01510-07

[37] Lee S, Lee B, Lee JW, Lee SK. Retinoid signaling and neurogenin2 function are coupled for the specification of spinal motor neurons through a chromatin modifier CBP. Neuron. 2009;62(5):641–654. doi: 10.1016/j.neuron.2009.04.025

[38] Lee S., Lee B., Lee J.W., Lee S.K. Retinoid signaling and neurogenin2 function are coupled for the specification of spinal motor neurons through a chromatin modifier CBP // Neuron. 2009. Vol. 62, N 5. P. 641–654. doi: 10.1016/j.neuron.2009.04.025

[39] Ma Q, Fode C, Guillemot F, Anderson DJ. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 1999;13(13):1717–1728. doi: 10.1101/gad.13.13.1717

[40] Ma Q., Fode C., Guillemot F., Anderson D.J. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia // Genes Dev. 1999. Vol. 13, N 13. P. 1717–1728. doi: 10.1101/gad.13.13.1717

[41] Kim WY. NeuroD regulates neuronal migration. Mol Cells. 2013;35(5):444–449. doi: 10.1007/s10059-013-0065-2

[42] Kim W.Y. NeuroD regulates neuronal migration // Mol Cells. 2013. Vol. 35, N 5. P. 444–449. doi: 10.1007/s10059-013-0065-2

[43] Schwab MH, Bartholomae A, Heimrich B, et al. Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. J Neurosci. 2000;20(10):3714–3724. Corrected and republished from: J Neurosci. 2000;20(21):8227. doi: 10.1523/JNEUROSCI.20-10-03714.2000

[44] Schwab M.H., Bartholomae A., Heimrich B., et al. Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus // Neurosci. 2000. Vol. 20, N 10. P. 3714–3724. Corrected and republished from: J Neurosci 2000. Vol. 20. P. 8227. doi: 10.1523/JNEUROSCI.20-10-03714.2000

[45] Olson JM, Asakura A, Snider L, et al. NeuroD2 is necessary for development and survival of central nervous system neurons. Dev Biol. 2001;234(1):174–187. doi: 10.1006/dbio.2001.0245

[46] Olson J.M., Asakura A., Snider L., et al. NeuroD2 is necessary for development and survival of central nervous system neurons // Dev Biol. 2001. Vol. 234, N 1. P. 174–187. doi: 10.1006/dbio.2001.0245

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