MADM-based analysis of Cdkn1c imprinting phenotypes

In order to determine the degree of cell-autonomy of imprinted Cdkn1c gene function in cortical development, we used genetic MADM paradigms17,18,19. To this end, we capitalize on two unique properties of the MADM system: (1) the cell-type-specific generation and visualization of uniparental chromosome disomy (UPD, somatic cells with two copies of the maternal or paternal chromosome) for the functional analysis of imprinted dosage-sensitive gene function; and (2) the sparseness of UPD generation for analyzing cell-autonomous phenotypes at single-cell resolution. Since the imprinted Cdkn1c locus, located on mouse chromosome 7 (Chr. 7), exhibits maternal expression11,12, MADM-labeled cells carrying maternal UPD (matUPD, two maternal chromosomes) are predicted to express two copies of Cdkn1c and cells with paternal UPD (patUPD, two paternal chromosomes) would not express Cdkn1c (Fig. 1a). Thus, the phenotypic consequences of Cdkn1c loss (patUPD) and gain (matUPD) of function can be assessed simultaneously in MADM-induced UPDs, which also express distinct fluorescent reporters (Fig. 1a). MADM-based generation of Chr. 7 UPD occurs only in a very small fraction of genetically defined cells18 and permits the analysis of postnatal stages since the sparseness of genetic mosaicism enables the bypassing of early lethality associated with loss of Cdkn1c function10,20.

Fig. 1: MADM-based analysis of imprinted Cdkn1c gene function at single-cell level. a MADM recombination events result in distinct fluorescent labeling of cells containing uniparental disomy (UPD). Yellow cells are control cells, green cells carry maternal uniparental chromosome disomy (matUPD) and red cells contain paternal uniparental chromosome disomy (patUPD). Cdkn1c is expressed from the maternal allele in yellow cells, which resembles the wild-type situation. In green cells (matUPD) Cdkn1c is expressed from both maternal alleles and predicted to result in growth/proliferation disadvantage (expression of two doses of a growth inhibitor). Red cells (patUPD) lack Cdkn1c expression and are expected to show growth/proliferation advantage due to lack of expression of a growth suppressor. b Schematic depicts green (GFP+), red (tdT+), yellow (GFP+/tdT+) and unlabeled MADM cells with UPD (red, green) and control cells (yellow, unlabeled). Parental origin of chromosome is indicated (M, maternal; P, paternal). Imprinting status of Cdkn1c (arrow, expression; ball on stick, repression) and predicted expression (0×, 1×, 2×) is indicated. c Relative Cdkn1c expression in matUPD (red bars) and patUPD (blue bars) at E13 and E16. Bars represent mean. *p < 0.05, ***p < 0.001 (Wald test). Data points indicate individual animals (n = 4–7). d MADM-labeling pattern in cerebral cortex of MADM-7 (MADM-7GT/TG;Emx1Cre/+) at P21. The parent from which the MADM cassettes were inherited is indicated in the respective genotypes in pink (mother) and blue (father). e Higher magnification of cortical cross-section (boxed area in (d)). f G/R ratio of single MADM-labeled cortical neurons is depicted as geometric mean. Note the equipotency of cells with matUPD and patUPD. Nuclei (d, e) were stained using 4',6-diamidino-2-phenylindole (DAPI, blue). Data points indicating individual animals (n = 3). Source data are provided as a Source Data file. Scale bar, 500 μm (d), 90 μm (e). Full size image

No cell-autonomous role for Cdkn1c in cortical neurogenesis

To generate mice with MADM-induced UPD of Chr. 7 (MADM-7) in proliferating cortical RGPs, we crossed female MADM-7TG/TG with male MADM-7GT/GT;Emx1Cre/+ (Supplementary Figs. 1a and 2a) to obtain experimental MADM-7GT/TG;Emx1Cre/+ mice. In MADM-7 mice, sparse green GFP+ cells carry matUPD (prediction: two copies of expressed Cdkn1c), red tdT+ patUPD (prediction: no Cdkn1c expression), yellow GFP+/tdT+ and unlabeled cells carry no UPD (prediction: 1× Cdkn1c expression) (Fig. 1b). In order to assess and validate whether the above MADM paradigm faithfully results in the predicted levels of Cdkn1c expression, we isolated cells carrying matUPD and patUPD, and control cells at embryonic day (E) 13 and E16 based on their fluorescence by FACS. Next the samples were processed for RNA sequencing (RNA-seq) and from the obtained sequences we quantified the relative levels of Cdkn1c expression in matUPD and patUPD, when compared to control (Fig. 1c, Supplementary Data 1, 2). As predicted from the MADM scheme the levels of Cdkn1c expression were (1) much lower in patUPD in comparison to matUPD; and (2) while Cdkn1c expression was nearly absent in patUPD at E16, the level in matUPD (two copies of Cdkn1c) reached approximately twofold of the level in control (one copy of Cdkn1c) (Fig. 1c). To further assess the experimental UPD paradigm and to corroborate the above results, we generated comprehensive coverage plots for the RNA reads in the Cdkn1c genomic locus (Supplementary Fig. 3a–c, f) and the larger Kcnq1 cluster region (Supplementary Fig. 4a–c, f). The in-depth analysis of these coverage plots revealed (1) no novel, previously unannotated, transcripts in the Cdkn1c locus; and (2) that the predicted Cdkn1c expression occurs faithfully in UPD as predicted from the MADM scheme (Fig. 1a). Specifically, cells with matUPD (two doses of Cdkn1c; red in Fig. 1c and Supplementary Figs. 3a and 4a) show about twofold higher Cdkn1c expression levels than control cells (one dose of Cdkn1c; yellow in Fig. 1a and dark yellow in Supplementary Figs. 3c and 4c) whereas cells with patUPD (zero dose of Cdkn1c; green in Fig. 1a and blue in Supplementary Figs. 3b and 4b) show a drastic reduction of Cdkn1c expression. Thus, these results conclusively validate the MADM approach resulting in distinct doses of expressed Cdkn1c transcripts depending on the UPD status.

Since Cdkn1c promotes growth inhibition and cell-cycle exit, and taking into consideration the imprinting and expression status of Cdkn1c, green matUPD (two doses of Cdkn1c) cells would be expected to show growth/proliferation disadvantage when compared to red patUPD (no Cdkn1c) cells (Fig. 1a). Perhaps surprisingly we however found a green/red (G/R), i.e. matUPD/patUPD ratio of ~1 and thus equal growth/proliferation potential of RGPs regardless of the UPD status (Fig. 1d–f). These results indicate that Cdkn1c has no prominent cell-autonomous role in cell-cycle regulation of proliferating cortical RGPs. To directly test this possibility, we first analyzed the expression levels of cell-cycle regulators in RNA-seq data. We could not find significant differences in the expression levels of a number of key cell-cycle regulators in cells with matUPD or patUPD, respectively (Fig. 2a, b, Supplementary Data 2). Next, we measured incorporation of EdU after 1 h of exposure at E13 in the embryonic neuroepithelium and found that MADM-labeled cells with matUPD, patUPD and control all displayed equal relative amounts with EdU label (Fig. 2c, d). Lastly, we monitored the fraction of proliferating cells that remained in cell-cycle (EdU+/Ki67+) in a 24-h time window. Again, we found similarly sized EdU+/Ki67+ fractions of MADM-labeled cells with matUPD, patUPD and control (Fig. 2e, f). Altogether these data indicate that individual RGPs with matUPD (two expressed doses of Cdkn1c) or patUPD (no expressed Cdkn1c), in an otherwise wild-type environment, exhibit similar proliferation behaviors. Based on the sparse induction of UPD in just very few cells (with a vastly normal background), these results show that Cdkn1c does not regulate RGP proliferation behavior cell-autonomously. This is also in agreement with the observed sparse expression pattern of p57KIP2 protein in RGPs (less than 12% of PAX6+ RGPs express p57KIP2)14. The results thus indicate that the observed cortical overgrowth observed in Cdkn1c−/− full knockout mice14 is mainly due to global non-cell-autonomous Cdkn1c function and/or community effects.

Fig. 2: Analysis of imprinted Cdkn1c cell-cycle properties based on MADM-UPD. a Schematic depicts green (GFP+), red (tdT+), yellow (GFP+/tdT+) and unlabeled MADM cells with UPD (red, green) and control cells (yellow, unlabeled). Parental origin of chromosome is indicated (M, maternal; P, paternal). Imprinting status of Cdkn1c (arrow, expression; ball on stick, repression) and predicted expression (0×, 1×, 2×) is indicated. b Expression levels of two representative cell-cycle genes (Cdk1, Pcna) in matUPD (red bars) and patUPD (blue bars) at E13 and E16. Data points indicate individual animals (n = 5−7). c EdU labeling (1 h chase) in MADM-7 (MADM-7GT/TG;Emx1Cre/+) cerebral cortex at E13 with GFP+ matUPD (green) and tdT+ patUPD (red) (top) and colabeled with EdU in white (bottom) double-positive cells are indicated by yellow arrows. d Fraction (%) of MADM+/EdU+ colabeled cells (red bar, matUPD (GFP+ cells); blue bar, patUPD (tdT+ cells); gray bar, control cells (GFP+/tdT+ cells)). Data points indicate individual animals (n = 6). e EdU+/Ki67+ colabeling in MADM-7 (MADM-7GT/TG;Emx1Cre/+) cerebral cortex with EdU injection (24 h chase) at E13. MADM-labeled (matUPD, GFP+; patUPD, tdT+ in top) Ki67+ (blue in middle) and EdU+ (white in bottom) triple-positive cells are indicated by yellow arrows. f Fraction (%) of MADM+/Ki67+/EdU+ cells of total MADM+/EdU+ cells after 24 h EdU chase (red bar, matUPD (GFP+); blue bar, patUPD (tdT+); gray bar, control cells (GFP+tdT+ cells)). Data points indicate individual animals (n = 4). All bars represent mean. Error bars represent SEM (d, f). Nuclei (c) were stained using DAPI (blue). NCX neocortex, HC hippocampus. Scale bar, 15 μm (c), 10 μm (c top and bottom), 25 μm (e), 20 μm (e top, middle, bottom). Source data are provided as a Source Data file. Full size image

No loss of imprinted Cdkn1c expression in cortical cells

An alternative explanation for our results indicating equipotency of Chr. 7 matUPD/patUPD could be the possible cell-type-specific loss of imprinting, a phenomenon which has been observed for imprinted Dlk1 in postnatal stem cells21. In other words, the paternal Cdkn1c allele would be de-repressed specifically in Emx1+ RGPs and/or their lineage. Thus cells with mat- and patUPD would both express similar doses of Cdkn1c and therefore show the same phenotype, i.e. the same number of projection neuron output from RGPs. To test this possibility we generated F1 C57BL/6J (B6)—CAST/EiJ (CAST) hybrids22 to qualitatively and quantitatively analyze allelic expression in RGPs and nascent neurons. We used two well-defined single nucleotide polymorphisms (SNPs) located in exon 2 and exon 4 of Cdkn1c (Supplementary Fig. 5a), and a SNP in Ndn (single exon gene which is paternally expressed) as control22. We first isolated genomic DNA from an individual B6/CAST hybrid embryo at E12 to confirm the presence of respective SNPs in Cdkn1c and Ndn. Sanger and deep sequencing of the genomic DNA confirmed the presence and ~50% abundance of each parental SNP as expected (Supplementary Fig. 5b–h, o). Next, we generated single-cell suspensions of E12 cortex from B6/CAST hybrids and used well-established FACS protocols23 to enrich for RGPs and projection neurons. From the obtained neuron and progenitor populations, we isolated RNA which we converted to cDNA, followed by Sanger and deep sequencing to determine allelic expression (Supplementary Fig. 5b–q). These experiments revealed highly skewed, almost exclusive allelic expression of paternally expressed Ndn, and maternally expressed Cdkn1c in both RGP and neuron cell populations. Importantly, paternal expression of Cdkn1c in neurons (<5%) and progenitors (<2%) was miniscule (Supplementary Fig. 5p, q). Thus imprinting and repression of the paternal Cdkn1c allele is intact in embryonic neurogenic RGPs and nascent neurons. In conclusion, the results from allelic expression experiments corroborate the above findings of MADM-based analysis of Chr. 7 UPD (Fig. 1). Together, these data demonstrate that Cdkn1c does not cell-autonomously control RGP-mediated neuron output and/or maturation, and that the observed macrocephaly in Cdkn1c−/− full knockout likely reflects global organism overgrowth.

Genetic Cdkn1c ablation results in microcephaly

To more directly assess Cdkn1c function in cortical RGPs at single-cell resolution, we next introduced a conditional Cdkn1c-flox allele24 into MADM-7 (MADM-7GT/TG,Cdkn1c-flox;Emx1Cre/+). When we introduced the Cdkn1c-flox allele from the mother, all cells (regardless of UPD status) of the Emx1+ lineage in F1 should in principle be equivalent to homozygous Cdkn1c−/− because of imprinting, i.e. deleted maternal expression and paternal silencing (Fig. 3a and Supplementary Figs. 1b and 2b). Indeed, we found that control cells, and cells with matUPD and patUPD, all displayed nearly undetectable relative Cdkn1c expression levels (Fig. 3b). However, contrary to our expectation of a cortical overgrowth phenotype due to Cdkn1c loss of function, and RGP equipotency with G/R of 1, we observed a dramatic reduction of GFP+ Cdkn1c−/− matUPD cells when compared to tdT+ Cdkn1c+/+ patUPD (although with two copies of silenced Cdkn1c) and severe microcephaly (Fig. 3c–f). This Cdkn1c conditional deletion phenotype is in stark contradiction with a growth suppressive function of Cdkn1c. Next, we evaluated whether the suspected growth-promoting function of Cdkn1c is subject to regulation by genomic imprinting. To this end we introduced the Cdkn1c-flox allele from the father (Fig. 3g and Supplementary Figs. 1b and 2c) and generated GFP+ Cdkn1c−/− patUPD cells which we compared to tdT+ Cdkn1c+/+ matUPD. Analysis of the relative Cdkn1c expression confirmed that patUPD express very low levels of Cdkn1c while matUPD (with two copies of Cdkn1c) show about twofold higher Cdkn1c expression in comparison to control cells (one copy of Cdkn1c) (Fig. 3h). Strikingly we observed again dramatic reduction of mutant Cdkn1c−/− cells when compared to Cdkn1c+/+ cells (Fig. 3i–l). This is unexpected because the imprinting status of Cdkn1c in patUPD already shows no expression of Cdkn1c due to homozygosity and complete silencing of both paternal Cdkn1c alleles (Fig. 3g, h). To further support our finding of a Cdkn1c growth-promoting function independent of parental Chr. 7 UPD status, we introduced the Cdkn1c-flox allele from both parents and generated a true Cdkn1c conditional knockout (cKO) but with sparse MADM-labeling for single-cell analysis (MADM-7GT,Cdkn1c-flox/TG,Cdkn1c-flox;Emx1Cre/+) (Fig. 3m and Supplementary Figs. 1c and 2d). In these cKO-Cdkn1c-MADM-7 mice control cells and cells with matUPD and patUPD all displayed nearly undetectable levels of Cdkn1c expression (Fig. 3n) similar to the paradigm with maternal Cdkn1c deletion (Fig. 3b). Consequently, the cKO-Cdkn1c-MADM-7 mice exhibit very strong microcephaly (Fig. 3o–q), similar to Cdkn1c-MADM-7 mice with maternal deletion (Fig. 3c–e), and slightly more severe than with paternal deletion (Fig. 3i–k). Importantly however, in cKO-Cdkn1c-MADM-7 mice we observed a G/R of ~1 unlike the very low G/R ratio in Cdkn1c-MADM-7 with maternal or paternal deletion, respectively (Fig. 3f, l, r). A G/R ratio of ~1 in cKO-Cdkn1c-MADM-7 cortex indicates equipotency of mutant Cdkn1c−/− neurogenic RGP cells regardless of the disomy status (i.e. green GFP+ are patUPD and red tdT+ are matUPD) similar like in MADM-7 where all cells are Cdkn1c+/+ although with UPD (Fig. 1).

Fig. 3: Cell-autonomous growth-promoting Cdkn1c function is independent of genomic imprinting status. Analysis of Cdkn1c-MADM-7 (MADM-7GT/TG,Cdkn1c;Emx1Cre/+) with maternal deletion (a–f), Cdkn1c-MADM-7 (MADM-7GT/TG,Cdkn1c;Emx1Cre/+) with paternal deletion (g–l), and cKO-Cdkn1c-MADM-7 (MADM-7GT,Cdkn1c/TG,Cdkn1c;Emx1Cre/+) (m–r). The parent from which the MADM cassettes with or without recombined Cdkn1c-flox allele (Cdkn1c) were inherited is indicated in the respective genotypes in pink (mother) and blue (father). Predicted Cdkn1c expression in GFP+ (green), tdT+ (red), GFP+/tdT+ (yellow) and unlabeled (gray) MADM-labeled cells (a, g, m); and true relative levels of Cdkn1c expression in GFP+ (green bar), tdT+, (red bar), GFP+/tdT+ (yellow bar) MADM-labeled cells (b, h, n) in Cdkn1c-MADM-7 with maternal deletion (a, b), Cdkn1c-MADM-7 with paternal deletion (g, h), and cKO-Cdkn1c-MADM-7 (m, n) at E16 are indicated (MM, matUPD; PP, patUPD; MP, control). Data points indicate individual animals (n = 5–7).Outlier values are not shown (see Source Data File). Bars indicate median. Imprinting of Cdkn1c (arrow, expression; ball on stick, repression) and predicted expression (0×, 1×, 2×) is indicated. Parental origin of chromosome is indicated (M, maternal; P, paternal) and conditional deletion of Cdkn1c is marked with red cross. MADM-labeling (GFP+, green; tdT+, red; GFP+/tdT+, yellow) (c, d, i, j, o, p), cortical width (μm) is shown with an error bar representing SEM (e, k, q), and G/R ratio (geometric mean) of single MADM-labeled cortical projection neurons (f, l, r) in Cdkn1c-MADM-7 with maternal deletion (c–f), Cdkn1c-MADM-7 with paternal deletion (i–l), and cKO-Cdkn1c-MADM-7 (o–r) at P21 are indicated. Boxed areas in overview images (c, i, o) show representative images of the extent of microcephaly at higher resolution (d, j, p). Nuclei were stained using DAPI (blue). Scale bar, 500 μm (overview (c, i, o)) and 90 μm (inset (d, j, p)). Note the decreased numbers of Cdkn1c−/− cells when compared to Cdkn1c+/+ cells in (f) and (l) but equipotency/equal numbers of Cdkn1c−/− cells in (r) regardless of the UPD status. Data points (e, f, k, l, q, r) indicating individual animals (n = 3). NCX neocortex, HC hippocampus. Source data are provided as a Source Data file. Full size image

Next, we analyzed the emergence of the microcephaly phenotype during development in a time course analysis. We found that the microcephaly phenotype was already evident from E13 onwards in Cdkn1c-MADM-7 with maternal and paternal deletion, respectively, and in cKO-Cdkn1c-MADM-7. The severity of microcephaly increased until E16 (Supplementary Fig. 6), persisted until birth and in postnatal mice up to P21 (latest time point of analysis).

In summary, our MADM-based analysis of UPD and in combination with conditional deletion of Cdkn1c revealed a growth-promoting function of Cdkn1c which is dominant over the imprinting status of the genomic Cdkn1c locus. Given that mosaic Cdkn1c-MADM-7 mice (all cells are Cdkn1cflox/+) with heterozygous Cdkn1c deletion in Emx1+ lineage show microcephaly similar like cKO-Cdkn1c-MADM-7 mice, we conclude that the growth-promoting function of Cdkn1c is also highly dosage sensitive. In other words, removal of one copy of Cdkn1c leads to haploinsufficiency with nearly identical phenotype to that observed when both copies were ablated.

Gene expression profile upon genetic deletion of Cdkn1c

To gain better insight into the putative mechanism of the growth-promoting Cdkn1c function, we first profiled global gene expression in cKO and compared to control mice. To this end we purified cells of the Emx1+ lineage in E16 cortices from control MADM-7 and cKO-Cdkn1c-MADM-7 mice by FACS. Next, the samples were processed for RNA-seq and global gene expression profiles were established for further analysis (Fig. 4a, Supplementary Data 3) (see Methods for details). First, we reduced the dimensionality of our data and identified similarities and differences in global gene expression between control and cKO-Cdkn1c-MADM-7 by principal component analysis (PCA). We found that the samples with distinct genotypes (i.e. control vs. cKO) segregated from each other (Fig. 4b). Next, we confirmed the loss of Cdkn1c expression in cKO-Cdkn1c-MADM-7 (Fig. 4c). To exclude the possibility of any remaining truncated partial Cdkn1c mRNA species and/or the residual presence of previously unannotated transcripts within the Cdkn1c genomic locus and broader Kcnq1 cluster region in cKO-Cdkn1c-MADM, we generated coverage plots for the obtained RNA reads (Supplementary Figs. 3d–f and 4d–f). This analysis confirmed (1) the drastic reduction of Cdkn1c transcripts in cKO-Cdkn1c-MADM and (2) did not reveal any novel unannotated transcripts that could hypothetically escape deletion in our conditional genetic experimental paradigm. These results thus conclusively validate our genetic deletion approach to conditionally ablate Cdkn1c expression in cortical Emx1+ lineage.

Fig. 4: Gene expression profile in cortex upon genetic deletion of Cdkn1c locus. a Experimental paradigm. b Principal component analysis of all samples used for analysis. Data points indicate individual animals (n = 3–7). c Relative levels of Cdkn1c in cKO and controls cells. Bars represent mean. Data points indicate individual animals (n (controls) = 7, n (cKO) = 3). d Number of up-, and downregulated genes in cKO/control comparisons (adjusted p value < 0.05, Wald test). e Significance scores of all informative genes within 2 Mbp up-, and downstream of Cdkn1c are shown (y axis). Each dot represents one gene with the position given as the midpoint of its genomic locus (mega base-pairs (Mbp), x axis). Horizontal lines mark limits for significant differential expression (corresponding to an adjusted p value of 0.05, Wald test). Gray box marks the putative maximum size of the Kcnq1 imprinted gene cluster. f Curated list of significantly enriched gene ontology (GO) terms in the lists of up-, downregulated genes shown in (d). The full list of significantly enriched GO terms is available in Supplementary Data 5. Source data are provided as a Source Data file. Full size image

To obtain a first pass measure of the extent of differential gene expression in cKO-Cdkn1c-MADM-7, we plotted the number of up- and downregulated genes (Fig. 4d, Supplementary Data 4). Since imprinted Cdkn1c is embedded in a larger cluster, the Kcnq1-cluster of imprinted genes on Chr.7, we analyzed whether the expression of neighboring genes could be affected in the cKO-Cdkn1c-MADM-7 mice with conditional Cdkn1c deletion (Fig. 4e). While the expression profile of Cdkn1c indicated drastically low, nearly absent, levels of expression, the genes flanking (2 Mbp up- and downstream) the Cdkn1c genomic locus displayed no significant differential expression (Fig. 4e). We next performed gene-ontology (GO) enrichment analysis of the differentially expressed genes in cKO-Cdkn1c-MADM-7. This analysis revealed a very high probability of cellular phenotypes associated with the downregulation of genes related to neurogenesis in general and with the upregulation of genes involved in cell death in particular (Fig. 4f, Supplementary Data 5).

Cdkn1c is cell-autonomously required for cellular survival

Based upon our findings from gene expression profiling and since previous studies showed that the hydrocephalus phenotype in Cdkn1c cKO induced with Nestin-Cre can be rescued by concomitant p53 (Trp53) ablation15, we next analyzed cell death parameters upon conditional loss of Cdkn1c. We thus stained cortex at E13 in MADM-7, Cdkn1c-MADM-7 and cKO-Cdkn1c-MADM-7 embryos for apoptotic cells with antibodies against Caspase-3 (Fig. 5). While in MADM-7 almost no cortical cells showed signs of apoptosis (Fig. 5a–e), high numbers (up to 20% of all cells) of Caspase-3+ cells were detected in Cdkn1c-MADM-7 (maternal and paternal deletion) and cKO-Cdkn1c-MADM-7 (Fig. 5f–t).

Fig. 5: Cdkn1c function is required for nascent cortical projection neuron survival. Analysis of developing cerebral cortex in MADM-7 (MADM-7GT/TG;Emx1Cre/+) (a–e), Cdkn1c-MADM-7 (MADM-7GT/TG,Cdkn1c;Emx1Cre/+) with maternal deletion (f–j), Cdkn1c-MADM-7 (MADM-7GT/TG,Cdkn1c;Emx1Cre/+) with paternal deletion (k–o), and cKO-Cdkn1c-MADM-7 (MADM-7GT,Cdkn1c/TG,Cdkn1c;Emx1Cre/+) (p–t) at E13. The parent, from which the MADM cassettes and with recombined Cdkn1c-flox allele (Cdkn1c) (f–t) was inherited, is indicated in the respective genotypes in pink (mother) and blue (father). Schematics (a, f, k, p) depict green (GFP+), red (tdT+), yellow (GFP+/tdT+) and unlabeled MADM cells with UPD (red, green) and control cells (yellow, unlabeled). Imprinting (arrow, expression; ball on stick, repression) and expression (0×, 1×, 2×) status is indicated. Conditional deletion of Cdkn1c is marked with red cross. Parental origin of chromosome is indicated (M maternal, P paternal). MADM-labeling in overview (GFP+, green; tdT+, red; GFP+/tdT+, yellow) (b, g, l, q) and G/R ratio (geometric mean) of single MADM-labeled cortical projection neurons (c, h, m, r) in MADM-7 (b, c), Cdkn1c-MADM-7 with maternal deletion (g, h), Cdkn1c-MADM-7 with paternal deletion (l, m), and cKO-Cdkn1c-MADM-7 (q, r) at E13 indicate emerging microcephaly. Note the equipotency of cells with matUPD and patUPD in (c) and (r) but decreased numbers of Cdkn1c−/− cells when compared to Cdkn1c+/+ cells in (h) and (m) regardless of the UPD status. Labeling of apoptotic Caspase-3+ cells (white) in MADM tissue (GFP+, green; tdT+, red; GFP+/tdT+, yellow) (d, i, n, s); and quantification of Caspase-3+/DAPI+ coloc (%) (e, j, o, t) in MADM-7 (d, e), Cdkn1c-MADM-7 with maternal deletion (i, j), Cdkn1c-MADM-7 with paternal deletion (n, o), and cKO-Cdkn1c-MADM-7 (s, t) at E13. Note that in MADM-7 almost no Caspase-3+ cells are detected. Yellow arrows indicate GFP+ remnants of Cdkn1c−/− mutant cells. All bars indicate mean. Error bars represent SEM (e, j, o, t). Data points indicate individual animals (n = 3). Nuclei were stained using DAPI (blue). NCX neocortex. Scale bar, 200 μm (b, g, l, q) and 20 μm (d, i, n, s). Source data are provided as a Source Data file. Full size image

Interestingly, at E13 the relative number of homozygous mutant cells, i.e. green Cdkn1c−/− cells, in Cdkn1c-MADM-7 (maternal and paternal deletion) was already reduced in comparison to red Cdkn1c+/+ wild-type cells (Fig. 5h, m). Note that again this reduction in cell number appeared irrespective of the chromosomal disomy status and thus independent of genomic imprinting. However, Cdkn1c-MADM-7 mice are genetic mosaics with green cells that show homozygous deletion of the Cdkn1c genomic locus, red cells with homozygous intact Cdkn1c locus and heterozygous yellow/unlabeled cells with one deleted copy of Cdkn1c locus. Therefore, we next analyzed whether the red cells (tdT+) with homozygous intact Cdkn1c locus in Cdkn1c-MADM-7 (maternal and paternal deletion) mice would have a survival advantage or disadvantage when compared to the heterozygous yellow or unlabeled (DAPI+) cells. Strikingly, far fewer red cells with homozygous intact Cdkn1c genomic locus were positive for Caspase-3 than unlabeled (DAPI+) heterozygous cells, regardless of the imprinting status and thus expressed dose of Cdkn1c transcript (Supplementary Fig. 7). Collectively, these results demonstrate that the genetic deletion of Cdkn1c genomic locus results in an increased probability of cell death in a highly dosage-dependent manner. In other words, removal of one copy of Cdkn1c results in haploinsufficiency with increased apoptosis when compared to wild-type cells with two intact copies of Cdkn1c.

In the course of our analysis of cell death upon genetic deletion of Cdkn1c, we noticed that Caspase-3+ cells in Cdkn1c-MADM-7 and cKO-Cdkn1c-MADM-7 at E13 seemed to appear less prominently in the ventricular zone (VZ) but more abundant in nascent neurons located in the emerging cortical plate (CP). To quantitatively assess the rate of cell death in ventricular RGPs and nascent neurons in the CP, we stained sections from MADM-7, Cdkn1c-MADM-7 and cKO-Cdkn1c-MADM-7 embryos with antibodies against Caspase-3 together with the RGP marker PAX6 or nascent neuron marker NEUROD2, respectively. Indeed, the absolute number of PAX6+/Caspase-3+ was significantly lower than the number of NEUROD2+/Caspase-3+ cells in Cdkn1c-MADM-7 and cKO-Cdkn1c-MADM-7 (Supplementary Fig. 8). Thus, not only nascent cortical projection neurons (NEUROD2+) show a higher incidence of cell death upon Cdkn1c ablation but also PAX6+ RGPs. Consequently, the overall number of actively proliferating RGPs is already reduced from early E13 embryonic stages onward (Supplementary Fig. 9). We also corroborated our findings by staining sections from MADM-7, Cdkn1c-MADM-7 and cKO-Cdkn1c-MADM-7 embryos with antibodies against p53 which marks cells that initiate the apoptotic pathway (Supplementary Fig. 10). Lastly, both upper layer callosal (SATB2+) and deep layer corticofugal (TBR1+) projection neuron populations were cell-autonomously prone to apoptosis and thus reduced in Cdkn1c-MADM-7 with maternal and paternal deletion, respectively (Fig. 6). Altogether, we conclude that the cell-autonomous growth-promoting Cdkn1c function discovered here is mainly acting to promote the survival of differentiating and maturing cortical projection neurons and (to a lesser extent) proliferating RGPs.