Microdissection and Single-Cell Suspension of Neocortical Layers From Ferret Brain for Single-Cell Assays

Overview

Journal Bio Protoc

Specialty Biology

Date 2024 Dec 30

PMID 39735300

Authors

Lucia Del-Valle-Anton

Salma Amin

Victor Borrell

Affiliations

Soon will be listed here.

Abstract

Brain development is highly complex and dynamic. During this process, the different brain structures acquire new components, such as the cerebral cortex, which builds up different germinal and cortical layers during its development. The genetic study of this complex structure has been commonly approached by bulk-sequencing of the entire cortex as a whole. Here, we describe the methodology to study this layered tissue in all its complexity by microdissecting two germinal layers at two developmental time points. This protocol is combined with a step-by-step explanation of tissue dissociation that provides high-quality cells ready to be analyzed by the newly developed single-cell assays, such as scRNA-seq, scATAC-seq, and TrackerSeq. Altogether, this approach increases the resolution of the genetic analyses from the cerebral cortex compared to bulk studies. It also facilitates the study of laboratory animal models that recapitulate human cortical development better than mice, like ferrets. Key features • Microdissection of individual germinal layers in the developing cerebral cortex from living brain slices. • Enzymatic and mechanical dissociation generates single-cell suspensions available for high-throughput single-cell assays. • Protocol optimized for embryonic and early postnatal ferret cortex.

References

Del-Valle-Anton L, Amin S, Cimino D, Neuhaus F, Dvoretskova E, Fernandez V . Multiple parallel cell lineages in the developing mammalian cerebral cortex. Sci Adv. 2024; 10(13):eadn9998. PMC: 10971412. DOI: 10.1126/sciadv.adn9998. View

Matsumoto N, Tanaka S, Horiike T, Shinmyo Y, Kawasaki H . A discrete subtype of neural progenitor crucial for cortical folding in the gyrencephalic mammalian brain. Elife. 2020; 9. PMC: 7173966. DOI: 10.7554/eLife.54873. View

Singh A, Del-Valle-Anton L, de Juan Romero C, Zhang Z, Ortuno E, Mahesh A . Gene regulatory landscape of cerebral cortex folding. Sci Adv. 2024; 10(23):eadn1640. PMC: 11152136. DOI: 10.1126/sciadv.adn1640. View

Martinez-Martinez M, de Juan Romero C, Fernandez V, Cardenas A, Gotz M, Borrell V . A restricted period for formation of outer subventricular zone defined by Cdh1 and Trnp1 levels. Nat Commun. 2016; 7:11812. PMC: 4897765. DOI: 10.1038/ncomms11812. View

Hansen D, Lui J, Parker P, Kriegstein A . Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature. 2010; 464(7288):554-561. DOI: 10.1038/nature08845. View

Reillo I, de Juan Romero C, Garcia-Cabezas M, Borrell V . A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex. Cereb Cortex. 2010; 21(7):1674-94. DOI: 10.1093/cercor/bhq238. View

Wang X, Tsai J, LaMonica B, Kriegstein A . A new subtype of progenitor cell in the mouse embryonic neocortex. Nat Neurosci. 2011; 14(5):555-61. PMC: 3083489. DOI: 10.1038/nn.2807. View

Pollen A, Nowakowski T, Chen J, Retallack H, Sandoval-Espinosa C, Nicholas C . Molecular identity of human outer radial glia during cortical development. Cell. 2015; 163(1):55-67. PMC: 4583716. DOI: 10.1016/j.cell.2015.09.004. View

Thomsen E, Mich J, Yao Z, Hodge R, Doyle A, Jang S . Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods. 2015; 13(1):87-93. PMC: 4869711. DOI: 10.1038/nmeth.3629. View

10.

Del-Valle-Anton L, Borrell V . Folding brains: from development to disease modeling. Physiol Rev. 2021; 102(2):511-550. DOI: 10.1152/physrev.00016.2021. View

11.

Pilz G, Shitamukai A, Reillo I, Pacary E, Schwausch J, Stahl R . Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type. Nat Commun. 2013; 4:2125. PMC: 3717501. DOI: 10.1038/ncomms3125. View

12.

Borrell V . In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation. J Neurosci Methods. 2009; 186(2):186-95. DOI: 10.1016/j.jneumeth.2009.11.016. View

13.

Fietz S, Kelava I, Vogt J, Wilsch-Brauninger M, Stenzel D, Fish J . OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling. Nat Neurosci. 2010; 13(6):690-9. DOI: 10.1038/nn.2553. View

14.

Shitamukai A, Konno D, Matsuzaki F . Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors. J Neurosci. 2011; 31(10):3683-95. PMC: 6622781. DOI: 10.1523/JNEUROSCI.4773-10.2011. View

15.

Betizeau M, Cortay V, Patti D, Pfister S, Gautier E, Bellemin-Menard A . Precursor diversity and complexity of lineage relationships in the outer subventricular zone of the primate. Neuron. 2013; 80(2):442-57. DOI: 10.1016/j.neuron.2013.09.032. View

16.

de Juan Romero C, Bruder C, Tomasello U, Sanz-Anquela J, Borrell V . Discrete domains of gene expression in germinal layers distinguish the development of gyrencephaly. EMBO J. 2015; 34(14):1859-74. PMC: 4547892. DOI: 10.15252/embj.201591176. View

17.

Bilgic M, Wu Q, Suetsugu T, Shitamukai A, Tsunekawa Y, Shimogori T . Truncated radial glia as a common precursor in the late corticogenesis of gyrencephalic mammals. Elife. 2023; 12. PMC: 10662950. DOI: 10.7554/eLife.91406. View

18.

Kalebic N, Gilardi C, Stepien B, Wilsch-Brauninger M, Long K, Namba T . Neocortical Expansion Due to Increased Proliferation of Basal Progenitors Is Linked to Changes in Their Morphology. Cell Stem Cell. 2019; 24(4):535-550.e9. DOI: 10.1016/j.stem.2019.02.017. View

19.

Li Z, Tyler W, Zeldich E, Santpere Baro G, Okamoto M, Gao T . Transcriptional priming as a conserved mechanism of lineage diversification in the developing mouse and human neocortex. Sci Adv. 2020; 6(45). PMC: 7673705. DOI: 10.1126/sciadv.abd2068. View

20.

Nowakowski T, Pollen A, Sandoval-Espinosa C, Kriegstein A . Transformation of the Radial Glia Scaffold Demarcates Two Stages of Human Cerebral Cortex Development. Neuron. 2016; 91(6):1219-1227. PMC: 5087333. DOI: 10.1016/j.neuron.2016.09.005. View