Illumination of neural development by in vivo clonal analysis

Single embryonic and adult neural stem cells (NSCs) are characterized by their self-renewal and differentiation potential. Lineage tracing via clonal analysis allows for specific labeling of a single NSC and tracking of its progeny throughout development. Over the past five decades, a plethora of clonal analysis methods have been developed in tandem with integration of chemical, genetic, imaging and sequencing techniques. Applications of these approaches have gained diverse insights into the heterogeneous behavior of NSCs, lineage relationships between cells, molecular regulation of fate specification and ontogeny of complex neural tissues. In this review, we summarize the history and methods of clonal analysis as well as highlight key findings revealed by single-cell lineage tracking of stem cells in developing and adult brains across different animal models. Keywords: In vivo clonal analysis, Lineage tracing, Neural stem cells, Neural development, Nuclear brain structur

In vivo clonal analysis reveals spatiotemporal regulation of thalamic nucleogenesis

<div><p>The thalamus, a crucial regulator of cortical functions, is composed of many nuclei arranged in a spatially complex pattern. Thalamic neurogenesis occurs over a short period during mammalian embryonic development. These features have hampered the effort to understand how regionalization, cell divisions, and fate specification are coordinated and produce a wide array of nuclei that exhibit distinct patterns of gene expression and functions. Here, we performed <i>in vivo</i> clonal analysis to track the divisions of individual progenitor cells and spatial allocation of their progeny in the developing mouse thalamus. Quantitative analysis of clone compositions revealed evidence for sequential generation of distinct sets of thalamic nuclei based on the location of the founder progenitor cells. Furthermore, we identified intermediate progenitor cells that produced neurons populating more than one thalamic nuclei, indicating a prolonged specification of nuclear fate. Our study reveals an organizational principle that governs the spatial and temporal progression of cell divisions and fate specification and provides a framework for studying cellular heterogeneity and connectivity in the mammalian thalamus.</p></div

Intravenous Administration of Adipose-Derived Stem Cell Protein Extracts Improves Neurological Deficits in a Rat Model of Stroke

Treatment of adipose-derived stem cell (ADSC) substantially improves the neurological deficits during stroke by reducing neuronal injury, limiting proinflammatory immune responses, and promoting neuronal repair, which makes ADSC-based therapy an attractive approach for treating stroke. However, the potential risk of tumorigenicity and low survival rate of the implanted cells limit the clinical use of ADSC. Cell-free extracts from ADSC (ADSC-E) may be a feasible approach that could overcome these limitations. Here, we aim to explore the potential usage of ADSC-E in treating rat transient middle cerebral artery occlusion (tMCAO). We demonstrated that intravenous (IV) injection of ADSC-E remarkably reduces the ischemic lesion and number of apoptotic neurons as compared to other control groups. Although ADSC and ADSC-E treatment results in a similar degree of a long-term clinical beneficial outcome, the dynamics between two ADSC-based therapies are different. While the injection of ADSC leads to a relatively mild but prolonged therapeutic effect, the administration of ADSC-E results in a fast and pronounced clinical improvement which was associated with a unique change in the molecular signature suggesting that potential mechanisms underlying different therapeutic approach may be different. Together these data provide translational evidence for using protein extracts from ADSC for treating stroke

Clonal lineage tracing of basal progenitor cells shows prolonged nuclear specification of thalamic cells.

<p>(<b>A</b>) Left: sample images of frontal sections from E18.5 <i>Neurog1^CreERT2;ZSGreen</i> mouse brains to show labeling of progeny of <i>Neurog1</i>-expressing progenitor cells when tamoxifen is administered at E11.5, E12.5, or E13.5. Sections at three different dorso-ventral levels are shown for each stage of tamoxifen administration. (<b>B</b>) Sample image of a <i>Neurog1^CreERT2</i> clone at E18.5. The colonization of 2 nuclei within a single clone is shown. Scale bar: 300 μm. (<b>C</b>) Bar graph showing the number of nuclei populated for <i>Neurog1^CreERT2</i>-labeled clones with 2 to 4 cells. (<b>D</b>) Bar graph showing the percentage of clones populating single or multiple nuclei for <i>Neurog1</i>-labeled clones with 2 to 4 cells. The left bar indicates clones that are activated at E11.5 or E12.5, and the right bar indicates clones activated at E13.5 or E14.5. (<b>E</b>) Pie chart showing the percentage of clones populating single or multiple nuclei for clones undergoing terminal divisions. The breakdown of nuclei populated for clones colonizing multiple nuclei is shown. CM, centromedian; dLG, dorsal lateral geniculate; LD, lateral dorsal; LHb, lateral habenula; LP, lateral posterior; MD, mediodorsal; MGv, ventral medial geniculate; MHb, medial habenula; PF, parafascicular; Po, posterior; PV, paraventricular; Re, reuniens; VL, ventrolateral; VP, ventral posterior; VPm, ventral posteromedial.</p

Outside-in temporal specification of thalamic nucleogenesis.

<p>(<b>A</b>) A schematic of the TH in the caudal diencephalon of the mouse embryo. In the sagittal schematic (left two panels), the embryo is facing left. The thalamus is bordered by the pretectum caudally, the habenula dorsally, the basal plate ventrally, and by the ZLI rostrally. The three dashed lines represent the planes of sections shown in the frontal schematic (right two panels). In this study, we used Cre drivers of 3 genes, <i>Gli1</i>, <i>Olig3</i>, and <i>Neurog1</i>. Within the color-labeled regions of the diencephalon, <i>Gli1</i> is expressed in pTH-C (rostro-ventral region near the pTH-R domain, shown in darker green) and the prethalamus (light blue) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005211#pbio.2005211.ref006" target="_blank">6</a>]. <i>Olig3</i> is expressed in pTH-C (green) and pTH-R (pink) domains of the thalamus as well as in the ZLI (dark blue) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005211#pbio.2005211.ref003" target="_blank">3</a>]. <i>Neurog1</i> is expressed in the habenula (yellow), pTH-C (green), and ZLI (dark blue) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005211#pbio.2005211.ref003" target="_blank">3</a>]. (<b>B</b>) Frontal sections of the forebrain showing EdU birthdating in the neonatal mouse thalamus with EdU injections at E10.5, E11.5, E12.5, E13.5, or E14.5. Three representative levels (dorsal, middle, and ventral) are shown for each stage of EdU injection. In all three levels, there is a general trend in which neurons of laterally located nuclei are born before neurons of more medially located nuclei, demonstrating the outside-in pattern of neurogenesis. Scale bar: 500 μm. 3V, third ventricle; Cx, cortex; EdU, ethynyldeoxyuridine; HB, habenula; Hyp, hypothalamus; LV, lateral ventricle; M, midbrain; PT, pretectum; PTH, prethalamus; pTH-C, caudal thalamic progenitor domain; pTH-R, rostral thalamic progenitor domain; RP, roof plate; TH, thalamus; Tel, telencephalon; ZLI, zona limitans intrathalamica.</p

Temporal change of cell divisions during thalamic nucleogenesis.

<p>(<b>A</b>) Bar graph showing a breakdown of the cell number per clone for all <i>Neurog1^CreERT2</i>-labeled clones. (<b>B</b>) Top: bar graphs showing breakdown of the cell number per clone for all <i>Gli1^CreERT2</i> and <i>Olig3^CreERT2</i>-labeled clones. Bottom: bar graphs showing breakdown of the cell number per clone for all <i>Gli1^CreERT2</i> and <i>Olig3^CreERT2</i>-labeled G/R hemiclones. (<b>C</b>) A schematic depicting the three defined types of clonal division patterns: symmetric proliferative (“Sym Pro”), asymmetric neurogenic (“Asym Neuro”), and other (“Sym Neuro”). (<b>D</b>) Bar graph showing the percentage of division pattern within clones labeled using <i>Gli1</i> and <i>Olig3</i> driver lines with tamoxifen administration at E9.5 for <i>Gli1</i> and E10.5/E11.5 for <i>Olig3</i>. (<b>E</b>) Bar graph showing the distribution of the size of minority hemiclones in asymmetric neurogenic clones of <i>Gli1</i> and <i>Olig3</i> MADM brains. (<b>F</b>) Graph showing the distribution of the numbers of G and R cells of symmetric neurogenic clones of <i>Gli1</i> and <i>Olig3</i> MADM brains. G, green; R, red.</p

Clonal lineage tracing of thalamic progenitors using MADM.

<p>(<b>A</b>) Sample images of frontal sections from <i>Gli1^CreERT2;H2B-GFP</i>, <i>Olig3^CreERT2;ZSGreen</i>, and <i>Neurog1^CreERT2;ZSGreen</i> mouse brains counterstained with DAPI to show labeling of progenitor cells or their progeny. The left three panels show labeling 1 d after tamoxifen administration (E10.5 for <i>Gli1^CreERT2</i>, E11.5 for <i>Olig3^CreERT2</i>, and E12.5 for <i>Neurog1^CreERT2</i>). Scale bar: 500 μm. (<b>B</b>) Bar graphs showing regional distribution of labeled clones in the forebrain of <i>Gli1^CreERT2</i>, <i>Olig3^CreERT2</i>, <i>and Neurog1^CreERT2</i> mouse lines crossed with <i>MADM11</i> reporter mice. Brains were analyzed 1 d after tamoxifen administration. (<b>C</b>) Bar graphs showing frequencies of thalamic clones (G/R or Y) per hemisphere labeled 1 d after tamoxifen administration for the <i>Gli1^CreERT2;MADM11</i>, <i>Olig3^CreERT2;MADM11</i>, and <i>Neurog1^CreERT2;MADM11</i> mice. More than 75% of hemispheres had no recombined clones. The curves represent the best non-linear fit. G/R, green/red; Hypo, hypothalamus; MADM, mosaic analysis with double markers; Telen, telencephalon; Tha, thalamus; Y, yellow.</p

RGC position dictates domain-specific distribution of clones.

<p>(<b>A</b>) The four micrographs show frontal sections of E18.5 forebrain, in which colored solid bars indicate the 4 zones—DR, DM, DC, and V—of RGC positions. Scale bar: 500 μm. Bottom: four faying surface plots illustrate representative shapes of the respective clones. (<b>B</b>) 2D shape of a representative clone for each location type shown in Fig 6A. The red dot indicates the position of the residual RGC. (<b>C</b>) Clustering analysis of clones based on the position of RGCs. AD, anterodorsal; CLPC, centrolateral, paracentral; CM, centromedian; DC, dorsal caudal; dLG, dorsal lateral geniculate; DM, dorsal middle; DR, dorsal rostral; eml, external medullary lamina; IGL, intergeniculate leaflet; IMD, intermediodorsal; LD, lateral dorsal; LP, lateral posterior; MD, mediodorsal; MG, medial geniculate; MGv, ventral medial geniculate; MVF, medial ventral field; PF, parafascicular; Po, posterior; PV, paraventricular; Re/Rh, reuniens/rhomboid; RGC, radial glial cell; V, ventral; VL, ventrolateral; vLG, ventral lateral geniculate; VM, ventromedial; VP, ventral posterior; ZI, zona incerta.</p

Cell division patterns reveal the fate specification of early-born neurons in thalamus.

<p>(<b>A</b>) Brains were reconstructed from serial sections cumulatively containing all labeled cells within a single clone using IMARIS. Shown is a sample representative 2D projection (left panel) and 3D reconstruction (right panel) of thalamic clone derived from a <i>Gli1^CreERT2</i> MADM E18.5 brain. Violescent stereostructure sketches the contours of the hemisphere, the green filament shows the process of a residual radial glial cell, and green and red spots represent labeled GFP+ and tdTomato+ cells, respectively. Scale bar: 300 μm. (<b>B</b>) Bar graph showing the distribution of the size (cell number) of asymmetric clones. (<b>C</b>) Left: bar graph showing the average number of nuclei populated in symmetric and asymmetric G/R clones. Symmetric proliferative clones contributed to more thalamic nuclei than asymmetric neurogenic clones. Right: bar graph showing the average number of nuclei populated in majority (“major”) and minority (“minor”) hemiclones within asymmetric clones. (<b>D</b>) Bar graph showing the percentage of clones (left panel) and number of cells (right panel) populating PSN in majority (“major”) and minority (“minor”) hemiclones within asymmetric clones. dLG, dorsal lateral geniculate; eml, external medullary lamina; G, green; GFP, green fluorescent protein; LP, lateral posterior; MADM, mosaic analysis with double markers; PSN, principal sensory nuclei; PVN, paraventricular nucleus; R, red; RT, reticular nucleus; td, tandem dimer; vLG, ventral lateral geniculate; ZI, zona incerta.</p