- Email: [email protected]
Formation of dorsal–ventral axis of the pallium derived from mouse embryonic stem cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells Makoto Nasu a, *, Kenji Shimamura b, Shigeyuki Esumi a, Nobuaki Tamamaki a a
Department of Morphological Neural Science, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku, Kumamoto, 860-8556, Japan b Department of Brain Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, 2-2-1, Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2019 Accepted 14 January 2020 Available online xxx
The telencephalon is one of the most-elaborated tissues. A broad variety of cell types is produced by spatiotemporally regulated mechanisms and is involved, in different combinations, in subregional formation. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsaleventral (DeV) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP, respectively). An in vitro differentiation system has been achieved using mouse embryonic stem cells, and major telencephalic neurons can be obtained in this way; however, in using the in vitro differentiation system, many telencephalic neuron subtypes remain undifferentiated, although some of them are related to neuronal diseases. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction. A continuous arrangement of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/ Pax6þ cells was achieved in Foxg1þ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1þ cells resided in the VP fraction. These findings suggested that the DeV axis of the pallium was recapitulated in the in vitroederived pallium. © 2020 Elsevier Inc. All rights reserved.
Keywords: Embryonic stem cell In vitro differentiation The pallium The dorsaleventral axis
1. Introduction The telencephalon is one of the most-elaborated tissues. A broad variety of cell types are produced by spatiotemporally regulated mechanisms and are involved, in different combinations, in subregional formation. Irregular proportions of neurons composing the telencephalon cause mental disorders [1e5]. Animal models are required to elucidate the pathophysiology of such disorders; however, there are some problems in establishing animal models. Some mental disorders are human-specific, and animal models are insufficiently complex to reproduce mental disorders. Progress in stem cell biology using embryonic stem (ES) cells and inducedpluripotent stem (iPS) cells is expected to resolve these problems [6,7]. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsaleventral (DeV) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP,
* Corresponding author. E-mail address: [email protected] (M. Nasu).
respectively), giving rise to the archicortex (hippocampus), the neocortex, the paleocortex (piriform cortex), and migratory cells, including amygdaloid and CajaleRetzius cells [8e10]. Since the development of in vitro differentiation system using mouse ES cells [11,12], differentiation methods have been developed for a broad range of telencephalic cells, such as neocortical layer-specific neurons [13,14]; dorsal midline tissues, including hippocampal neurons [13,15]; and the subpallium (the ventral half of the telencephalon)-derived GABAergic neurons [16e18]. Although these methods can be used to obtain major telencephalic neurons, many telencephalic neuron subtypes remain undifferentiated when using the in vitro differentiation system, although some of these are related to neuronal diseases [19,20]. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction using Sox1::EGFP ES cells. We then examined, using Foxg1:venus ES cells, whether DeV axis formation was achieved in the in vitroederived pallium. Our results showed that continuous arrangement of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/Pax6þ cells was achieved in some Foxg1þ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1þ cells resided in the VP
https://doi.org/10.1016/j.bbrc.2020.01.070 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
2
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 1. Inhibition of the TGFbeta signal was efficient for the neural induction of Sox1::EGFP ES cells. (a,b) Whole-mount fluorescence images on day 5 merged with phase contrast images. Sox1::EGFP ES cells were cultured in the absence [a, TGFi()] and presence [b, TGFi(þ)] of SB 431542. (c) Relative size of aggregates in the absence [TGFi()] and presence [TGFi(þ)] of SB 431542. The average size of TGFi() aggregates was set as 1. (d) Relative fluorescence intensity of aggregates in the absence [TGFi()] and presence [TGFi(þ)] of SB 431542. The average intensity of TGFi() aggregates was set as 1. Values are expressed as mean ± standard error of the mean (SEM). Scale bars, 200 mm *P < 0.05.
fraction. These findings suggested that the DeV axis of the pallium was recapitulated in the in vitroederived pallium. 2. Material and methods 2.1. ES cell culture Mouse ES cells [Foxg1:venus [13] and Sox1::EGFP [21], kindly gifted by Dr. Austin Smith (Cambridge University)] were maintained as described previously [22]. In brief, ES cells were cultured on feeder-free, gelatin-coated dishes with a maintenance medium comprising Glasgow Minimum Essential Medium supplemented with 10% Knockout Serum Replacement (KSR; Thermo), 1% foetal calf serum (FCS), 1 mM pyruvate (Sigma-Aldrich), 0.1 mM
nonessential amino acids (Thermo), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), and leukaemia inhibitory factor. With minor modifications, we used the same in vitro differentiation conditions for the 3D culture as described in our previous study [22]. In brief, ES cells were dissociated in 0.25% trypsin and re-aggregated rapidly by plating on 96-well low cell adhesion culture plates (Sumilon) in differentiation medium (5,000 cells per 100 ml/well). The differentiation medium was a growth factor-free chemically defined medium (gfCDM) comprising Iscove’s modified Dulbecco’s medium (IMDM; FUJIFILM Wako)/Ham’s F12 medium (FUJIFILM Wako) 1:1, 1 Chemically Defined Lipid Concentrate (Thermo), 450 mM monothioglycerol (Sigma-Aldrich), 5 mg/ml purified bovine serum albumin, 5.5 mg/ml apo-transferrin, 6.7 ng/ml selenium, and 10 mg/ ml insulin. We denoted the ES cell seeding day as differentiation
Fig. 2. Process of neural induction of Sox1::EGFP ES cells. (a) Snapshots of time-lapse imaging of Sox1::EGFP ES cells under the TGFi condition during days 1e6. (b) Snapshots of time-lapse imaging of Sox1::EGFP ES cells under the TGFi and WNTi condition during days 1e6. (c) Arbitrary unit (A.U.) of fluorescence intensity of Sox1::EGFP ES cells cultured under the TGFi (solid line) and TGFi/WNTi (dashed line) conditions. Time interval, 4 h. (d) Perimeter (mm) of Sox1::EGFP ES cell aggregates cultured under the TGFi (solid line) and TGFi/WNTi (dashed line) conditions. Time interval, 4 h. Values are expressed as mean ± SEM. Scale bars, 200 mm ***P < 0.001.
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 3. DeV axis formation within the pallium. (a,b) In vivo pattern of expression of Emx1 (a) and Pax6 (b) at E12. (c) In vitroederived Foxg1þ neuroepithelium, which is a telencephalic tissue. (d,e) Foxg1þ neuroepithelium was positive for pallial markers Emx1 (d) and Pax6 (e). Triangles indicate virtual expressional gradients. Hoechst 33342 was used to counterstain nuclei (blue). Lv, lateral ventricle; Pa, pallium; MP/DP/LP/VP, medial/dorsal/lateral/ventral pallium; Sp, subpallium. Scale bars, 200 mm (a,b); 100 mm (cee). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
day 0. On day 5, the cell aggregates were transferred to bacterialgrade, non-coated dishes with Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12 medium supplemented with N2 supplement and apo-transferrin (FUJIFILM Wako). The TGF inhibitor SB 431542 (TGFi) was added at a final concentration of 5 mM on day 0. The Wnt inhibitors IWP2 and IWR1e (WNTi) were added on day 0 at respective final concentrations of 1 mM and 2 mM. The final one percent of Matrigel (Corning) was added on day 1. Sonic hedgehog (Shh) was added at final concentrations of 10 nM (days 3e5) and 30 nM (days 5e12).
at the indicated dilutions: anti-Ascl1/Mash1 (mouse, 1:200; BD Biosciences), anti-Dbx1 (guinea pig, 1:2,000; custom made [23]), anti-Emx1 (guinea pig, 1:1,000; TaKaRa), anti-Neurog2 (goat, 1:1,000; Santa Cruz), anti-Pax6 (rabbit, 1:2,000; abcam), and antiTbr2 (rabbit, 1:1,000; abcam). Images were obtained via a fluorescence microscope (OLYMPUS, KEYENCE) and laser-scanning confocal microscope (OLYMPUS, Carl Zeiss) and analysed using ImageJ. All animal experiments were performed in accordance with institutional (Kumamoto University) guidelines, and they were approved by the animal care and use committee of Kumamoto University.
2.2. Time-lapse imaging 2.4. Statistical analyses Time-lapse imaging was performed during days 1e6 using the IncuCyte Zoom live cell imaging system (Essen Bioscience). Aggregates were transferred to a 48-well plate on day 1 and embedded with Matrigel (Corning) for fixing. The differentiation medium was mixed with an equivalent volume of fresh gfCDM. We acquired time-lapse images of phase contrast and green fluorescence at 4-h intervals. Four and three aggregates were recorded under two distinct conditions, the presence and absence of WNTi with SB 431542, respectively. Aggregate size was represented by measurement of the perimeters (mm). Fluorescent intensity was standardised by the area of aggregates and expressed as the arbitrary unit (A.U.) of fluorescent intensity. 2.3. Immunostaining We sacrificed Jcl:ICR mice at either embryonic day 12 (E12) or embryonic day 14 (E14). All mice were anaesthetised with Medetomidine/Midazolam/Butorphanol tartrate/PBS (final dose, 0.3 mg/ kg; 4 mg/kg; 5 mg/kg, respectively), perfused with 4% paraform aldehyde and post-fixed with 4% paraform aldehyde for 1 h. Frozen tissues were sliced coronally at 12 mm. Immunohistochemistry was conducted as described. We used the following primary antibodies
Student’s t-tests were conducted to compare the size and fluorescence intensity of aggregates under distinct culture conditions. Two-way ANOVAs were conducted to compare the time-lapse images. P < 0.05 (two-tailed) was considered significant for all tests. 3. Results 3.1. Efficient conditions for neural induction Previously, we ascertained minimal, efficient conditions for inducing the telencephalon using gfCDM [22]. In this study, we further examined whether those conditions were efficient for neural induction to assess the effect of a TGFb signal inhibitor (SB431542, TGFi) and WNT signal inhibitors (IWP2 and IWR1endo, WNTi). We used Sox1::EGFP ES cells expressing EGFP specifically in neural lineage cells, as Sox1 is well-known as a marker for early neural lineage cells [24,25]. Initially, we studied the neural induction rate in either the presence or the absence of TGFi from the beginning of the differentiation culture. Although the size of aggregates was almost independent of the presence or absence of TGFi on day 5, EGFP expression by Sox1::EGFP ES cell aggregates
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
4
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. VP-derived neurons. (a) Dbx1 expresses in the VP at E12. (b,c) Ngn2 (b) and Tbr2 (c) express in the whole pallium at E12. (d) Ascl1 expresses in the subpallium. (e) Pax6 expresses in the whole pallium at E14. The Pax6þ/Ngn2þ/Tbr2þ pallium and Ascl1þ subpallium adjoin each other at the pallialesubpallial boundary (PSB) (indicated by arrows). In the vicinity of the boundary, Dbx1 expresses in a small portion of the Ngn2þ fraction. (fen) Serial sections of in vitroederived Foxg1þ neuroepithelia. (feh) In the Foxg1þ fraction (f), Ngn2 (g) and Tbr2 (h) expressions indicated the pallial cells. (iek) In the Foxg1þ neuroepithelium (i), Ngn2þ (j) and Ascl1þ (k) fractions adjoined at the PSB. (len) In the Foxg1þ/Pax6þ fraction (l,m), Dbx1 expressions (n, indicated by white arrowheads) were observed in the vicinity of the PSB. Hoechst 33342 was used to counterstain nuclei (blue). Scale bars, 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
was higher under the TGFi condition (Fig. 1aed), in consistent with previous findings [12,26]. We then carried out time-lapse imaging of Sox1::EGFP ES cells from days 1e6 to observe the neural induction process under either the TGFi or the TGFi/WNTi conditions. Sox1::EGFP fluorescence started becoming visible over the aggregate around day 3, and its intensity strengthened with the lapse of time (Fig. 2aec; Supplementary movie 1,2). WNTi administration was neutral for neural induction under the TGFi condition, although the aggregates were significantly smaller under the TGFi/ WNTi condition than under the condition without WNTi (Fig. 2c and d). Inhibiting TGFb and WNT signalling had no effect on the timing of neural lineage commitment. The growth curves of the perimeter were linear to time and independent of the neuralisation, which began from day 3. Thus, we found that inhibiting the TGFbeta signal was efficient for neural induction. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2020.01.070.
3.2. Formation of pallial subdivisions Previously, we made a boundary formation of multi-lineage cells in a continuous neuroepithelium by utilising an extracellular matrix, such as the cortex-hem boundary, giving rise to the hem, the choroid plexus and the hem-derived CajaleRetzius cells, along with the pallialesubpallial boundary (PSB) [22]. Although this
implied the establishment of the DeV gradient in the pallial lineage, this is not obvious thus far. Emx1 and Pax6 are progenitor markers for the pallium [27e30]. Though they co-localise in the greater portion of the pallium, their expressions are complementary in the dorsal and ventral edge regions; Emx1 expresses in a high medial to low ventral gradient, and Pax6 expresses in a high ventral/lateral to low medial gradient (Fig. 3a and b). Thus, three lineages of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/Pax6þ cells were arranged continuously along the DeV axis of the pallium, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. Using Emx1 and Pax6 for the DeV markers within the pallium and Foxg1:venus ES cells expressing fluorescent venus specifically in the telecephalon, we examined whether DeV axis formation was achieved in the in vitroederived pallium. Our findings showed that there was continuous arrangement of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/Pax6þ cells in some Foxg1þ neuroepithelia (Fig. 3cee; Supplementary Fig. 1a). This suggested that the DeV axis of the pallium was recapitulated in the in vitroederived pallium (from left to right in Supplementary Fig. 1a). The MP, DP, and LP contribute to cortical formation, and the VP produces characteristic cells, including amygdaloid and CajaleRetzius cells, which migrate cells from their point of origin and are derived from Dbx1þ progenitors (Fig. 4a and b; Supplementary Fig. 1b) [31,32]. To verify the DeV formation in vitro,
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
we studied the differentiation of these VP-derived cells. The VP is the ventral-most part of the pallium (Pax6þ/Neurog2þ), and it adjoins the subpallium (Ascl1/Mash1þ) ventrally at the PSB (Fig. 4bee; Supplementary Figs. 4c and d). Thus, we searched the Foxg1þ neuroepithelium containing the boundary of Neurog2þ and Ascl1þ cells at day 12. In consistent with our previous findings, administering Sonic hedgehog (Shh) induced not only the ventral aspect of telencephalon but also the PSB-containing neuroepithelium (Fig. 4fek; Supplementary Figs. 1e and f) [22]. In some of the PSB-containing neuroepithelia, we found a small portion of Dbx1þ cells in the Foxg1þ/Pax6þ pallium (Fig. 4len; Supplementary Figs. 1g and h). Moreover, the position of Dbx1þ cells was well recapitulated in vivo; Dbx1þ cells were apart from the ventral lumen, where apical progenitors reside. 4. Discussion We showed that ES cellederived aggregates in a 3D selforganising culture retained the continuous neuroepithelium containing multi-lineage cells, including the boundary formation and DeV axis formation within the pallium, which are seen in vivo. The neocortex includes multiple domains and has a key role in highorder functions, including memory, speech, value judgements and sociality, through different combinations of those multi-functional domains. Examining neurodevelopmental disorders in a cellular base more deeply will necessitate more elaborate modelling. Even under the conditions of our study, we could obtain a small portion of subregional cells and more complex organoids, comprising a broad range of cell types. Improvement of the in vitroederived organoid model will facilitate examination of the pathophysiology of mental disorders. Author contributions M.N. conceived and designed the research, performed all experiments and analyses, and wrote the manuscript. K.S., E.S. and N.T. helped performing the experiments. Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP15K18957 (MN) and funded by Center for Metabolic Regulation of Healthy Aging, Kumamoto University. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.070 Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.070. References [1] C.L. Beasley, Z.J. Zhang, I. Patten, G.P. Reynolds, Selective deficits in prefrontal cortical GABAergic neurons in schizophrenia defined by the presence of calcium-binding proteins, Biol. Psychiatry 52 (2002) 708e715. [2] T. Hashimoto, D.W. Volk, S.M. Eggan, K. Mirnics, J.N. Pierri, Z. Sun, A.R. Sampson, D.A. Lewis, Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia, J. Neurosci. 23
5
(2003) 6315e6326. [3] P. Levitt, K.L. Eagleson, E.M. Powell, Regulation of neocortical interneuron development and the implications for neurodevelopmental disorders, Trends Neurosci. 27 (2004) 400e406. [4] E. Rossignol, Genetics and function of neocortical GABAergic interneurons in neurodevelopmental disorders, Neural Plast. 2011 (2011) 30e40. [5] M. Tsubomoto, R. Kawabata, X. Zhu, Y. Minabe, K. Chen, D.A. Lewis, T. Hashimoto, Expression of transcripts selective for GABA neuron subpopulations across the cortical visuospatial working memory network in the Healthy state and schizophrenia, Cerebr. Cortex 29 (2019) 3540e3550. [6] Y. Sasai, Next-generation regenerative medicine: organogenesis from stem cells in 3D culture, Cell Stem Cell 12 (2013) 520e530. [7] K. Ardhanareeswaran, J. Mariani, G. Coppola, A. Abyzov, F.M. Vaccarino, Human induced pluripotent stem cells for modelling neurodevelopmental disorders, Nat. Rev. Neurol. 13 (2017) 265e278. [8] L. Puelles, E. Kuwana, E. Puelles, J.L.R. Rubenstein, Comparison of the mammalian and avian telencephalon from the perspective of gene expression data, Eur. J. Morphol. 37 (1999) 139e150. [9] L. Puelles, E. Kuwana, E. Puelles, a Bulfone, K. Shimamura, J. Keleher, S. Smiga, J.L. Rubenstein, Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1, J. Comp. Neurol. 424 (2000) 409e438. n, Development and evolution of the pallium, Semin. Cell [10] L. Medina, A. Abella Dev. Biol. 20 (2009) 698e711. [11] H. Kawasaki, K. Mizuseki, S. Nishikawa, S. Kaneko, Y. Kuwana, S. Nakanishi, S.I. Nishikawa, Y. Sasai, Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity, Neuron 28 (2000) 31e40. [12] K. Watanabe, D. Kamiya, A. Nishiyama, T. Katayama, S. Nozaki, H. Kawasaki, Y. Watanabe, K. Mizuseki, Y. Sasai, Directed differentiation of telencephalic precursors from embryonic stem cells, Nat. Neurosci. 8 (2005) 288e296. [13] M. Eiraku, K. Watanabe, M. Matsuo-Takasaki, M. Kawada, S. Yonemura, M. Matsumura, T. Wataya, A. Nishiyama, K. Muguruma, Y. Sasai, Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals, Cell Stem Cell 3 (2008) 519e532. [14] N. Gaspard, T. Bouschet, R. Hourez, J. Dimidschstein, G. Naeije, J. van den Ameele, I. Espuny-Camacho, A. Herpoel, L. Passante, S.N. Schiffmann, A. Gaillard, P. Vanderhaeghen, An intrinsic mechanism of corticogenesis from embryonic stem cells, Nature 455 (2008) 351e357. [15] H. Sakaguchi, T. Kadoshima, M. Soen, N. Narii, Y. Ishida, M. Ohgushi, J. Takahashi, M. Eiraku, Y. Sasai, Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue, Nat. Commun. 6 (2015) 8896. [16] A.M. Maroof, K. Brown, S.H. Shi, L. Studer, S.A. Anderson, Prospective isolation of cortical interneuron precursors from mouse embryonic stem cells, J. Neurosci. 30 (2010) 4667e4675. [17] T. Danjo, M. Eiraku, K. Muguruma, K. Watanabe, M. Kawada, Y. Yanagawa, J.L.R. Rubenstein, Y. Sasai, Subregional specification of embryonic stem cellderived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals, J. Neurosci. 31 (2011) 1919e1933. [18] S. Cambray, C. Arber, G. Little, A.G. Dougalis, V. de Paola, M.A. Ungless, M. Li, T.A. Rodríguez, Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors, Nat. Commun. 3 (2012) 841. [19] K. Ishii, K.I. Kubo, K. Nakajima, Reelin and neuropsychiatric disorders, Front. Cell. Neurosci. 10 (2016) 1e13. [20] X. Zhang, T.T. Ge, G. Yin, R. Cui, G. Zhao, W. Yang, Stress-induced functional alterations in amygdala: implications for neuropsychiatric diseases, Front. Neurosci. 12 (2018) 1e9. [21] J. Aubert, M.P. Stavridis, S. Tweedie, M. O’Reilly, K. Vierlinger, M. Li, P. Ghazal, T. Pratt, J.O. Mason, D. Roy, A. Smith, Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 11836e11841. [22] M. Nasu, N. Takata, T. Danjo, H. Sakaguchi, T. Kadoshima, S. Futaki, K. Sekiguchi, M. Eiraku, Y. Sasai, Robust formation and maintenance of continuous stratified cortical neuroepithelium by laminin-containing matrix in mouse ES cell culture, PLoS One 7 (2012) 13e14. [23] A. Shiraishi, K. Muguruma, Y. Sasai, Generation of thalamic neurons from mouse embryonic stem cells, Development 144 (2017) 1211e1220. [24] M. Li, L. Pevny, R. Lovell-Badge, A. Smith, Generation of purified neural precursors from embryonic stem cells by lineage selection, Curr. Biol. 8 (1998) 971e974. [25] L.H. Pevny, S. Sockanathan, M. Placzek, R. Lovell-Badge, A role for SOX1 in neural determination, Development 125 (1998) 1967e1978. [26] S. Parisi, D. D’Andrea, C.T. Lago, E.D. Adamson, M.G. Persico, G. Minchiotti, Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells, J. Cell Biol. 163 (2003) 303e314. rant, E. Boncinelli, M. Wassef, Expression of the [27] A.S. Fernandez, C. Pieau, J. Repe Emx-1 and Dlx-1 homeobox genes define three molecularly distinct domains in the telencephalon of mouse, chick, turtle and frog embryos: implications for the evolution of telencephalic subdivisions in amniotes, Development 125 (1998) 2099e2111. [28] K.M. Bishop, J.L.R. Rubenstein, D.D.M. O’Leary, Distinct actions of Emx1, Emx2, and Pax6 in regulating the specification of areas in the developing neocortex, J. Neurosci. 22 (2002) 7627e7638. [29] D.D.M. O’Leary, S.J. Chou, S. Sahara, Area patterning of the mammalian cortex,
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
6
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Neuron 56 (2007) 252e269. [30] U. Borello, A. Pierani, Patterning the cerebral cortex: traveling with morphogens, Curr. Opin. Genet. Dev. 20 (2010) 408e415. ^me, S. Vigneau, M. Sigrist, S. Arber, [31] F. Bielle, A. Griveau, N. Narboux-Ne M. Wassef, A. Pierani, Multiple origins of Cajal-Retzius cells at the borders of
the developing pallium, Nat. Neurosci. 8 (2005) 1002e1012. [32] T. Hirata, P. Li, G.M. Lanuza, L. a Cocas, M.M. Huntsman, J.G. Corbin, Identification of distinct telencephalic progenitor pools for neuronal diversity in the amygdala, Nat. Neurosci. 12 (2009) 141e149.
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells Makoto Nasu a, *, Kenji Shimamura b, Shigeyuki Esumi a, Nobuaki Tamamaki a a
Department of Morphological Neural Science, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku, Kumamoto, 860-8556, Japan b Department of Brain Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, 2-2-1, Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2019 Accepted 14 January 2020 Available online xxx
The telencephalon is one of the most-elaborated tissues. A broad variety of cell types is produced by spatiotemporally regulated mechanisms and is involved, in different combinations, in subregional formation. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsaleventral (DeV) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP, respectively). An in vitro differentiation system has been achieved using mouse embryonic stem cells, and major telencephalic neurons can be obtained in this way; however, in using the in vitro differentiation system, many telencephalic neuron subtypes remain undifferentiated, although some of them are related to neuronal diseases. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction. A continuous arrangement of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/ Pax6þ cells was achieved in Foxg1þ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1þ cells resided in the VP fraction. These findings suggested that the DeV axis of the pallium was recapitulated in the in vitroederived pallium. © 2020 Elsevier Inc. All rights reserved.
Keywords: Embryonic stem cell In vitro differentiation The pallium The dorsaleventral axis
1. Introduction The telencephalon is one of the most-elaborated tissues. A broad variety of cell types are produced by spatiotemporally regulated mechanisms and are involved, in different combinations, in subregional formation. Irregular proportions of neurons composing the telencephalon cause mental disorders [1e5]. Animal models are required to elucidate the pathophysiology of such disorders; however, there are some problems in establishing animal models. Some mental disorders are human-specific, and animal models are insufficiently complex to reproduce mental disorders. Progress in stem cell biology using embryonic stem (ES) cells and inducedpluripotent stem (iPS) cells is expected to resolve these problems [6,7]. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsaleventral (DeV) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP,
* Corresponding author. E-mail address: [email protected] (M. Nasu).
respectively), giving rise to the archicortex (hippocampus), the neocortex, the paleocortex (piriform cortex), and migratory cells, including amygdaloid and CajaleRetzius cells [8e10]. Since the development of in vitro differentiation system using mouse ES cells [11,12], differentiation methods have been developed for a broad range of telencephalic cells, such as neocortical layer-specific neurons [13,14]; dorsal midline tissues, including hippocampal neurons [13,15]; and the subpallium (the ventral half of the telencephalon)-derived GABAergic neurons [16e18]. Although these methods can be used to obtain major telencephalic neurons, many telencephalic neuron subtypes remain undifferentiated when using the in vitro differentiation system, although some of these are related to neuronal diseases [19,20]. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction using Sox1::EGFP ES cells. We then examined, using Foxg1:venus ES cells, whether DeV axis formation was achieved in the in vitroederived pallium. Our results showed that continuous arrangement of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/Pax6þ cells was achieved in some Foxg1þ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1þ cells resided in the VP
https://doi.org/10.1016/j.bbrc.2020.01.070 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
2
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 1. Inhibition of the TGFbeta signal was efficient for the neural induction of Sox1::EGFP ES cells. (a,b) Whole-mount fluorescence images on day 5 merged with phase contrast images. Sox1::EGFP ES cells were cultured in the absence [a, TGFi()] and presence [b, TGFi(þ)] of SB 431542. (c) Relative size of aggregates in the absence [TGFi()] and presence [TGFi(þ)] of SB 431542. The average size of TGFi() aggregates was set as 1. (d) Relative fluorescence intensity of aggregates in the absence [TGFi()] and presence [TGFi(þ)] of SB 431542. The average intensity of TGFi() aggregates was set as 1. Values are expressed as mean ± standard error of the mean (SEM). Scale bars, 200 mm *P < 0.05.
fraction. These findings suggested that the DeV axis of the pallium was recapitulated in the in vitroederived pallium. 2. Material and methods 2.1. ES cell culture Mouse ES cells [Foxg1:venus [13] and Sox1::EGFP [21], kindly gifted by Dr. Austin Smith (Cambridge University)] were maintained as described previously [22]. In brief, ES cells were cultured on feeder-free, gelatin-coated dishes with a maintenance medium comprising Glasgow Minimum Essential Medium supplemented with 10% Knockout Serum Replacement (KSR; Thermo), 1% foetal calf serum (FCS), 1 mM pyruvate (Sigma-Aldrich), 0.1 mM
nonessential amino acids (Thermo), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), and leukaemia inhibitory factor. With minor modifications, we used the same in vitro differentiation conditions for the 3D culture as described in our previous study [22]. In brief, ES cells were dissociated in 0.25% trypsin and re-aggregated rapidly by plating on 96-well low cell adhesion culture plates (Sumilon) in differentiation medium (5,000 cells per 100 ml/well). The differentiation medium was a growth factor-free chemically defined medium (gfCDM) comprising Iscove’s modified Dulbecco’s medium (IMDM; FUJIFILM Wako)/Ham’s F12 medium (FUJIFILM Wako) 1:1, 1 Chemically Defined Lipid Concentrate (Thermo), 450 mM monothioglycerol (Sigma-Aldrich), 5 mg/ml purified bovine serum albumin, 5.5 mg/ml apo-transferrin, 6.7 ng/ml selenium, and 10 mg/ ml insulin. We denoted the ES cell seeding day as differentiation
Fig. 2. Process of neural induction of Sox1::EGFP ES cells. (a) Snapshots of time-lapse imaging of Sox1::EGFP ES cells under the TGFi condition during days 1e6. (b) Snapshots of time-lapse imaging of Sox1::EGFP ES cells under the TGFi and WNTi condition during days 1e6. (c) Arbitrary unit (A.U.) of fluorescence intensity of Sox1::EGFP ES cells cultured under the TGFi (solid line) and TGFi/WNTi (dashed line) conditions. Time interval, 4 h. (d) Perimeter (mm) of Sox1::EGFP ES cell aggregates cultured under the TGFi (solid line) and TGFi/WNTi (dashed line) conditions. Time interval, 4 h. Values are expressed as mean ± SEM. Scale bars, 200 mm ***P < 0.001.
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 3. DeV axis formation within the pallium. (a,b) In vivo pattern of expression of Emx1 (a) and Pax6 (b) at E12. (c) In vitroederived Foxg1þ neuroepithelium, which is a telencephalic tissue. (d,e) Foxg1þ neuroepithelium was positive for pallial markers Emx1 (d) and Pax6 (e). Triangles indicate virtual expressional gradients. Hoechst 33342 was used to counterstain nuclei (blue). Lv, lateral ventricle; Pa, pallium; MP/DP/LP/VP, medial/dorsal/lateral/ventral pallium; Sp, subpallium. Scale bars, 200 mm (a,b); 100 mm (cee). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
day 0. On day 5, the cell aggregates were transferred to bacterialgrade, non-coated dishes with Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12 medium supplemented with N2 supplement and apo-transferrin (FUJIFILM Wako). The TGF inhibitor SB 431542 (TGFi) was added at a final concentration of 5 mM on day 0. The Wnt inhibitors IWP2 and IWR1e (WNTi) were added on day 0 at respective final concentrations of 1 mM and 2 mM. The final one percent of Matrigel (Corning) was added on day 1. Sonic hedgehog (Shh) was added at final concentrations of 10 nM (days 3e5) and 30 nM (days 5e12).
at the indicated dilutions: anti-Ascl1/Mash1 (mouse, 1:200; BD Biosciences), anti-Dbx1 (guinea pig, 1:2,000; custom made [23]), anti-Emx1 (guinea pig, 1:1,000; TaKaRa), anti-Neurog2 (goat, 1:1,000; Santa Cruz), anti-Pax6 (rabbit, 1:2,000; abcam), and antiTbr2 (rabbit, 1:1,000; abcam). Images were obtained via a fluorescence microscope (OLYMPUS, KEYENCE) and laser-scanning confocal microscope (OLYMPUS, Carl Zeiss) and analysed using ImageJ. All animal experiments were performed in accordance with institutional (Kumamoto University) guidelines, and they were approved by the animal care and use committee of Kumamoto University.
2.2. Time-lapse imaging 2.4. Statistical analyses Time-lapse imaging was performed during days 1e6 using the IncuCyte Zoom live cell imaging system (Essen Bioscience). Aggregates were transferred to a 48-well plate on day 1 and embedded with Matrigel (Corning) for fixing. The differentiation medium was mixed with an equivalent volume of fresh gfCDM. We acquired time-lapse images of phase contrast and green fluorescence at 4-h intervals. Four and three aggregates were recorded under two distinct conditions, the presence and absence of WNTi with SB 431542, respectively. Aggregate size was represented by measurement of the perimeters (mm). Fluorescent intensity was standardised by the area of aggregates and expressed as the arbitrary unit (A.U.) of fluorescent intensity. 2.3. Immunostaining We sacrificed Jcl:ICR mice at either embryonic day 12 (E12) or embryonic day 14 (E14). All mice were anaesthetised with Medetomidine/Midazolam/Butorphanol tartrate/PBS (final dose, 0.3 mg/ kg; 4 mg/kg; 5 mg/kg, respectively), perfused with 4% paraform aldehyde and post-fixed with 4% paraform aldehyde for 1 h. Frozen tissues were sliced coronally at 12 mm. Immunohistochemistry was conducted as described. We used the following primary antibodies
Student’s t-tests were conducted to compare the size and fluorescence intensity of aggregates under distinct culture conditions. Two-way ANOVAs were conducted to compare the time-lapse images. P < 0.05 (two-tailed) was considered significant for all tests. 3. Results 3.1. Efficient conditions for neural induction Previously, we ascertained minimal, efficient conditions for inducing the telencephalon using gfCDM [22]. In this study, we further examined whether those conditions were efficient for neural induction to assess the effect of a TGFb signal inhibitor (SB431542, TGFi) and WNT signal inhibitors (IWP2 and IWR1endo, WNTi). We used Sox1::EGFP ES cells expressing EGFP specifically in neural lineage cells, as Sox1 is well-known as a marker for early neural lineage cells [24,25]. Initially, we studied the neural induction rate in either the presence or the absence of TGFi from the beginning of the differentiation culture. Although the size of aggregates was almost independent of the presence or absence of TGFi on day 5, EGFP expression by Sox1::EGFP ES cell aggregates
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
4
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. VP-derived neurons. (a) Dbx1 expresses in the VP at E12. (b,c) Ngn2 (b) and Tbr2 (c) express in the whole pallium at E12. (d) Ascl1 expresses in the subpallium. (e) Pax6 expresses in the whole pallium at E14. The Pax6þ/Ngn2þ/Tbr2þ pallium and Ascl1þ subpallium adjoin each other at the pallialesubpallial boundary (PSB) (indicated by arrows). In the vicinity of the boundary, Dbx1 expresses in a small portion of the Ngn2þ fraction. (fen) Serial sections of in vitroederived Foxg1þ neuroepithelia. (feh) In the Foxg1þ fraction (f), Ngn2 (g) and Tbr2 (h) expressions indicated the pallial cells. (iek) In the Foxg1þ neuroepithelium (i), Ngn2þ (j) and Ascl1þ (k) fractions adjoined at the PSB. (len) In the Foxg1þ/Pax6þ fraction (l,m), Dbx1 expressions (n, indicated by white arrowheads) were observed in the vicinity of the PSB. Hoechst 33342 was used to counterstain nuclei (blue). Scale bars, 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
was higher under the TGFi condition (Fig. 1aed), in consistent with previous findings [12,26]. We then carried out time-lapse imaging of Sox1::EGFP ES cells from days 1e6 to observe the neural induction process under either the TGFi or the TGFi/WNTi conditions. Sox1::EGFP fluorescence started becoming visible over the aggregate around day 3, and its intensity strengthened with the lapse of time (Fig. 2aec; Supplementary movie 1,2). WNTi administration was neutral for neural induction under the TGFi condition, although the aggregates were significantly smaller under the TGFi/ WNTi condition than under the condition without WNTi (Fig. 2c and d). Inhibiting TGFb and WNT signalling had no effect on the timing of neural lineage commitment. The growth curves of the perimeter were linear to time and independent of the neuralisation, which began from day 3. Thus, we found that inhibiting the TGFbeta signal was efficient for neural induction. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2020.01.070.
3.2. Formation of pallial subdivisions Previously, we made a boundary formation of multi-lineage cells in a continuous neuroepithelium by utilising an extracellular matrix, such as the cortex-hem boundary, giving rise to the hem, the choroid plexus and the hem-derived CajaleRetzius cells, along with the pallialesubpallial boundary (PSB) [22]. Although this
implied the establishment of the DeV gradient in the pallial lineage, this is not obvious thus far. Emx1 and Pax6 are progenitor markers for the pallium [27e30]. Though they co-localise in the greater portion of the pallium, their expressions are complementary in the dorsal and ventral edge regions; Emx1 expresses in a high medial to low ventral gradient, and Pax6 expresses in a high ventral/lateral to low medial gradient (Fig. 3a and b). Thus, three lineages of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/Pax6þ cells were arranged continuously along the DeV axis of the pallium, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. Using Emx1 and Pax6 for the DeV markers within the pallium and Foxg1:venus ES cells expressing fluorescent venus specifically in the telecephalon, we examined whether DeV axis formation was achieved in the in vitroederived pallium. Our findings showed that there was continuous arrangement of Emx1þ/Pax6, Emx1þ/Pax6þ, and Emx1/Pax6þ cells in some Foxg1þ neuroepithelia (Fig. 3cee; Supplementary Fig. 1a). This suggested that the DeV axis of the pallium was recapitulated in the in vitroederived pallium (from left to right in Supplementary Fig. 1a). The MP, DP, and LP contribute to cortical formation, and the VP produces characteristic cells, including amygdaloid and CajaleRetzius cells, which migrate cells from their point of origin and are derived from Dbx1þ progenitors (Fig. 4a and b; Supplementary Fig. 1b) [31,32]. To verify the DeV formation in vitro,
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
we studied the differentiation of these VP-derived cells. The VP is the ventral-most part of the pallium (Pax6þ/Neurog2þ), and it adjoins the subpallium (Ascl1/Mash1þ) ventrally at the PSB (Fig. 4bee; Supplementary Figs. 4c and d). Thus, we searched the Foxg1þ neuroepithelium containing the boundary of Neurog2þ and Ascl1þ cells at day 12. In consistent with our previous findings, administering Sonic hedgehog (Shh) induced not only the ventral aspect of telencephalon but also the PSB-containing neuroepithelium (Fig. 4fek; Supplementary Figs. 1e and f) [22]. In some of the PSB-containing neuroepithelia, we found a small portion of Dbx1þ cells in the Foxg1þ/Pax6þ pallium (Fig. 4len; Supplementary Figs. 1g and h). Moreover, the position of Dbx1þ cells was well recapitulated in vivo; Dbx1þ cells were apart from the ventral lumen, where apical progenitors reside. 4. Discussion We showed that ES cellederived aggregates in a 3D selforganising culture retained the continuous neuroepithelium containing multi-lineage cells, including the boundary formation and DeV axis formation within the pallium, which are seen in vivo. The neocortex includes multiple domains and has a key role in highorder functions, including memory, speech, value judgements and sociality, through different combinations of those multi-functional domains. Examining neurodevelopmental disorders in a cellular base more deeply will necessitate more elaborate modelling. Even under the conditions of our study, we could obtain a small portion of subregional cells and more complex organoids, comprising a broad range of cell types. Improvement of the in vitroederived organoid model will facilitate examination of the pathophysiology of mental disorders. Author contributions M.N. conceived and designed the research, performed all experiments and analyses, and wrote the manuscript. K.S., E.S. and N.T. helped performing the experiments. Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP15K18957 (MN) and funded by Center for Metabolic Regulation of Healthy Aging, Kumamoto University. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.070 Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.070. References [1] C.L. Beasley, Z.J. Zhang, I. Patten, G.P. Reynolds, Selective deficits in prefrontal cortical GABAergic neurons in schizophrenia defined by the presence of calcium-binding proteins, Biol. Psychiatry 52 (2002) 708e715. [2] T. Hashimoto, D.W. Volk, S.M. Eggan, K. Mirnics, J.N. Pierri, Z. Sun, A.R. Sampson, D.A. Lewis, Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia, J. Neurosci. 23
5
(2003) 6315e6326. [3] P. Levitt, K.L. Eagleson, E.M. Powell, Regulation of neocortical interneuron development and the implications for neurodevelopmental disorders, Trends Neurosci. 27 (2004) 400e406. [4] E. Rossignol, Genetics and function of neocortical GABAergic interneurons in neurodevelopmental disorders, Neural Plast. 2011 (2011) 30e40. [5] M. Tsubomoto, R. Kawabata, X. Zhu, Y. Minabe, K. Chen, D.A. Lewis, T. Hashimoto, Expression of transcripts selective for GABA neuron subpopulations across the cortical visuospatial working memory network in the Healthy state and schizophrenia, Cerebr. Cortex 29 (2019) 3540e3550. [6] Y. Sasai, Next-generation regenerative medicine: organogenesis from stem cells in 3D culture, Cell Stem Cell 12 (2013) 520e530. [7] K. Ardhanareeswaran, J. Mariani, G. Coppola, A. Abyzov, F.M. Vaccarino, Human induced pluripotent stem cells for modelling neurodevelopmental disorders, Nat. Rev. Neurol. 13 (2017) 265e278. [8] L. Puelles, E. Kuwana, E. Puelles, J.L.R. Rubenstein, Comparison of the mammalian and avian telencephalon from the perspective of gene expression data, Eur. J. Morphol. 37 (1999) 139e150. [9] L. Puelles, E. Kuwana, E. Puelles, a Bulfone, K. Shimamura, J. Keleher, S. Smiga, J.L. Rubenstein, Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1, J. Comp. Neurol. 424 (2000) 409e438. n, Development and evolution of the pallium, Semin. Cell [10] L. Medina, A. Abella Dev. Biol. 20 (2009) 698e711. [11] H. Kawasaki, K. Mizuseki, S. Nishikawa, S. Kaneko, Y. Kuwana, S. Nakanishi, S.I. Nishikawa, Y. Sasai, Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity, Neuron 28 (2000) 31e40. [12] K. Watanabe, D. Kamiya, A. Nishiyama, T. Katayama, S. Nozaki, H. Kawasaki, Y. Watanabe, K. Mizuseki, Y. Sasai, Directed differentiation of telencephalic precursors from embryonic stem cells, Nat. Neurosci. 8 (2005) 288e296. [13] M. Eiraku, K. Watanabe, M. Matsuo-Takasaki, M. Kawada, S. Yonemura, M. Matsumura, T. Wataya, A. Nishiyama, K. Muguruma, Y. Sasai, Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals, Cell Stem Cell 3 (2008) 519e532. [14] N. Gaspard, T. Bouschet, R. Hourez, J. Dimidschstein, G. Naeije, J. van den Ameele, I. Espuny-Camacho, A. Herpoel, L. Passante, S.N. Schiffmann, A. Gaillard, P. Vanderhaeghen, An intrinsic mechanism of corticogenesis from embryonic stem cells, Nature 455 (2008) 351e357. [15] H. Sakaguchi, T. Kadoshima, M. Soen, N. Narii, Y. Ishida, M. Ohgushi, J. Takahashi, M. Eiraku, Y. Sasai, Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue, Nat. Commun. 6 (2015) 8896. [16] A.M. Maroof, K. Brown, S.H. Shi, L. Studer, S.A. Anderson, Prospective isolation of cortical interneuron precursors from mouse embryonic stem cells, J. Neurosci. 30 (2010) 4667e4675. [17] T. Danjo, M. Eiraku, K. Muguruma, K. Watanabe, M. Kawada, Y. Yanagawa, J.L.R. Rubenstein, Y. Sasai, Subregional specification of embryonic stem cellderived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals, J. Neurosci. 31 (2011) 1919e1933. [18] S. Cambray, C. Arber, G. Little, A.G. Dougalis, V. de Paola, M.A. Ungless, M. Li, T.A. Rodríguez, Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors, Nat. Commun. 3 (2012) 841. [19] K. Ishii, K.I. Kubo, K. Nakajima, Reelin and neuropsychiatric disorders, Front. Cell. Neurosci. 10 (2016) 1e13. [20] X. Zhang, T.T. Ge, G. Yin, R. Cui, G. Zhao, W. Yang, Stress-induced functional alterations in amygdala: implications for neuropsychiatric diseases, Front. Neurosci. 12 (2018) 1e9. [21] J. Aubert, M.P. Stavridis, S. Tweedie, M. O’Reilly, K. Vierlinger, M. Li, P. Ghazal, T. Pratt, J.O. Mason, D. Roy, A. Smith, Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 11836e11841. [22] M. Nasu, N. Takata, T. Danjo, H. Sakaguchi, T. Kadoshima, S. Futaki, K. Sekiguchi, M. Eiraku, Y. Sasai, Robust formation and maintenance of continuous stratified cortical neuroepithelium by laminin-containing matrix in mouse ES cell culture, PLoS One 7 (2012) 13e14. [23] A. Shiraishi, K. Muguruma, Y. Sasai, Generation of thalamic neurons from mouse embryonic stem cells, Development 144 (2017) 1211e1220. [24] M. Li, L. Pevny, R. Lovell-Badge, A. Smith, Generation of purified neural precursors from embryonic stem cells by lineage selection, Curr. Biol. 8 (1998) 971e974. [25] L.H. Pevny, S. Sockanathan, M. Placzek, R. Lovell-Badge, A role for SOX1 in neural determination, Development 125 (1998) 1967e1978. [26] S. Parisi, D. D’Andrea, C.T. Lago, E.D. Adamson, M.G. Persico, G. Minchiotti, Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells, J. Cell Biol. 163 (2003) 303e314. rant, E. Boncinelli, M. Wassef, Expression of the [27] A.S. Fernandez, C. Pieau, J. Repe Emx-1 and Dlx-1 homeobox genes define three molecularly distinct domains in the telencephalon of mouse, chick, turtle and frog embryos: implications for the evolution of telencephalic subdivisions in amniotes, Development 125 (1998) 2099e2111. [28] K.M. Bishop, J.L.R. Rubenstein, D.D.M. O’Leary, Distinct actions of Emx1, Emx2, and Pax6 in regulating the specification of areas in the developing neocortex, J. Neurosci. 22 (2002) 7627e7638. [29] D.D.M. O’Leary, S.J. Chou, S. Sahara, Area patterning of the mammalian cortex,
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070
6
M. Nasu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Neuron 56 (2007) 252e269. [30] U. Borello, A. Pierani, Patterning the cerebral cortex: traveling with morphogens, Curr. Opin. Genet. Dev. 20 (2010) 408e415. ^me, S. Vigneau, M. Sigrist, S. Arber, [31] F. Bielle, A. Griveau, N. Narboux-Ne M. Wassef, A. Pierani, Multiple origins of Cajal-Retzius cells at the borders of
the developing pallium, Nat. Neurosci. 8 (2005) 1002e1012. [32] T. Hirata, P. Li, G.M. Lanuza, L. a Cocas, M.M. Huntsman, J.G. Corbin, Identification of distinct telencephalic progenitor pools for neuronal diversity in the amygdala, Nat. Neurosci. 12 (2009) 141e149.
Please cite this article as: M. Nasu et al., Formation of dorsaleventral axis of the pallium derived from mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.070