Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs

MADM-ML, a Mouse Genetic Mosaic System with Increased Clonal Efficiency

<div><p>Mosaic Analysis with Double Markers (MADM) is a mouse genetic system that allows simultaneous gene knockout and fluorescent labeling of sparse, clonally-related cells within an otherwise normal mouse, thereby circumventing embryonic lethality problems and providing single-cell resolution for phenotypic analysis <i>in vivo</i>. The clonal efficiency of MADM is intrinsically low because it relies on Cre/loxP-mediated mitotic recombination <i>between</i> two homologous chromosomes rather than within the same chromosome, as in the case of conditional knockout (CKO). Although sparse labeling enhances <i>in vivo</i> resolution, the original MADM labels too few or even no cells when a low-expressing Cre transgene is used or a small population of cells is studied. Recently, we described the usage of a new system, MADM-ML, which contains three mutually exclusive, self-recognizing loxP variant sites as opposed to a single loxP site present in the original MADM system (referred to as MADM-SL in this paper). Here we carefully compared the recombination efficiency between MADM-SL and MADM-ML using the same Cre transgene, and found that the new system labels significantly more cells than the original system does. When we established mouse medulloblastoma models with both the original and the new MADM systems, we found that, while the MADM-SL model suffered from varied tumor progression and incomplete penetrance, the MADM-ML model had consistent tumor progression and full penetrance of tumor formation. Therefore MADM-ML, with its higher recombination efficiency, will broaden the applicability of MADM for studying many biological questions including normal development and disease modeling at cellular resolution <i>in vivo</i>.</p> </div

The MADM system.

<p>MADM generates sibling green mutant and red wildtype daughter cells from a colorless heterozygous progenitor via Cre-loxP mediated mitotic recombination followed by X segregation. MADM can also generate yellow and colorless heterozygous cells when mitotic recombination is followed by Z segregation.</p

The design of MADM with multiple loxP sites (MADM-ML).

<p>(A) Mutually exclusive loxP sites (wildtype loxP and loxP variants lox2272 and lox5171) do not recombine with each other <i>in </i><i>cis</i>. (B) These loxP variants recombine <i>in </i><i>trans</i>, but only with their identical sites, but not with other variants. </p

MADM allows visualization of early lesions and detailed tumor organization.

<p>(A) A representative brain section from a MADM-generated medulloblastoma. The GFP-labeling of mutant cells amidst sparse background labeling of granule neurons clearly identifies early lesion sites consisting of only hundreds to thousands of cells in a P90 mouse. (B) Tumor cells in the small lesion are GFP+, suggesting that p53 loss is critical for malignant transformation. (C) Co-staining of GFP with proliferation or differentiation markers allows us to perform detailed analysis of tumor organization. Top panels show many actively dividing tumor cells that are Ki67+ GFP+ (inset is a zoomed-in image showing clear GFP/Ki67 overlap). Bottom panels show NeuN+ GFP+ cells within the tumor mass, suggesting that some tumor cells differentiate into neurons (inset is a zoomed-in image showing clear GFP/NeuN overlap).</p

MADM-ML has significantly higher recombination efficiency than MADM-SL, and maintains the productive G2-X segregation pattern.

<p>(A) Representative images of cerebella in MADM Math1-Cre mice at P5 and P14 with quantification areas outlined (EGL for P5, IGL for P14). MADM-ML has significantly more labeled cells than MADM-SL does, for both P5 and P14 data set. P5 data (n=5 for ML/ML, n=4 for SL/SL, and n=5 ML/SL) are analyzed by ANOVA and multiple comparisons with Tukey HSD. There is no significant difference between SL/SL and ML/SL. P14 data (n=5 for ML/ML and n=7 for SL/SL) are analyzed by an independent samples t-test with unequal variance. (B) There is no significant difference in G2-X segregation frequency between the two MADM systems, as measured by percent of green and red cells among total labeled cells. n=5 for ML/ML and n=7 for SL/SL. Error bars are one standard deviation.</p

Two possible mechanisms for increased recombination efficiency of MADM-ML.

<p>(A) Hypothesis #1: increased recombination efficiency could result from higher local Cre recombinase concentration retained by multiple loxP sites. If this is the case, then ML/SL should have an intermediate recombination efficiency between SL/SL and ML/ML. (B) Hypothesis #2: increased recombination efficiency could be due to increased substrate concentration, i.e. three times more loxP site pairing. If this is the case, then ML/SL should have the same recombination efficiency as SL/SL has.</p

The generation of a medulloblastoma model with the MADM system.

<p>(A) Breeding scheme for generating medulloblastoma mouse model. (B) The correlation between fluorescent protein labeling and genotype of cells generated by MADM in this model. All cells in the animal are heterozygous for <i>Ptc</i> mutation but <i>p53</i> status varies. Green cells are homozygous null for <i>p53</i>; red cells are homozygous WT for <i>p53</i>; yellow and colorless cells are heterozygous for <i>p53</i> (KO/WT). (C) Left, a normal adult brain contains a floxed-stop Rosa-tdTomato Cre reporter and <i>Math1</i>-Cre. Middle, CKO medulloblastoma model contains the same Cre line and reporter, heterozygous <i>Ptc</i> mutation, and homozygous <i>p53flox</i> alleles. Right, an early tumor formed using our MADM mudulloblastoma model. T: tumor. Scale bar: 500um.</p

MADM-ML medulloblastoma model generates tumors at full penetrance with a more consistent timeline of tumor progression.

<p>(A) Representative images used for scoring tumor size and penetrance. (B) Summary of different tumor sizes in MADM-SL (green stars) and MADM-ML (purple dots) models at various ages. MADM-SL does not have a predictable tumor progression time course and has less than 40% tumor penetrance even after P75. Conversely, MADM-ML has a relatively consistent timeline of tumor progression and full penetrance.</p

Astrocytic trans-differentiation completes a multicellular paracrine feedback loop required for medulloblastoma tumor growth

The tumor microenvironment (TME) is critical for tumor progression. However, the establishment and function of the TME remain obscure because of its complex cellular composition. Using a mouse genetic system called mosaic analysis with double markers (MADMs), we delineated TME evolution at single-cell resolution in sonic hedgehog (SHH)-activated medulloblastomas that originate from unipotent granule neuron progenitors in the brain. First, we found that astrocytes within the TME(TuAstrocytes) were trans-differentiated from tumor granule neuron precursors (GNPs), which normally never differentiate into astrocytes. Second, we identified that TME-derived IGF1 promotes tumor progression. Third, we uncovered that insulin-like growth factor 1 (IGF1) is produced by tumor-associated microglia in response to interleukin-4 (IL-4) stimulation. Finally, we found that IL-4 is secreted by TuAstrocytes. Collectively, our studies reveal an evolutionary process that produces a multi-lateral network within the TME of medulloblastoma: a fraction of tumor cells trans-differentiate into TuAstrocytes, which, in turn, produce IL-4 that stimulates microglia to produce IGF1 to promote tumor progression