Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Developmentally primed cortical neurons maintain fidelity of differentiation and establish appropriate functional connectivity after transplantation

Abstract

Repair of complex CNS circuitry requires newly incorporated neurons to become appropriately, functionally integrated. One approach is to direct differentiation of endogenous progenitors in situ, or ex vivo followed by transplantation. Prior studies find that newly incorporated neurons can establish long-distance axon projections, form synapses and functionally integrate in evolutionarily old hypothalamic energy-balance circuitry. We now demonstrate that postnatal neocortical connectivity can be reconstituted with point-to-point precision, including cellular integration of specific, molecularly identified projection neuron subtypes into correct positions, combined with development of appropriate long-distance projections and synapses. Using optogenetics-based electrophysiology, experiments demonstrate functional afferent and efferent integration of transplanted neurons into transcallosal projection neuron circuitry. Results further indicate that ‘primed’ early postmitotic neurons, including already fate-restricted deep-layer projection neurons and/or plastic postmitotic neuroblasts with partially fate-restricted potential, account for the predominant population of neurons capable of achieving this optimal level of integration.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Developmentally primed immature neurons integrate as distinct molecular projection neuron subtypes.
Fig. 2: Transplanted neurons assuming positions in cortical layers II–VI progressively mature and successfully extend long-distance subcerebral axon projections.
Fig. 3: Double birthdating of donor neurons before transplantation to investigate the stage and origin of cells mediating successful cortical integration.
Fig. 4: Newly incorporated neurons of combinatorial subcerebral molecular identity establish anatomical subcerebral output connectivity with high fidelity.
Fig. 5: Newly incorporated neurons of combinatorial callosal molecular identity establish anatomic interhemispheric output connectivity with high fidelity.
Fig. 6: Newly incorporated neurons establish point-to-point long-distance connectivity.
Fig. 7: Newly incorporated neurons afferently integrate into long-distance transcallosal circuitry.
Fig. 8: Transplanted neurons establish functional efferent local and transcallosal synaptic connectivity with ipsi- and contralateral recipient-derived neurons.

Similar content being viewed by others

References

  1. Czupryn, A. et al. Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice. Science 334, 1133–1137 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Southwell, D. G. et al. Interneurons from embryonic development to cell-based therapy. Science 344, 1240622 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Baraban, S. C. et al. Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice. Proc. Natl. Acad. Sci. USA 106, 15472–15477 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Calcagnotto, M. E. et al. Effect of neuronal precursor cells derived from medial ganglionic eminence in an acute epileptic seizure model. Epilepsia 51, 71–75 (2010). Suppl 3.

    Article  PubMed  Google Scholar 

  5. Martínez-Cerdeño, V. et al. Embryonic MGE precursor cells grafted into adult rat striatum integrate and ameliorate motor symptoms in 6-OHDA-lesioned rats. Cell Stem Cell 6, 238–250 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Southwell, D. G., Froemke, R. C., Alvarez-Buylla, A., Stryker, M. P. & Gandhi, S. P. Cortical plasticity induced by inhibitory neuron transplantation. Science 327, 1145–1148 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zipancic, I., Calcagnotto, M. E., Piquer-Gil, M., Mello, L. E. & Alvarez-Dolado, M. Transplant of GABAergic precursors restores hippocampal inhibitory function in a mouse model of seizure susceptibility. Cell Transplant. 19, 549–564 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. De la Cruz, E. et al. Interneuron progenitors attenuate the power of acute focal ictal discharges. Neurotherapeutics 8, 763–773 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Tanaka, D. H., Toriumi, K., Kubo, K., Nabeshima, T. & Nakajima, K. GABAergic precursor transplantation into the prefrontal cortex prevents phencyclidine-induced cognitive deficits. J. Neurosci. 31, 14116–14125 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Hunt, R. F., Girskis, K. M., Rubenstein, J. L., Alvarez-Buylla, A. & Baraban, S. C. GABA progenitors grafted into the adult epileptic brain control seizures and abnormal behavior. Nat. Neurosci. 16, 692–697 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Stanfield, B. B. & O’Leary, D. D. Fetal occipital cortical neurones transplanted to the rostral cortex can extend and maintain a pyramidal tract axon. Nature 313, 135–137 (1985).

    Article  CAS  PubMed  Google Scholar 

  12. Hernit-Grant, C. S. & Macklis, J. D. Embryonic neurons transplanted to regions of targeted photolytic cell death in adult mouse somatosensory cortex re-form specific callosal projections. Exp. Neurol. 139, 131–142 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Shin, J. J. et al. Transplanted neuroblasts differentiate appropriately into projection neurons with correct neurotransmitter and receptor phenotype in neocortex undergoing targeted projection neuron degeneration. J. Neurosci. 20, 7404–7416 (2000).

    CAS  PubMed  Google Scholar 

  14. Fricker-Gates, R. A., Shin, J. J., Tai, C. C., Catapano, L. A. & Macklis, J. D. Late-stage immature neocortical neurons reconstruct interhemispheric connections and form synaptic contacts with increased efficiency in adult mouse cortex undergoing targeted neurodegeneration. J. Neurosci. 22, 4045–4056 (2002).

    CAS  PubMed  Google Scholar 

  15. Magavi, S. S., Leavitt, B. R. & Macklis, J. D. Induction of neurogenesis in the neocortex of adult mice. Nature 405, 951–955 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Gaillard, A. et al. Reestablishment of damaged adult motor pathways by grafted embryonic cortical neurons. Nat. Neurosci. 10, 1294–1299 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Chen, J., Magavi, S. S. & Macklis, J. D. Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice. Proc. Natl. Acad. Sci. USA 101, 16357–16362 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gaspard, N. et al. An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature 455, 351–357 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Ideguchi, M., Palmer, T. D., Recht, L. D. & Weimann, J. M. Murine embryonic stem cell-derived pyramidal neurons integrate into the cerebral cortex and appropriately project axons to subcortical targets. J. Neurosci. 30, 894–904 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Espuny-Camacho, I. et al. Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. Neuron 77, 440–456 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Michelsen, K. A. et al. Area-specific reestablishment of damaged circuits in the adult cerebral cortex by cortical neurons derived from mouse embryonic stem cells. Neuron 85, 982–997 (2015).

    Article  CAS  PubMed  Google Scholar 

  23. Falkner, S. et al. Transplanted embryonic neurons integrate into adult neocortical circuits. Nature 539, 248–253 (2016).

    Article  PubMed  Google Scholar 

  24. Custo Greig, L. F., Woodworth, M. B., Galazo, M. J., Padmanabhan, H. & Macklis, J. D. Molecular logic of neocortical projection neuron specification, development and diversity. Nat. Rev. Neurosci. 14, 755–769 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Leyva-Díaz, E. & López-Bendito, G. In and out from the cortex: development of major forebrain connections. Neuroscience 254, 26–44 (2013).

    Article  PubMed  Google Scholar 

  26. Arlotta, P. et al. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207–221 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Alcamo, E. A. et al. Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57, 364–377 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Britanova, O. et al. Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57, 378–392 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Galazo, M. J., Emsley, J. G. & Macklis, J. D. corticothalamic projection neuron development beyond subtype specification: Fog2 and intersectional controls regulate intraclass neuronal diversity. Neuron 91, 90–106 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Molyneaux, B. J., Arlotta, P., Hirata, T., Hibi, M. & Macklis, J. D. Fezl is required for the birth and specification of corticospinal motor neurons. Neuron 47, 817–831 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Chen, B., Schaevitz, L. R. & McConnell, S. K. Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc. Natl. Acad. Sci. USA 102, 17184–17189 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Breunig, J. J., Arellano, J. I., Macklis, J. D. & Rakic, P. Everything that glitters isn’t gold: a critical review of postnatal neural precursor analyses. Cell Stem Cell 1, 612–627 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Alvarez-Dolado, M. et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425, 968–973 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Wagers, A. J. & Weissman, I. L. Plasticity of adult stem cells. Cell 116, 639–648 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Goldman, S. A. Stem and progenitor cell-based therapy of the central nervous system: hopes, hype, and wishful thinking. Cell Stem Cell 18, 174–188 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Oki, K. et al. Human-induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain. Stem Cells 30, 1120–1133 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Tornero, D. et al. Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery. Brain 136, 3561–3577 (2013).

    Article  PubMed  Google Scholar 

  38. Davies, S. J., Field, P. M. & Raisman, G. Long interfascicular axon growth from embryonic neurons transplanted into adult myelinated tracts. J. Neurosci. 14, 1596–1612 (1994).

    CAS  PubMed  Google Scholar 

  39. Sadegh, C. & Macklis, J. D. Established monolayer differentiation of mouse embryonic stem cells generates heterogeneous neocortical-like neurons stalled at a stage equivalent to midcorticogenesis. J. Comp. Neurol. 522, 2691–2706 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sances, S. et al. Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nat. Neurosci. 19, 542–553 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ladewig, J., Koch, P. & Brüstle, O. Auto-attraction of neural precursors and their neuronal progeny impairs neuronal migration. Nat. Neurosci. 17, 24–26 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Ziemba, K. S., Chaudhry, N., Rabchevsky, A. G., Jin, Y. & Smith, G. M. Targeting axon growth from neuronal transplants along preformed guidance pathways in the adult CNS. J. Neurosci. 28, 340–348 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Péron, S. et al. A delay between motor cortex lesions and neuronal transplantation enhances graft integration and improves repair and recovery. J. Neurosci. 37, 1820–1834 (2017).

    Article  PubMed  Google Scholar 

  44. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Hirata, T. et al. Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons. Dev. Dyn. 230, 546–556 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Catapano, L. A., Arnold, M. W., Perez, F. A. & Macklis, J. D. Specific neurotrophic factors support the survival of cortical projection neurons at distinct stages of development. J. Neurosci. 21, 8863–8872 (2001).

    CAS  PubMed  Google Scholar 

  47. Molyneaux, B. J. et al. DeCoN: genome-wide analysis of in vivo transcriptional dynamics during pyramidal neuron fate selection in neocortex. Neuron 85, 275–288 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Vega, C. J. & Peterson, D. A. Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination. Nat. Methods 2, 167–169 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Petreanu, L., Mao, T., Sternson, S. M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Saito, T. & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237–246 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Franks, K. M. & Isaacson, J. S. Synapse-specific downregulation of NMDA receptors by early experience: a critical period for plasticity of sensory input to olfactory cortex. Neuron 47, 101–114 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Albeanu, D. F., Soucy, E., Sato, T. F., Meister, M. & Murthy, V. N. LED arrays as cost effective and efficient light sources for widefield microscopy. PLoS One 3, e2146 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank P. Davis, D. Schuback and K. Yee for superb technical assistance; B. Goetze at the Harvard Center for Biological Imaging for excellent assistance with confocal spectral unmixing; M. Eichner at the Institute for Clinical Epidemiology and Applied Biometry at University of Tübingen for statistical advice; C. Lois at Caltech, R. Hevner at University of Washington, M. Hibi at Nagoya University) and S.K. McConnell at Stanford University for generous sharing of mice, antibodies and reagents; and current and past members of the Macklis laboratory for helpful suggestions. This work was partially supported by grants to J.D.M. from the National Institutes of Health-NINDS NS041590 and NS049553, with additional infrastructure support from NS045523 and NS075672; from the ALS Association to J.D.M.; from The Regeneration Project to J.D.M. and from DFG grant Wu 590/2-1 to T.V.W. F.M. was partially supported from a grant to V.N.M. (DC011291). H.P. was partially supported by an International Brain Research Organization (IBRO) Research Fellowship and by a fellowship from The Regeneration Project.

Author information

Authors and Affiliations

Authors

Contributions

T.V.W. and J.D.M. designed the overall experimental directions and specific analyses, and T.V.W., H.P. and J.D.M. wrote and edited the manuscript. T.V.W. also performed all non-electrophysiology-related experiments and data analysis, generated all experimental animals including those for electrophysiology, assisted with electrophysiology and performed post hoc immunocytochemical analysis of tissue sections used for electrophysiological evaluation. F.M. performed all electrophysiological recordings and data analysis and participated in manuscript editing. H.P. co-performed in utero electroporation. A.P.W. assisted with mouse breeding and surgeries, immunocytochemistry and microscopy. T.V.W., J.D.M., H.P. and V.N.M. contributed to data analysis and biological interpretation.

Corresponding author

Correspondence to Jeffrey D. Macklis.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Time course of subcerebral and transcallosal axon extension by transplanted neurons.

Transplanted neurons progressively extend long-distance axon projections and reach pons and more distal subcerebral targets as well as the contralateral cortex (f and g) by 5-6 days post-transplantation (dpt). Subcerebral axon projections: 4 dpt (a [striatum] and b [cerebral peduncle]), 5 dpt (c [pons] and d [proximal medulla oblongata]), 6 dpt (e [distal medulla oblongata]). Trans-callosal axon projections: 4 dpt (h), 5 dpt (i), 6 dpt (j). eGFP (green); L1 (red). Scale bars: a – e, 200 µm; h, – j, 100 µm.

Supplementary Figure 2 (related to Fig. 2) Transplanted neurons assuming positions in cortical layers II–-VI progressively mature and successfully extend transcallosal long-distance axon projections.

(a) Schematic illustrating the experimental outline. Micro-transplantation of eGFP+ single cell suspension into P0/P1 recipient sensorimotor cortex. Stereotaxic injection of retrograde axonal tracer (CTB555) into the contralateral hemisphere at P6. Analysis at P7 and P14. (b, c) A subset of transplanted neurons project trans-callosally. These neurons progressively mature from a DCX+ and NeuN-/low (b) to a DCX-/low and NeuN+ state (c). Compare the DCX expression of the recipient cortex at both time points. DCX decreases dramatically on a very similar time scale in the recipient cortex as for the transplanted neurons. The positions of the boxes in low magnification panels (b, c) correspond to the cortical area examined in high magnification panels Arrows indicate transplanted neurons. eGFP (green), CTB555 (red), DCX (immature neuronal marker; purple), NeuN (mature neuronal marker; blue). Scale bars: b, c (low magnification), 50 µm; b, c (high magnification), 10 µm.

Supplementary Figure 3 (related to Fig. 2) Long-distance axon projections of transplanted neurons remain stable long-term.

(a) Schematic illustrating the experimental outline. Micro-transplantation of eGFP+ single cell suspension into P0/P1 recipient sensorimotor cortex. Stereotaxic injection of retrograde axonal tracer (CTB555) into the contralateral hemisphere at P35 and analysis at P37/P38. (b) A subset of transplanted neurons establish trans-callosal axon projections, remaining stable long-term, without pruning or retraction for periods as long as analyzed (5 weeks). These neurons progressively mature to a DCX- and NeuN+ state. The position of the box in the low magnification panel (b) corresponds to the cortical area examined in high magnification panels. Arrows indicate a transplanted neuron. eGFP (green), CTB555 (red), DCX (immature neuronal marker; purple), NeuN (mature neuronal marker; blue). Scale bars: b (low magnification), 50 µm; b (high magnification), 10 µm.

Supplementary Figure 4 (related to Fig. 3) Double birthdating of donor neurons prior to transplantation identifies the stage and origin of cells undergoing successful cortical integration.

(a) Recipient cortex after transplantation and retrograde labeling from the cp. The eGFP+ transplanted neurons (boxed areas in low magnification panels) and recipient SCPN are retrogradely labeled with CTB555 from the cp. There are three additional eGFP+ birthdated, transplanted, but retrograde CTB555 negative neurons (inset in high magnification CldU panel, top row, displays the CldU signal) in deeper layers of the same displayed section. These three neurons were double-positive for IdU and CldU, demonstrating that both birthdating and ICC were successful, and indicating that these three neurons were likely to be still mitotic or only very early postmitotic (less than 16 hours) at the time of transplantation. Analysis of eGFP+ and CTB555+ transplanted neurons shows that they are positive for IdU, but not CldU (arrows), demonstrating that these neurons were postmitotic for more than 16 hours before transplantation. The position of the boxes in the low magnification panels corresponds to the cortical area examined in the high magnification panels. eGFP (green), CTB555 (red), IdU (blue), CldU (purple). (b) Quantification from transplanted neurons analyzed with combined double IdU/CldU birthdating plus retrograde labeling (n=4). Double birthdating data are presented for three transplanted and retrogradely labeled SCPN (IdU+/CldU-; green in graphical representation); primary data from one additional neuron (shown here in black) is presented in Fig. 3. All analyzed neurons were IdU+/CldU-. These data indicate that early postmitotic neurons, encompassing already fate-restricted deep layer projection neurons and/or plastic post-mitotic neuroblasts with partially fate-restricted potential, account for the predominant population of neurons capable of achieving optimal level integration, and establishing subcerebral axon projections after transplantation.

Supplementary Figure 5 (related to Fig. 4) Newly incorporated neurons of combinatorial subcerebral molecular identity establish anatomical subcerebral output connectivity with high fidelity.

(a) High power confocal images of 3 eGFP+ (green) transplanted neurons retrogradely labeled with LumaFluor microspheres or CTB555 (red) from the cerebral peduncle demonstrate SCPN identity by high-level CTIP2 (purple) expression (as opposed to low-level expression, a characteristic of corticothalamic projection neurons) and by only barely detectable or completely absent callosal projection neuron identifier SATB2 (blue). Insets display retrogradely labeled layer V recipient SCPN within the same sections, and their respective CTIP2 (purple) expression levels for comparison. (b) High power confocal images of 3 additional eGFP+ (green) transplanted neurons retrogradely labeled with CTB555 (red) from the cerebral peduncle confirm SCPN identity and exclude identity of closely related corticofugal projection neuron populations by being negative for the corticothalamic identifier FOG2 (purple). DAPI (blue). Scale bars: a, b, 10 µm. (c) Quantification from transplanted neurons analyzed with combined molecular projection neuron subtype identity markers plus retrograde labeling (n=13). Molecular identity data are presented for six transplanted and retrogradely labeled SCPN (green in graphical representation); primary data from seven additional neurons (shown here in black) are presented in Figs. 4 and 6; Suppl. Figs. 6, 8, and 12. Each row of dots matches with the same row molecular descriptions to their right. Taken together, these data reveal a remarkable level of molecular-to-projection fidelity, even compared with the very closely related corticofugal CThPN, since neither SATB2+ nor FOG2+ transplanted neurons projected subcerebrally.

Supplementary Figure 6 (related to Fig. 4) Combined epifluorescence and confocal CTIP2 expression level analysis confirms SCPN identity of transplanted and subcerebrally projecting neurons.

Analysis and comparison of CTIP2 expression levels (by epifluorescence and confocal microscopy) of CTB555 (red) retrogradely labeled recipient layer V SCPN and an eGFP+ (green) transplanted and subcerebrally projecting neuron, being localized in close vicinity at the border between layers IV and V. (a) Epifluorescence montage of upper cortical layers and adjacent layer V. The eGFP+ transplanted neuron and a recipient SCPN (indicated by arrow) with their respective nuclei are boxed and magnified in (b, c and g, h). Imaging was performed at the same exposure settings, demonstrating comparable high-level CTIP2 expression, in contrast to low-level CTIP2 expression as typical for related CFuPN populations including CThPN. (d – f) Single optical confocal section of the eGFP+ (green) transplanted neuron (boxed in (a)), unequivocally demonstrating that the analyzed high-level CTIP2 positive nucleus (purple) belongs to the transplanted and CTB555 (red) retrogradely labeled neuron. Scale bars: a, 50 µm; b – h, 10 µm. (i) Quantification from transplanted neurons analyzed with combined molecular projection neuron subtype identity markers plus retrograde labeling (n=13). Molecular identity data are presented for one transplanted and retrogradely labeled SCPN (green in graphical representation); primary data from 12 additional neurons (shown here in black) are presented in Figs. 4 and 6; Suppl. Figs. 5, 8, and 12. Each row of dots matches with the same row molecular descriptions to their right. Taken together, these data indicate specific SCPN, rather than broad corticofugal projection neuron (CFuPN), identity.

Supplementary Figure 7 (related to Fig. 5) Newly incorporated neurons of combinatorial callosal molecular identity establish anatomic interhemispheric output connectivity with high fidelity.

(a) High power confocal images of 3 eGFP+ (green) transplanted neurons retrogradely labeled with CTB555 (red) from the contralateral hemisphere demonstrate CPN identity by expression of the CPN identifier SATB2 (blue) and being negative for or only exhibiting very low-level expression of CTIP2 (purple). (b) High power confocal images of 3 additional eGFP+ (green) transplanted and with CTB555 (red) from the contralateral hemisphere retrogradely labeled neurons further confirm CPN identity by SATB2 (blue) expression and being negative for FOG2 (purple). Scale bars: a, b, 10 µm. (c) Quantification from transplanted neurons analyzed with combined molecular projection neuron subtype identity markers plus retrograde labeling (n=9). Molecular identity data are presented for six transplanted and retrogradely labeled CPN (green in graphical representation); primary data from three additional neurons (shown here in black) are presented in Fig. 5. Each row of dots matches with the same row molecular descriptions to their right. Taken together, all analyzed transplanted and trans-callosally projecting neurons possess CPN molecular identity, and no transplanted neurons of SCPN or CThPN molecular identity were retrogradely labeled from the contralateral hemisphere.

Supplementary Figure 8 Transplanted primed immature neurons rebuild subcerebral axon projections in Fezf2-knockout recipient cortex.

(a) Micro-transplantation of eGFP+ single cell suspension into Fezf2-/- P0/P1 recipient sensorimotor cortex. Ultrasound-guided injection of retrograde axonal tracer (CTB555) into the cerebral peduncle (cp) at P6. Analysis at P8. (b) Epifluorescence montage of a sagittal section of recipient Fezf2-/- cortex, including the adjacent part of the striatum (indicated by asterisk). There are 3 eGFP+ transplanted neurons in cortical layer VI (boxed area in in layer VI). CTB555 retrogradely labels the center neuron (arrow) from the cerebral peduncle (cp) and medium spiny neurons (MSNs; asterisk) in the striatum (due to the anatomical close proximity of the cp and MSN projection targets in the substantia nigra). Note the absence of retrograde label and high-level CTIP2 expression in layer V, demonstrating the absence of endogenous layer V SCPN in the Fezf2-/- recipient cortex (for comparison: the 3-ways split inset in the right upper corner displays control cortex after retrograde labeling from the cp; note the high-level CTIP2 staining and CTB555 labeling of layer V neurons (SCPN) and striatal MSN (asterisk)). Retrograde label of MSN in the Fezf2-/- recipient ascertains correct targeting of the cp with CTB555. The position of the box in layer VI corresponds to the cortical area examined in the adjacent confocal panel (first panel, top row; merge: eGFP, CTB555, CTIP2, SATB2) and to the inset in the right upper corner of the confocal panel (CTIP2 only) demonstrating high-level CTIP2 expression of the transplanted and retrogradely labeled neuron (arrow) in comparison to adjacent recipient-derived layer VI neurons. The position of the box in the confocal panel (first panel, top row) corresponds to the cortical area examined in high magnification panels, indicating high-level CTIP2 expression and absence of SATB2 expression in the retrogradely labeled transplanted neuron (arrow) (note: one of the two flanking (not retrogradely labeled) transplanted neurons also exhibits high-level CTIP2 expression, while the other one is negative for CTIP2). eGFP (green), CTB555 (red), SATB2 (blue), CTIP2 (purple). Scale bars: b (epifluorescence montage and first confocal panel, top row), 50 µm; b (high magnification confocal panels), 10 µm. (c) Quantification from transplanted neurons analyzed with combined molecular projection neuron subtype identity markers plus retrograde labeling (n=13). Molecular identity data are presented for one retrogradely labeled SCPN after transplantation into Fezf2-/- recipient cortex (green in graphical representation); primary data from 12 additional neurons (shown here in black) are presented in Figs. 4 and 6; Suppl. Figs. 5, 6, and 12. Each row of dots matches with the same row molecular descriptions to their right. Taken together, these data not only reveal a remarkable level of molecular-to-projection fidelity, but, quite strikingly, demonstrate that newly incorporated (transplanted) neurons of SCPN molecular subtype identity extend subcerebral axon projections even in the absence of the endogenous pyramidal tract, thereby reconstituting otherwise entirely absent long-distance connectivity in the recipient brain.

Supplementary Figure 9 Spines of transplanted neurons are juxtaposed to recipient-derived presynaptic synaptophysin, suggesting synaptic integration with the recipient cortex.

(a) Confocal stack 3D reconstructions of a transplanted eGFP+ neuron surrounded by synaptophysin-positive recipient-derived presynaptic terminals. (b) Epifluorescence image of the transplanted neuron. The boxed areas are magnified on the right (merge 3D reconstruction of a confocal stack; and high power magnification of single optical sections), showing a synaptophysin-positive recipient-derived presynaptic terminal flanked from two sides by eGFP+ synaptic spines on the apical dendrite of the transplanted neuron (indicated by arrows). These data are highly suggestive of synaptic integration of the transplanted neuron into the recipient cortex. eGFP (green), synaptophysin (red). Scale bars: a, 20 µm; b (epifluorescence image), 20 µm; b (merge 3D reconstruction), 10 µm; b (high power single optical sections), 10 µm.

Supplementary Figure 10 (related to Fig. 7 and Fig. 8) ChR2-Venus expression in recipient-derived neurons or donor neurons renders neurons light-sensitive.

(a) tdTomato is used to visualize in utero-electroporated layer II/III neurons by epifluorescence in an acute slice preparation without exciting co-electroporated channelrhodopsin-2-Venus (gray scale panel). One neuron is patched (indicated by the arrows) and subsequently photo-stimulated with a blue LED. A biocytin-containing internal solution allowed for post hoc ICC analysis. A low power collapsed confocal stack (first panel) gives an overview of in utero-electroporated recipient-derived neurons in layers II/III (td-tomato+ and channelrhodopsin-2-Venus+). The position of the box corresponds to the cortical area examined in the high magnification panels giving a detailed view of the biocytin filled neuron. Note this neuron is positive for biocytin, cytoplasmic tdTomato and membrane-bound channelrhodopsin-2-Venus. Representative traces are shown on the right. Blue bars indicate light pulses. Top trace: Each light pulse robustly elicits a series of action potentials, thereby demonstrating sufficient expression of channelrhodopsin-2-Venus by in utero-electroporated neurons. Bottom trace: Corresponding action currents mediated by channlerhodopsin-2-Venus. The arrow points out an action current at a smaller time scale. Channelrhodopsin-2-Venus (green, membrane bound), tdTomato (red, cytoplasmic), biocytin (blue, cytoplasmic). Scale bars: a, 50 µm. (b, c) Two examples of patch clamp recordings of transplanted and channelrhodopsin-2-Venus and tdTomato in vitro Amaxa electroporated neurons are shown; transplanted neurons are visualized by epifluorescence of tdTomato in the acute slice preparation (gray scale panels). Depolarization of the transplanted neurons by current injection through the patch pipette elicits action potential trains, thereby confirming neuronal identity (middle panels). Photo-stimulation (blue bars) results in a robust depolarization and elicits several action potential spikes, thereby demonstrating sufficient expression of channelrhodopsin-2-Venus by in vitro Amaxa electroporated and transplanted neurons (right panels; top traces). Corresponding channelrhodopsin-2-Venus mediated action currents (right panels; bottom traces). Arrows point out action currents at a smaller time scale. Scale bars: b, c, 50 µm.

Supplementary Figure 11 Rare cell-fusion events do not account for the observed results.

Transplantations of either control eGFP+/Fezf2+/- neuronal suspensions (contain all projection neuron subtypes) or eGFP+/Fezf2-/- suspensions (contain all projection neuron subtypes but SCPN, which were genetically removed from the mix) were performed and the axon projection pattern was analyzed. While transplanted Fezf2+/- neurons (a) send axons to corticofugal/subcortical targets (b [striatum], c [thalamus], d [tectum], e [pons]), Fezf2-/- neurons (f) reach (g) the striatum but fail to innervate more distal subcortical targets including (h) thalamus, (i) tectum and (j) pons (arguing against potential cell fusion events between transplanted and recipient derived SCPN, in which case a similar axon projection pattern would have been postulated). The inset in the top right corner in (f) demonstrates that Fezf2-/- neurons are capable of sending trans-callosal axons (the hemisphere contralateral to the transplanted cortex is shown on the right side; the boxed area is magnified in the left part of the inset, visualizing eGFP+ axons projecting from the corpus callosum up into the cortex). eGFP (green), L1 (red), NeuN (blue). Scale bars: a, f, 1000 µm; b – e and g – j, 250 µm; inset (right panel), 1000 µm; inset (left panel), 100 µm.

Supplementary Figure 12 Rare cell-fusion events do not account for the observed results.

(a) Micro-transplantation of eGFP+ single cell suspension into P0/P1 Fezf2+/LacZ recipient sensorimotor cortex. Ultrasound-guided injection of retrograde axonal tracer (CTB555) into the cerebral peduncle (cp) at P6. Five-channel confocal (with spectral unmixing) analysis at P8. (b) The eGFP+ transplanted neuron (arrow) in layer V and adjacent recipient SCPN (arrowheads) in the same field of view are retrogradely labeled with CTB555 from the cerebral peduncle and express comparable high levels of CTIP2; the transplanted neuron is negative for Satb2 and Fezf2-b-gal while a large number of recipient SCPN in the same optical plane are Fezf2-b-gal positive. The position of the boxes corresponds to the cortical area examined in high magnification panels. The position of the inset boxes in the low magnification panels is magnified and shown as an inset in the high magnification Fezf2-b-gal panel for comparison. Fezf2-b-gal negativity of the transplanted neuron in comparison to positivity of recipient-derived neurons argues against fusion of eGFP+ transplant-derived cells with recipient-derived SCPN. eGFP (green), CTB555 (red), SATB2 (blue), CTIP2 (cyan), Fezf2-b-gal (purple). Scale bars: b (low magnification), 50 µm; b (high magnification), 10 µm; b (inset in the high magnification Fezf2-b-gal panel), 10 µm. (c) Quantification from transplanted neurons analyzed with combined molecular projection neuron subtype identity markers plus retrograde labeling (n=13). Molecular identity data are presented for one transplanted and retrogradely labeled SCPN (green in graphical representation); primary data from 12 additional neurons (shown here in black) are presented in Figs. 4 and 6; Suppl. Figs. 5, 6, and 8. Each row of dots matches with the same row molecular descriptions to their right. Taken together, these data reveal a remarkable level of molecular-to-projection fidelity, even compared with the very closely related corticofugal CThPN, since neither SATB2+ nor FOG2+ transplanted neurons projected subcerebrally.

Supplementary Figure 13 Quantification of molecular identity data.

For analysis of molecular identity (CTIP2, SATB2, FOG2 ICC) combined with retrograde labeling (from cerebral peduncle or contralateral hemisphere), all neurons identified throughout all experiments and complying with the a priori criteria (neurons located in cortical gray matter separated from white matter by at least one layer of recipient-derived cortical neurons, and retrogradely labeled from target sites) were included in analysis; no neurons meeting these criteria were excluded. Representative neurons are depicted in the main figures (Figs. 4, 5, 6), and all additional analyzed neurons in the Supplementary Figures (Suppl. Figures 5, 6, 7, 8, 12). Each of these neurons is represented by a solid dot in the graphical representation. In total, n=13 transplanted neurons projecting to subcerebral targets (CSMN/SCPN) and n=9 neurons projecting to the contralateral hemisphere (CPN) (projection target is indicated at the bottom) were analyzed, and all display appropriate molecular identity for SCPN or CPN, respectively (molecular identity are indicated on the right side). Each row of dots matches with the same row molecular descriptions to their right. No transplanted and retrogradely labeled neurons were identified to have mismatching projection target and molecular identity (indicated by Ø). Fisher’s exact test was applied for statistical analysis, comparing the frequency of expression of a marker combination (SCPN or CPN) by neurons projecting to expected targets (cerebral peduncle or contralateral hemisphere) with the frequency of expression of a marker combination (SCPN or CPN) by neurons projecting to the alternative (mismatching) targets (contralateral hemisphere or cerebral peduncle): p<0.00001, demonstrating an extremely high level of molecular-to-projection fidelity. Fisher’s exact test is explicitly valid when a chi-squared test-based approximation is inadequate, since sample sizes are small, or the data are very unequally distributed among the cells of the table.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wuttke, T.V., Markopoulos, F., Padmanabhan, H. et al. Developmentally primed cortical neurons maintain fidelity of differentiation and establish appropriate functional connectivity after transplantation. Nat Neurosci 21, 517–529 (2018). https://doi.org/10.1038/s41593-018-0098-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-018-0098-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing