Name: lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (madm)
Uploaded: 2020-05-08T14:30:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM at JoVE.com

We thank all members of the Hippenmeyer laboratory for discussion, the Bioimaging Facility, Life Science Facility, and Pre-Clinical Facility at IST Austria for technical support. This work was supported by IST Austria institutional funds; R.B. received support from Austrian Science Fund (FWF) Lise-Meitner program (M 2416); N.A received support from Austrian Science Fund (FWF) Firnberg-Programm (T 1031); GC received support from the European Union&#39;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement No. 754411 as an ISTplus postdoctoral fellow; A.H. received support from an &#214;AW DOC (Doctoral Fellowship of the Austrian Academy of Sciences). This study was also supported by the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation programme (grant agreement No 725780 LinPro) to S.H.

The authors have nothing to disclose.

A method to use MADM to track cell lineage of individual RGPs in vivo in the developing neocortex is described. When combined with TM-inducible CreERT2, MADM events can be precisely timed, providing a highly qualitative and quantitative visual readout of stem cell division patterns at the single cell level. By titrating the dose of TM delivered, in an ideal situation an average of less than one clone per cortical hemisphere can be obtained, providing adequate spatial separation to unambiguously distinguish individual clones. By maintaining tissue integrity, this method also captures essential information regarding position, morphology, and absolute cell numbers. MADM cassettes on Chr. 117,11,12,46,56,57, on Chr. 751, and the original MADM at Rosa2647,53,59 have been used in MADM clonal analysis studies. The high-resolution of individual cells provides unprecedented insight into both morphology and the clonal relationship of daughter cells and permits the live imaging of proliferating stem cells and emerging clones46,52.
Caesarean section and fostering of pups for analysis of clones at postnatal timepoints is a necessary and critical step in the protocol. Depending on the health status of the TM-treated pregnant dam, it may not be necessary to perform a Caesarean section. However, raising the pups with a foster mother is still required, because the TM-treated mother may have trouble lactating. No differences have been observed in the need for fostering with different CreERT2 drivers. Both MADM lines and foster mothers are maintained on an outbred CD-1 background. If Caesarean section is not necessary, the TM-treated pregnant dam used to generate experimental pups may be reused for additional experimental breedings in accordance to principles of 3R (note that this can only be done if animal experimental licenses approve of this practice). Foster mothers can be used for fostering pups within 2 days after they give birth, but higher success rates have been observed when foster mothers give birth on the same day as the experimental mice that must be fostered. Therefore, it is important to set up timed matings for foster mothers in parallel to experimental matings in step 1.1. Maintaining a similar litter number as the original foster mother&#39;s litter can improve the survival rate of fostered pups, and therefore removal of some to all of the original litter may be necessary. Additional steps that may improve fostering includes rubbing the experimenter&#39;s gloves with litter and food (to remove the scent of the gloves); rubbing the pups gently after the Caesarean section with fragments of the foster mother&#39;s soiled litter and nest; and placement of the pups in close contact with the foster mother&#39;s pups prior to their placement in the foster mouse cage.
As in other reporter-based lineage tracing methods, careful consideration must be taken when choosing the optimal CreERT2 driver for MADM clonal experiments. First, the promoter used must express the recombinase both temporally and spatially in the progenitor population of interest. Finding this promoter can be challenging, because some promoters may change expression patterns or become silenced at different stages of development. To improve cell type specificity multiple site-specific recombinases, each driven by separate promoters, have been used. When one or both recombinases are expressed in the same cell, this labels the cell and its progeny with a fluorescent reporter74,75,76,77. In summary, it is important to choose a CreERT2 driver that is specific to the population of progenitors being analyzed.
The most critical step in this method is the identification of a clone, because all cells must be derived unambiguously from a single recombination event (step 8.1). Titration of TM concentration ensures less than one cluster of red/green cells per brain hemisphere and maximizes the probability of analyzing a single clone (step 2.2)7,11. Clones should be discarded if neighboring clusters of cells occur within 500 &#181;m of the clone of interest. Therefore, it is important to examine several sections before and after the appearance of a clone to ensure that there are no additional recombination events nearby. Due to weaker signal of the fluorophores, it is necessary to perform immunohistochemistry for eGFP and tdT in embryonic clones (see section 6). This is only recommended in adult clones if additional antigens will be colabeled. When imaging clones, it is important to capture the entire width of the cortex where the clone is located (i.e., from pial surface to the corpus callosum; see step 8.4) to not miss any cells. This also facilitates image alignment during image processing (section 9). Section 8 of the protocol requires an inverted confocal microscope but can be adapted depending on the microscope setup available. Epifluorescence microscopy can be used, but confocal microscopy is recommended because this leads to a decrease in light contamination from outside the focus plane. It is also important that the laser intensity and gain is adjusted so that green, red, and yellow cells can be unambiguously identified. Regardless of the setup, it is recommended to use an objective of at least 20x to ensure full spatial separation of closely positioned cells. In addition to recording the cortical depth of all cells (step 8.6), cortical regions where the clones are located must be identified using a brain atlas such as the Allen Brain atlas or other stereotaxic coordinate maps. A file naming paradigm should also be adopted to make sure clone images are easily identifiable. The following information could be included in the file naming: unique image ID, date image was taken, genotype of animal, age of induction, age of analysis, image number in relation to the rest of the images from the same clone.
Introduction of a mutation distal to one MADM cassette distinctively allows the generation of genetic mosaics71 and permits the dissection of molecular regulators of lineage and cell type diversity at the clonal level7,11,46,62. To generate a genetic mosaic with MADM, the MADM cassettes must be meiotically linked to the same chromosome as the gene of interest (see Figure 2 for breeding scheme). This limits current clonal analysis with MADM to genes located on Chr. 751, Chr. 1146, Chr. 1251, and Chr. 6 distal to the Rosa26 locus47. Future studies will use MADM cassettes targeted to any chromosome, permitting the mosaic analysis of virtually all genes of the mouse genome at the clonal level.
Finally, MADM is not limited to the analysis of progenitor cells in the developing neocortex. The study of many stem cell niches could benefit from the ability to resolve spatiotemporal arrangements of clonally related cells. By applying MADM to other regions of the brain, disease conditions (e.g., cancer), or in other tissues47,50,51,52,53,54,55,56,57,58,59, studies revealed lineage relationships in clones derived from diverse classes of progenitor and stem cells (see Table 1 for current list of MADM clonal studies). Another interesting future application of MADM is to combine it with additional functional or subcellular reporters, which would increase the degree of information that can be acquired from clones.

The cerebral cortex is a highly organized structure composed of six distinct layers. The cortex contains a diverse array of cell types including neurons and glia, which interact to form functional neural circuits. Most, if not all, cortical excitatory projection neurons and glia are derived from a common pool of neural stem cells (NSCs) known as the radial glial progenitors (RGPs)1,2,3. RGPs themselves are derived from neuroepithelial stem cells (NESCs) composing the early embryonic neuroepithelium. By embryonic day 9 (E9) in mice, NESCs begin to transition into RGPs4. RGP lineage progression requires precise temporal and spatial regulation, and when this process is hindered, severe neurological disorders such as megalencephaly, microcephaly, lissencephaly, or impairments such as schizophrenia and autism can result5,6. At E10, most RGPs undergo symmetric proliferative divisions, resulting in an expansion of the neural progenitor pool4,7. RGPs eventually begin to divide asymmetrically, producing cortical projection neurons in a temporally defined manner. Through consecutive waves of neurogenesis, newborn neurons migrate into the cortical plate forming cortical laminae with early born neurons occupying deep layers and late born neurons residing in the superficial layers8,9,10. Because clonally related pyramidal neurons migrate radially into the cortex with very little tangential dispersion, daughter cells tend to form a column or cone shaped structure referred to as a neuronal radial unit4,11,12,13. By E17, embryonic neurogenic expansion is complete in mice14. RGPs can also produce ependymal cells and some classes of glia, including astrocytes and oligodendrocytes1,15,16,17,18,19. The potential of RGPs to give rise to both neurons and astrocytes seems to be consistent across all cortical regions18, with approximately 1/6 of neurogenic RGPs also producing glia11.
Currently, the genetic and epigenetic factors regulating temporal progression of a stem cell along its lineage are mostly unknown. Temporal patterns of gene expression may have substantial impact on lineage decisions in RGPs20,21,22,23,24. How this tightly knit relationship between temporal and spatial patterning leads to the molecular diversity of adult neuronal types across cortical areas is not known. Likewise, how the individual stem cell potential and its cellular output is modulated at the cellular and molecular level is an important unanswered question. Future studies will hopefully address some of these questions, ultimately expanding our understanding of functional cortical circuit formation.
Developmental neurobiology seeks to understand the lineage relationship that cells in the brain share with one another. Initially, very few research tools were available for this, and many early studies relied on visual observations of division patterns in transparent organisms such as Caenorhabditis elegans25. Recent decades have seen a dramatic increase in the number and sophistication of techniques available13,26,27,28,29. The emergence of the CRISPR-Cas9 genome editing system allows for synthetic reconstruction of cell lineage relationships by introducing evolving DNA barcodes27,30. Two recent examples of barcoding strategies include the use of homing guide RNA that directs CRISPR-Cas9 to specific DNA barcode loci or a cytidine deaminase fused with nickase Cas9 to target endogenous interspersed repeat regions31,32. These technologies provide highly multiplexed approaches through the introduction of barcodes that progressively and stably accumulate unique mutations over time. Genome editing approaches are highly valuable because they allow retroactive analysis of the relationship between any two cells based on the shared inheritance of these barcodes. However, in order to read the barcodes in individual cells, the tissue usually must be disrupted, and therefore information regarding position, morphology, and absolute cell numbers from an individual progenitor is lost.
Combinatorial labeling paradigms preserve spatial information and in principle also allow for the distinction between closely localized or even overlapping clones33,34. For a lineage tracing method to be informative it must label individual progenitors and their progeny in a sparse and indelible manner. Notably, the Brainbow35 and Confetti36,37 approaches use stochastic multicolor Cre recombinase-based reporters that express a combination of fluorescent proteins from a single locus. The extensive number of simultaneous color combinations that can be achieved in vivo make this a powerful tool when tracing cortical RGP clones and astrocytes34. Transposon-based systems providing stable genomic integration of transgenes encoding fluorescent reporters and permitting lineage tracing of cortical progenitors have also been developed33,38,39,40,41. Transposon-based systems have an added advantage in that the reporter constructs stably integrate into the genome, and thereby reliably label lineally related daughter cells. To trace astrocyte lineages specifically, a number of methods have been developed that involve electroporation of piggyBac transposases including Star Track, which makes use of a combination of constructs encoding different fluorescent proteins40,42. Another approach, MAGIC markers, introduces Brainbow vectors as transposable transgenes. This has been successfully used to trace embryonic neural and astrocyte progenitors34,43. Recently, mosaic analysis by dual recombinase-mediated cassette exchange (MADR) was found to stably label mutant cells expressing transgenic elements from precisely defined chromosomal loci44. These powerful in vivo combinatorial labeling techniques have provided numerous insights into the lineage dynamics of progenitor cells. However, these analyses are performed on fixed tissue, providing a snapshot of individual clones at a defined developmental stage. In order to observe changes in the lineage dynamics of single clones over time, chronic in vivo imaging methods similar to those performed in the adult dentate gyrus need to be applied45.
Mosaic analysis with double markers (MADM) is a powerful dual color labeling method that enables in vivo lineage tracing of individual progenitor cells in mice46,47. Two components are necessary for MADM labeling events to occur: First, MADM cassettes must be targeted to identical loci on homologous chromosomes. Cassettes consist of two chimeric fluorescent reporter genes, eGFP (green, [G]) and tandem dimer Tomato (red, tdT[T]). The GT cassette contains the N-terminus of eGFP and the C-terminus of tdT, separated by an intron containing a loxP site. The TG cassette is constructed inversely, with the N-terminus of tdT and the C-terminus of eGFP. Second, the expression of Cre recombinase in the same cell containing the targeted MADM cassettes is essential. In the absence of Cre, the chimeric cassettes do not express functional eGFP or tdT because their coding sequences are disrupted. The loxP sites serve as the target for Cre-mediated interchromosomal recombination, resulting in the reconstitution of both expression cassettes simultaneously. If recombination occurs during the G2 phase of cell cycle followed by X segregation (G2-X), the two daughter cells will each express one of the two fluorescent proteins. Temporal regulation of CreERT2 activity using tamoxifen (TM) provides precise information on birth date of MADM clones and the division patterns of their progeny (Figure 1A)29,46,47.
MADM can potentially systematically label individual clones with high single cell resolution in the mouse brain similar to traditional but nonspecific and laborious methods like Golgi staining48 or dye filling49. Because only the promoter driving CreERT2 determines the cell type specificity of clonal MADM labeling, MADM can in principle be applied for clonal lineage tracing throughout any murine organ and tissue47,50,51,52. Indeed, studies have already used MADM to reveal lineage relationships in clones derived from diverse tissues47,50,51,52,53,54,55,56,57,58,59. MADM experimental paradigms have been applied to study lineage in cortical projection neurons, glia, and postnatal stem cells in the developing neocortex7,11,12,46,60,61,62,63,64,65. MADM has also been used to study cell lineage in the adult dentate gyrus, thalamus, cerebellar granule cells, and interneurons at the clonal level (see Table 1 for a complete list)47,53,54,56,57,66.
A unique feature of MADM is the ability to genetically link mutations distal to one MADM cassette, thereby creating a genetic mosaic (Figure 1B and Figure 2). This results in wild type daughter cells labeled with one fluorescent marker (tdT in Figure 1B) and homozygous mutant siblings with the other (eGFP in Figure 1B) in an unlabeled heterozygous environment. MADM is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Originally, MADM cassettes were targeted into the Rosa26 locus47, but MADM analysis of gene function was limited to genes distal to the locus. To overcome (at least partly) this limitation and expand the possibilities for MADM-based gene analyses, MADM cassettes were knocked in close to the centromeres of Chr. 751, Chr. 1146, and Chr. 1251. Targeting all 19 mouse autosomes with MADM cassettes is in progress and will allow for virtually any gene to be studied in the future, providing an unparalleled platform for the study of developmental lineage relationships in combination with functional genetic analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL tuberculin syringe (Omnifix Luer Lock)</td>
			<td>Braun</td>
			<td>9204512N</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-diazabicyclooctane (DABCO)</td>
			<td>Roth</td>
			<td>0718.2</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe (Omnifix Luer Lock)</td>
			<td>Braun</td>
			<td>8508429N</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge</td>
			<td>Sarstedt</td>
			<td>65.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>24 multi-well dishes</td>
			<td>Roth/Greiner Bio-one</td>
			<td>CE56.1</td>
			<td></td>
		</tr>
		<tr>
			<td>27- gauge x 3/4 needle (Sterican)</td>
			<td>Braun</td>
			<td>16010256E</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma</td>
			<td>C8267-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips (24 x 60 mm #1)</td>
			<td>Thermo Fisher Scientific (Menzel)</td>
			<td>15747592</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat Cryostar NX70</td>
			<td>Thermo Fisher Scientific</td>
			<td>957000H</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Pen (Wax marker)</td>
			<td>Agilent</td>
			<td>S200230-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable microtome blade (MX35 Ultra)</td>
			<td>Thermo Fisher Scientific</td>
			<td>705830</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps (Dumont #5)</td>
			<td>Fine Science Tools (FST)</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass anti-roll plate</td>
			<td>Histocom</td>
			<td>M 449980</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 800 Confocal</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td></td>
			<td></td>
			<td>25 mg/mL DAPCO, 6 g Glycerol, 2.4 g Mowiol 4-88, 6 mL dH2O, 12 mL 0.2 M Tris-HCl (pH 8.5)</td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Roth</td>
			<td>0713.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Innovative Research</td>
			<td>IGDNSER100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>441244-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump 323E/D 400RPM</td>
			<td>Watson-Marlow</td>
			<td>036.3124.00A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S8501-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus glass slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>J1800AMNT</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding mold T-12 (22mm square)</td>
			<td>Polysciences Inc.</td>
			<td>18986-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma</td>
			<td>93363</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P9416-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and Plugins:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>1.52p</td>
			<td><a href="http://fiji.sc/" target="_blank">Fiji</a></td>
			<td></td>
		</tr>
		<tr>
			<td>MultiStackReg</td>
			<td>1.45</td>
			<td><a href="http://bradbusse.net/sciencedownloads.html" target="_blank">Download link</a></td>
			<td></td>
		</tr>
		<tr>
			<td>TurboReg</td>
			<td></td>
			<td><a href="http://bigwww.epfl.ch/thevenaz/turboreg/" target="_blank">EPFL Bioimaging</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Blue</td>
			<td>2.6</td>
			<td><a href="http://www.zeiss.com/microscopy/en_us/products/microscope-software/zen.html#introduction" target="_blank">Zeiss</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental Models: Organisms/Strains:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: Emx1-CreER</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:027784</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: MADM-11-GT</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:013749</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: MADM-11-TG</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:013751</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFP 1:500</td>
			<td>Aves Labs</td>
			<td>GFP-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-tdTomato 1:500</td>
			<td>Sicgen Antibodies</td>
			<td>AB8181-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-RFP 1:500</td>
			<td>MBL</td>
			<td>PM005</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Chicken Alexa Fluor 488 1:500</td>
			<td>Jackson Immuno Research</td>
			<td>715-475-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Goat Cy3 1:500</td>
			<td>Jackson Immuno Research</td>
			<td>705-165-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit Cy3 1:500</td>
			<td>Jackson Immuno Research</td>
			<td>711-165-152</td>
			<td></td>
		</tr>
	</tbody>
</table>

lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (madm)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

MADM results in the reconstitution of functional green and red fluorescent proteins with two daughter cells each expressing one of the two fluorescent proteins upon G2-X chromosome segregation events (Figure 1A). Because MADM events result in permanent and distinct labeling of the two descendent lineages, quantifiable assessment of green and red daughter cell lineages (subclones) can be performed. Variables including division pattern (e.g., symmetric versus asymmetric) and potential (e.g., the number of progeny) of the original progenitor can be determined. &#160;Quantifying each fluorescently labeled subclone is informative when retroactively determining if the original progenitor cell is undergoing symmetric proliferative divisions, or asymmetric, neurogenic divisions at the time of TM induction. Previous studies grouped Emx1-CreERT2 or Nestin-CreERT2 derived excitatory projection clones in the cortex into two broad classes7,11,46. The first, termed &#34;symmetric proliferative clones&#34;, are composed on average of a considerable number of neurons, with both green and red subclones containing four or more neurons each. The second group, &#34;asymmetric clones&#34; defines a class of clones where the &#34;minority&#34; subclone contains fewer than three neurons and the &#34;majority&#34; subclone, four or more11. These definitions are specific to cortical RGPs and may need to be revisited for other brain regions and tissues. For both classes of cortical clones, progeny will be distributed throughout the superficial and deep layers.
When designing MADM clonal studies there are a number of aspects that must be taken into consideration. The time when MADM events are induced by administration of TM is a key consideration (Figure 3). For cortical excitatory projection neuron MADM clones (i.e., using Emx1-CreERT2 or Nestin-CreERT2) at E10, nearly all RGPs were still undergoing symmetric divisions11. Therefore, induction at E10 with TM captured multiple rounds of proliferative RGP amplification and resulted in clones with high neuron numbers. However, the number of RGPs at E10 was generally small and thus TM administration generated very few MADM events (sometimes less than one per brain). The majority of RGPs switched from symmetric to asymmetric neurogenic divisions at around E12. To target strictly asymmetric neurogenic clones, it was best to induce at E12 or later (Figure 3). The time between TM induction and observing MADM recombination events in the cortex tended to be less than 24 h. IP injections were the preferred method for administering TM at embryonic stages for this method because it led to greater reproducibility in clonal induction. It is also important to keep the TM dose to a minimum for two reasons. First, if the MADM recombination rate increases, the probability of inducing multiple, perhaps overlapping, clones is higher. Second, if too much TM is delivered, an increased rate of abortion, embryo reabsorption, and smaller litter sizes may be observed. Abortions in approximately half of all pregnant dams was observed when TM injections were delivered at E10. This frequency decreased from E11 onwards and diminished to approximately 1/3 of pregnant dams aborting. For a summary of TM doses, induction times, and CreERT2 drivers used in previous MADM studies, refer to Table 1. Reporter activity in the absence of TM was observed with some TM-inducible CreERT2 drivers69. Ectopic expression or MADM recombination events in the absence of TM was not observed with the Emx1-CreERT2 of Nestin-CreERT2 drivers. This may be partially due to the fact that TM-mediated chromosomal trans-recombinations occur at approximately 1:1,000 to 1:10,000 lower frequency than cis-recombinations, reducing the probability of ectopic MADM labeling.
Another factor to consider when planning a MADM clonal analysis experiment is the duration of the study. Varying the length of the time between TM induction and when the experiment was analyzed (A) (time window) displays stem cell dynamics over time64. Short embryonic time windows (i.e., TM/E11&#8722;A/E13; TM/E11&#8722;A/E16) captured the dynamics of embryonic neurogenesis (Figure 4). Comparing clones from two or more time windows provides quantitative insight into the number of cells produced and how neuron distribution varies at different stages of lineage progression64. To capture the entire potential of individual clones, it is necessary to extend the time window analyzed into postnatal or adult timepoints7,11,12. Examples of neocortical clones induced in the embryo and analyzed in the adult are shown in Figure 5. Of note, cortical neurogenesis is mostly completed and gliogenesis increases by E17. Approximately 1/6 neurogenic RGP also proceed to generate astrocytes and/or oligodendrocytes11.
Symmetric clones occur when RGPs undergo one or more rounds of proliferative division11. RGP clones induced between E10&#8722;E12 were on average larger in size and provided more spatial features of the final neuron distribution (Figure 4A-C). Clones with neurons relatively equally distributed throughout deep and superficial layers took on a &#34;cylinder&#34; shape while clones with neurons more dispersed in superficial layers than deeper layers developed a &#34;cone&#34; shape11. To fully capture the spatial and morphological information of a clone, it was necessary to computationally reconstruct each clone using sequential images. To measure clonal dispersion, the maximal lateral dispersion (measured in all dimensions) in superficial layers (LII&#8722;VI) of a clone was compared to neuron dispersion in deep layers (LV/LIV). This ratio (distribution upper:distribution lower) provided a quantifiable readout of the overall clone shape.
Asymmetric clones, where the minority subclone was three or less, provided insight into the neuronal output of a single RGP (Figure 4D-F and Figure 5A-F)7,11,12. The majority population (large subclone) could be labeled either in red or green, with an average of approximately seven excitatory projection neurons per clone when induced using an Emx1-CreERT2 or Nestin-CreERT2(Figure 5G)7,11,12. The total number of cells in a MADM clone could be further dissected by analyzing the distribution of neurons in the large subclone across superficial and deep layers. The minority population (small subclone) was labeled by the reciprocal color and was on average 1&#8722;2 cells per clone (Figure 5H). The total &#34;unit size&#34;, which was on average 8&#8722;9 neurons, could be calculated by adding the small and large subclones together (Figure 5I)7,11,12. It is important to note that while the neuronal output of RGPs was highly predictable, there was a degree of clonal heterogeneity12,70.
Introduction of a mutation distal to the MADM cassette enables the generation of genetic mosaics, providing a unique method to dissect the molecular regulators of stem cell lineage progression. As such, MADM provides an unparalleled experimental platform to study the cell-autonomous function of a gene (e.g., its association to microcephaly or macrocephaly). By comparing clones induced in a MADM genetic mosaic to clones induced in a control MADM, a highly quantitative readout of changes in neuron numbers and distribution can be generated. Previous MADM-based studies quantified the cell-autonomous function of Otx1 in microcephaly formation at the clonal level (see Figure 6A-E for a representative example)11. In another study, MADM clonal analysis demonstrated that Ndel1 does not cell-autonomously regulate projection neuron numbers, but instead the ability of newborn neurons to enter or migrate within the cortical plate, which later forms the adult cortex46. These studies demonstrated the highly quantitative nature of MADM clonal analysis in studying the cell-autonomous functions of genes regulating cortical development. There are currently no examples in the literature using MADM to study genes implicated in macrocephaly at the clonal level. However, in future studies analysis of genes relevant to the control of cortical size in general can provide highly desirable insights at the molecular and cellular level.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61147/61147fig1.jpg" /> 
Figure 1: The MADM principle for lineage tracing and clonal analysis at single stem cell level. (A) To perform lineage tracing and clonal analysis with MADM, two components must be present. First, MADM cassettes must be targeted to identical loci on homologous chromosomes. Cassettes consist of two chimeric fluorescent reporter genes, eGFP (green, [G]) and tandem dimer Tomato (red, tdT[T]). The GT cassette contains the N-terminus of eGFP and the C-terminus of tdT, separated by an intron containing a loxP site. The TG cassette is constructed inversely, with the N-terminus of tdT and the C-terminus of eGFP. Second, the expression of Cre recombinase must occur in the cell containing the targeted MADM cassettes. The loxP sites serve as a target for Cre-mediated interchromosomal recombination, resulting in the reconstitution of both expression cassettes simultaneously. If recombination occurs during the G2 phase of the cell cycle followed by X segregation (G2-X), the two daughter cells will express one of the two fluorescent proteins. (B) MADM principle for genetic mosaic analysis at a single clone level. Mutant alleles (point mutations, deletions, insertions, loxP-flanked conditional alleles as depicted in Figure 1B, etc.) can be introduced distal to the TG-MADM cassette via meiotic recombination (see Figure 2 and Hippenmeyer et al.46 for details on how to introduce mutant alleles into the MADM system). If a G2-X Cre recombinase-mediated interchromosomal trans-recombination occurs between MADM cassettes it results in one GFP+ homozygous mutant cell (GeneX-/-) for the gene of interest and one tdT+ homozygous wild type cell (GeneX+/+) in an unlabeled heterozygous environment46,47,71. Alternate labeling outcomes not used in the clonal analysis (i.e., yellow cells) have been previously described in detail11,46,47. <a href="https://www.jove.com/files/ftp_upload/61147/61147fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61147/61147fig2.jpg" /> 
Figure 2: Breeding schemes for generation of experimental MADM mice for lineage tracing. Breeding scheme for the generation of control MADM (A) and Gene X MADM (B) experimental MADM mice for clonal analysis. For more information regarding MADM breeding paradigms see Beattie et al.7 and Hippenmeyer et al.7,46. <a href="https://www.jove.com/files/ftp_upload/61147/61147fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61147/61147fig3.jpg" /> 
Figure 3: Time course paradigms for MADM-based clonal lineage analysis. Schematic of the experimental design time windows. For longitudinal sampling paradigms, the timepoint of clone induction remained constant and the length of time before analysis varied. In progressive interval sampling, the timepoint of analysis remained constant, but the time of induction varied. A combination of one or both approaches can be used depending on the questions addressed. <a href="https://www.jove.com/files/ftp_upload/61147/61147fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61147/61147fig4.jpg" /> 
Figure 4: MADM clonal analysis in the developing and adult neocortex. TM-mediated MADM clone induction in symmetrically proliferative (TM at E10) (A-C) and asymmetrically neurogenic (TM at E12) (D-F) dividing RGPs. Depicted are individual MADM clones in vivo in the developing (TM/E10&#8722;A/E16 and TM/E12&#8722;A/E16) (B,E) and adult (TM/E10&#8722;A/P21 and TM/E12&#8722;A/P21) (C,F) in MADM-11GT/TG; Nestin-CreERT2+/- (B,E) and MADM-11GT/TG; Emx1-CreERT2+/- (C,F). Neuron output was independent of subclone color and green majority/minority subclones could be compared to red majority/minority subclones under control conditions7,11. Approximately 1/6 of adult clones also contained astrocytes and/or oligodendrocytes, indicated by white asterisks. Panels B and F are reproduced with permission from Hippenmeyer et al.46 and Rulands and Simons72, respectively. CP = Cortical plate. <a href="https://www.jove.com/files/ftp_upload/61147/61147fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61147/61147fig5a.jpg" /> 
Figure 5: MADM clonal analysis to quantify RGP-mediated neuron output. Analysis of excitatory neuron (unit) production by individual neurogenic RGPs at the clonal level using MADM7,11. (A) Experimental paradigm for inducing mostly asymmetric MADM clones in the developing cortex. (B) Possible asymmetric clone outcomes with the majority subclone labeled in either green or red (C) Representative consecutive sections spanning a single neurogenic asymmetric clone (D,E) 3D reconstruction images of representative asymmetric G2-X MADM clones with majority population in red (D) or green (E) in MADM-11GT/TG; Emx1-CreERT2+/- with TM induction at E12 and analysis at P21. Note both green and red labeled cells are wild type. (F) Schematic indicating the two possible experimental MADM clone outcomes. (G) Quantification of the size of the majority population arising from renewing RGPs in MADM-11 clones. (H) Quantification of the size of the minority population arising from renewing RGPs in MADM-11 clones. (I) Quantification of the unitary size of asymmetric neurogenic MADM-11 clones. Hypothetical values could represent mean &#177; SEM. Scale bar = 100 &#181;m (D and E). TM = Tamoxifen. <a href="https://www.jove.com/files/ftp_upload/61147/61147fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61147/61147fig6.jpg" /> 
Figure 6: MADM clonal analysis to study genes that lead to microcephaly and macrocephaly. Hypothetical MADM clonal analysis results when performing functional genetic dissection of candidate genes that lead to microcephaly or macrocephaly. To dissect the cell-autonomous functions of a gene of interest (Gene X) on neuron output, MADM requires mutant alleles to be introduced distal to the MADM cassettes via meiotic recombination (for details how to introduce mutant alleles into the MADM system see also Figure 2, Hippenmeyer et al.46, and Laukoter et al.46,73). (A,B) Schematic indicating experimental MADM paradigm for functional analysis of clonal RGP units. The mutant subclone can either form the minority (A) or majority (B) population. (C-E) Hypothetical MADM clonal analysis results when quantifying control MADM (white bars), Gene-X MADM microcephaly (gray bars) and Gene-X MADM macrocephaly black bars) asymmetric clones. (C) Quantification of the size of the majority population. (D) Quantification of the size of the minority population. (E) Quantification of the unitary size of asymmetric neurogenic clones. Hypothetical values could represent mean &#177; SEM. S = Hypothetical scenario where difference in subclone cell number could reach significance, relative to control. <a href="https://www.jove.com/files/ftp_upload/61147/61147fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: MADM clonal studies in the literature. Summary of studies in the literature containing MADM clonal lineage experiments, including CreERT2 driver used, TM dose, and time of injection. <a href="https://www.jove.com/files/ftp_upload/61147/61147_table1.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>

Watch this Scientific Journal Video about Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM at JoVE.com

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...

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