Research ArticleDEVELOPMENTAL BIOLOGY

PRDM15 loss of function links NOTCH and WNT/PCP signaling to patterning defects in holoprosencephaly

See allHide authors and affiliations

Science Advances  10 Jan 2020:
Vol. 6, no. 2, eaax9852
DOI: 10.1126/sciadv.aax9852
  • Fig. 1 PRDM15 mutations in patients with SRNS and microcephaly/HPE.

    (A) Schematic representation of the PRDM15 mutation positions and the affected domains. (B) Alkaline phosphatase (AP) staining of ESCs; the respective genotypes are indicated in the lower panel. Data are average of four independent cell cultures (n = 4) ± SD. Statistical tests were applied on differences observed in the percentage of completely undifferentiated colonies. Student’s t test (two sided) was used to determine significance. (C) Heat map of differentially expressed genes in ESCs upon the indicated genetic manipulations. (D) mRNA levels of Rspo1 in ESCs; the respective genotypes are indicated by color code. Expression levels were normalized to Ubiquitin (Ubb), and Prdm15fl/fl (empty vector) was used as reference. Data shown are from three independent experiments (n = 3). (E) Enrichment of PRDM15 binding on promoter regions of the target gene (Rspo1) in ESCs—respective genotypes are indicated by color code—as measured by ChIP-qPCR. Depicted is the average enrichment [data from three independent cell cultures (n = 3)] over percent of input. In (B) to (E), the endogenous mouse Prdm15 has been deleted by the addition of OHT (50 nM) after ectopic expression of WT or mutant human PRDM15 (hPR15). In (D) and (E), center values, mean; error bars, SD. Student’s t test (two sided) was used to determine significance.

  • Fig. 2 Genetic deletion of Prdm15 leads to brain malformations and midgestation lethality in mice.

    (A) Genetic distribution of embryos from Prdm15+/LacZ intercrosses, indicating lethality between E12.5 and E14.5. (B) Phenotypic continuum of brain defects in E12.5 Prdm15lacZ/lacZ KO embryos. (C) Hematoxylin and eosin (H&E) staining of serial coronal sections of E12.5 brains from Prdm15+/+ WT (upper panel) and Prdm15lacZ/lacZ KO (lower panel) embryos. The mutants lack the complex organization of the anterior forebrain, including the lateral (LGE) and medial ganglionic eminences (MGE), the epithalamic and dorsal thalamic neuropeithelium (NE), and eyes. (D) Nestin-Cre–meditated deletion of Prdm15 in neuronal precursors does not affect brain development. Representative images are shown in (B) to (D). LGE/MGE, lateral and medial ganglionic eminences; NE, neuropeithelium; NCX, neocortex; E, eye; LV, lateral ventricle; V, ventricle; TOT, total. (B and D) Photo credit: Messerschmidt and Mzoughi.

  • Fig. 3 PRDM15 controls the AME specification and A/P patterning of the neural plate.

    (A) Schematic of the signaling centers governing A/P patterning in the mouse embryo. (B) At E6.5, Foxa2 is expressed in the AVE (red line) and APS (red asterisk). At E7.5, Lhx1 transcripts label the visceral endoderm (VE) overlying the epiblast including the AVE as well nascent mesoderm and midline axial mesendoderm. In Prdm15 mutants (mut), Foxa2 expression is confined to the distal VE, with little enrichment in the prospective AVE. Lhx1 is detected in the VE and mesoderm of the middle Prdm15 mutant, but only in the VE of the one on the right. (C) Expression of T, Lefty2, Foxa2, Chordin, and Shh in WT and Prdm15lacZ/lacZ embryos at E7.5. In Prdm15 mutants, T is expressed normally in the PS; Lefty2 transcripts are down-regulated in nascent mesoderm; Foxa2 and Chordin expression remains high distally in the region of the APS (angled black-dashed line) but does not extend anteriorly in the midline axial mesendoderm (am); and Shh expression is similarly weak in the anterior midline (asterisk). n, node. (D) Expression of Six3/Shh or Otx2/Shh in WT (upper) and Prdm15lacZ/lacZ KO (lower) embryos at E8.5. Six3 and Otx2 expression highlights the reduction in anterior forebrain (fb) development (angled black dashed lines) in Prdm15lacZ/lacZ KO mutants. no, notochord; mb, midbrain; DVE, Distal Visceral Endoderm. Representative images are shown in (B) to (D). (C and D) Photo credit: Dun and Ong.

  • Fig. 4 PRDM15 orchestrates transcriptional programs governing A/P patterning and brain development in the mouse embryo.

    (A) Unbiased clustering heat map of the entire transcriptome in WT (n = 8) versus Prdm15lacz/lacz KO (n = 10) E6.5 embryos, analyzed by RNA sequencing. Heat maps of differentially expressed genes from the indicated GO categories (B) and KEGG pathway (C) identified as top hits in the RNA sequencing. Light and dark blue rectangles on the right side indicate genes whose promoter region is directly bound by PRDM15 in ESCs only or both in ESCs and E6.5 embryos, respectively. (D) Snapshots of representative PRDM15 ChIP tracks (UCSC genome browser). Examples of conserved target genes (binding sites) between E6.5 embryos (blue) and ESCs (orange) are shown.

  • Fig. 5 PRDM15 is a master transcriptional regulator of the NOTCH and WNT/PCP pathways, and its targets are mutated in a large cohort of patients with HPE.

    (A) Functional groups identified by protein association network analysis of PRDM15 target genes mutated in patients with HPE using STRING. (B) mRNA levels of the indicated genes in ESCs; the respective genotypes are indicated in the lower panel. Expression levels were normalized to Ubiquitin (Ubb), and Prdm15fl/fl (empty vector) was used as reference. Rspo1 expression levels were used as positive control in Fig. 1D. Data shown are from three independent experiments (n = 3). (C) Enrichment of PRDM15 binding on promoter regions of the indicated target genes in ESCs—respective genotypes are indicated in the lower panel—as measured by ChIP-qPCR. ChIP on the Rspo1 promoter was used as a positive control for PRDM15 binding. Depicted is the average enrichment [data from three independent cell cultures (n = 3)] over percent of input. In (B) and (C), the endogenous mouse Prdm15 has been deleted by the addition of OHT (50 nM) after ectopic expression of WT or mutant human PRDM15. In (B) and (C), center values, mean; error bars, SD. Student’s t test (two sided) was used to determine significance.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/2/eaax9852/DC1

    Fig. S1. Functional characterization of PRDM15 mutations associated with SRNS and microcephaly/HPE.

    Fig. S2. Constitutive and conditional Prdm15 KO alleles.

    Fig. S3. Sox2-Cre epiblast-specific deletion of Prdm15 recapitulates the Prdm15Lacz phenotype.

    Fig. S4. Identification of PRDM15 transcriptional targets in the pre-gastrula mouse embryo.

    Fig. S5. CRISPR-Cas9–mediated knock-in of the mouse C842 mutation in ESCs.

    Fig. S6. mC842Y/hC844Y substitution is an LOF mutation.

    Table S1. ChIP-sequencing and RNA-sequencing analysis.

    Table S2. Genetic variants of PRDM15 target genes associated with HPE.

    Table S3. Oligonucleotides used for site-directed mutagenesis and mutation knock-in.

    Table S4. qRT-PCR primers and ChIP-qPCR primers.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Functional characterization of PRDM15 mutations associated with SRNS and microcephaly/HPE.
    • Fig. S2. Constitutive and conditional Prdm15 KO alleles.
    • Fig. S3. Sox2-Cre epiblast-specific deletion of Prdm15 recapitulates the Prdm15Lacz phenotype.
    • Fig. S4. Identification of PRDM15 transcriptional targets in the pre-gastrula mouse embryo.
    • Fig. S5. CRISPR-Cas9–mediated knock-in of the mouse C842 mutation in ESCs.
    • Fig. S6. mC842Y/hC844Y substitution is an LOF mutation.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). ChIP-sequencing and RNA-sequencing analysis.
    • Table S2 (Microsoft Excel format). Genetic variants of PRDM15 target genes associated with HPE.
    • Table S3 (Microsoft Excel format). Oligonucleotides used for site-directed mutagenesis and mutation knock-in.
    • Table S4 (Microsoft Excel format). qRT-PCR primers and ChIP-qPCR primers.

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article