Fig 1.
HdhneoQ20/Hdhex4/5, HdhneoQ50/Hdhex4/5 and HdhneoQ111/Hdhex4/5 hypomorphic embryos.
A) Representative pictures of wild-type (WT), Hdhex4/5/+, HdhneoQ20/+, HdhneoQ50/+, HdhneoQ111/+, HdhneoQ20/Hdhex4/5, HdhneoQ50/Hdhex4/5 and HdhneoQ111/Hdhex4/5 embryos. The size and the shape of embryos with different genotypes is shown at E18.5 for animals generated by crossing HdhneoQ20/+ with Hdhex4/5/+ mice and HdhneoQ50/+ with Hdhex4/5/+ mice. For embryos generated by crossing HdhneoQ111/+ with Hdhex4/5/+ mice, representative pictures were taken at E 8.5. Dashed white line in the WT image outlines the rostral part of the forebrain headfolds and the white arrow points to the heart. The yellow dashed lines in the HdhneoQ111/Hdhex4/5 images depict anterior endoderm in the late stage streak embryo, typical of a mouse developmental stage of E7.5. Scale bar = 300μm. Tables 1–3 summarizes the appearances of all embryos analyzed and the penetrance of the phenotypes. B-C) Box-and-whisker graphs show quartiles of height in cm (cm) and weight in grams (g) of different genotypic groups of animals generated by crossing HdhneoQ20/+ with Hdhex4/5/+ mice. The median is indicated and whiskers indicate 1.5XIQR. The number (N) of animals for each group is indicated above. Error bars represent standard deviations from the mean. D-E) Box-and-whisker graphs show quartiles of height in cm (cm) and weight in grams (g) of different genotypic groups of animals generated by crossing HdhneoQ50/+ with Hdhex4/5/+ mice. The median is indicated and whiskers indicate 1.5XIQR. The number (N) of animals for each group is indicated above. A significant reduction in size of HdhneoQ50/Hdhex4/5 embryos is noted. P value < 0.05 (*). Error bars represent standard deviations from the mean.
Table 1.
Related to Fig 1 phenotypes.
The table summarizes the total number of animals per litter and the percentages of normal and abnormal embryos/animals obtained at each specific developmental stage from HdhneoQ20/+ x Hdhex4/5/+ crosses. Data are related to Fig 1.
Table 2.
Related to Fig 1 phenotypes.
The table summarizes the total number of animals per litter and the percentages of normal and abnormal embryos/animals obtained at each specific developmental stage from HdhneoQ50/+ x Hdhex4/5/+ crosses. Data are related to Fig 1.
Table 3.
Related to Fig 1 phenotypes.
The table summarizes the total number of animals per litter and the percentages of normal and abnormal embryos/animals obtained at each specific developmental stage from HdhneoQ111/+ x Hdhex4/5/+ crosses. Data are related to Fig 1.
Fig 2.
Skeletal defects in HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 hypomorphic embryos.
Representative images of alcian blue and alizarin red-stained skeletal preparations from wild-type (WT), HdhneoQ20/Hdhex4/5, and HdhneoQ50/Hdhex4/5 E 18.5 embryos. A) Lumbar to sacral transformations (L6 to S1) in HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 embryos, where sacral area with the iliac bones is enlarged in the inset, L1 to L6 indicate lumbar vertebrae, T13 is the 13th thoracic vertebra and skeletal transformation is in red text. B) Defects in the sternum and xyphoid process of HdhneoQ20/Hdhex4/5, HdhneoQ50/Hdhex4/5 mice, enlarged in the insets, showing xyphoid process fenestration in HdhneoQ20/Hdhex4/5 embryos, with thoracic ribs numbered (black text). C-D) Cervical vertebrae defects in HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 animals, with the latter having an increase in the gap between C1 and C2 vertebrae, seen in horizontal (C) and vertical side (D) views. E) Defects in the morphology [greater horns (*)] and spacing between hyoid bone and cartilage structures, evident in the HdhneoQ50/Hdhex4/5 embryos. Scale bars in (A) and (B) = 1mm; scale bars in (C), (D), (E) = 500μm. See Table 4 for detailed description of the litters and quantitative analysis of the phenotypes.
Table 4.
Related to Fig 2 Skeletal Phenotypes.
The table summarizes the total number and the percentages of abnormal embryos presenting skeletal abnormalities (L6 to S1 transformation, Inverted Sternum tip, Six Vertebrosternal ribs, Fenestrated xiphoid process, T13 Aborted ribs, C1 to C2 gap, C1 to C2, C7 to T1, Hyoid defect) obtained for each genotype. Data are related to Fig 2.
Fig 3.
External and middle ear defects in HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 hypomorphic embryos.
A) Representative images of wild-type (WT), HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 heads, showing altered morphology of external ear in HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 animals at E18.5. B) The region of the external ear is enlarged from (A) for each genotype, showing mis-shapen or missing external ears for the HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 animals. C) Representative images of alcian blue and alizarin red-stained middle ear preparations from wild-type (WT), HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 E18.5 embryos, showing severely altered middle ossicles in HdhneoQ50/Hdhex4/5 animals. Middle ear ossicles are indicated in the WT preparation: M = manubrium, PB = processus brevis. In HdhneoQ50/Hdhex4/5 animals, all the middle ear ossicles were absent, only the Meckel’s Cartilage (MC) was observed. D) Representative images of alcian blue and alizarin red-stained middle ear preparations from wild-type (WT), HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 E18.5 animals, showing hypoplastic squamous bone (SQ_B) in HdhneoQ20/Hdhex4/5 and HdhneoQ50/Hdhex4/5 animals. Black line delineates the squamosal bone. E) Whole mount in situ hybridization representative images showing altered Gsc mRNA localization in HdhneoQ50/Hdhex4/5 embryos at E11.5. The inset shows an enlarged view of the pharyngeal arches with Gsc transcript staining localized in the I-md and II arch in WT, but only localized in the first arch in HdhneoQ50/Hdhex4/5 embryos. Abbreviations: I-md = mandibular process of pharyngeal arch 1; I-mx = maxillary process of pharyngeal arch 1; II = pharyngeal arch 2. Dashed lines delineate pharyngeal arches. F) Whole mount in situ hybridization representative images showing altered Hoxa2 mRNA localization in HdhneoQ50/Hdhex4/5 embryos at E11.5. The arrows indicate ectopic expression of Hoxa2 transcript in the dorsal area of HdhneoQ50/Hdhex4/5 embryos. Scale bars, 1.5 mm for A-B; 400 μm for C-D; 1.5 mm for E-F.
Table 5.
Related to Fig 3 Ear Phenotypes.
The table summarizes the penetrance (percentages of abnormal embryos) of ear phenotypes (Hypoplastic External Ear, Loss of Middle Ear Structures, Hypoplastic Squamosal bone) obtained for each genotype. Data are related to Fig 3.
Fig 4.
Skin defects in HdhneoQ50/Hdhex4/5 hypomorphic embryos.
Representative images of wild-type (WT) and HdhneoQ50/Hdhex4/5 animals, at E18.5. A) At E18.5, following toluidine-blue dye exclusion assay, WT animal shows a functional epidermal barrier but HdhneoQ50/Hdhex4/5 animal has a compromised epidermal barrier on the ventral surface. B) Immunoblot analyses of WT, Hdhex4/5/+, HdhneoQ50/+ and HdhneoQ50/Hdhex4/5 skin protein extracts (E18.5). Bands of Pro-filaggrin (pro-Flg, 2X-Flg, 3X-Flg) at 50–70 KDa and higher molecular weight bands and processed filaggrin (Flg) at 27 KDa were detected in all samples but were decreased in HdhneoQ50/Hdhex4/5 extracts. The bands of Loricrin (Lor) and alpha-tubulin (Tuba1) were similar in all samples. C) Bar graphs summarizing skin-extract protein immunoblot band signal-intensity (relative to WT), expressed as standard deviation of 3 biologic replicates using 2 mice each, as quantified by Image J software. Unpaired t-test. P value < 0.05 (*), P < 0.0001 (***). D) Representative images of cryostat-sectioned skin tissue, with DAPI stained nuclei to show reduced staining of the PCNA proliferation marker in HdhneoQ50/Hdhex4/5 skin. Green = PCNA staining; Blue = DAPI-stained nuclei. Dashed lines depict the cornified basal epidermal layer. All images were taken using a fluorescent 20X objective. Scale bars: 50μm E) Representative images of cryostat-sectioned skin tissue stained with TUNEL assay to quantify apoptotic cell death (arrows) in WT and HdhneoQ50/Hdhex4/5 embryos. Dashed lines depict the cornified basal epidermal layer. All images were taken using a phase-contrast 20X objective. Scale bars: 50μm.
Fig 5.
Hematopoietic defects in HdhneoQ50/Hdhex4/5 hypomorphic embryos.
A, E) Representative images of the appearance of fetal liver in wild-type (WT) and HdhneoQ50/Hdhex4/5 embryos displaying dome shaped cranium and exencephaly at E14.5 and E18.5. Scale bars: 1 mm. B, F, G) Representative FACS profiles of fetal liver hematopoiesis in wild-type (WT) and HdhneoQ50/Hdhex4/5 embryos at E14.5 and E18.5, respectively. Erythroblasts were gated into fractions defined by CD71 and Ter119 expression, displaying transient anemia at E14.5, rescued by E18.5. Myeloid cells and B cell progenitors were identified as Mac-1+Gr-1– and B220+CD19–, respectively at E14.5 fetal liver T cells were stained with CD4 and CD8a cell surface markers. C, H) Bar graphs summarizing the absolute cell numbers of whole fetal liver at E14.5 and E18.5 for wild-type (WT) and HdhneoQ50/Hdhex4/5 embryos, displaying transient defects in hematopoiesis at E14.5. At E14.5, WT embryos N = 5, HdhneoQ50/Hdhex4/5 embryos N = 3. At E18.5, WT embryos N = 3, HdhneoQ50/Hdhex4/5 embryos N = 3. Error bars represent standard deviations from the mean. (**) and (*) indicate significant P value, with (**) P<0.01 and (*) P<0.05, respectively. D, I) Bar graphs summarizing the results of colony assays on wild-type (WT) and HdhneoQ50/Hdhex4/5 fetal liver indicate a dramatic depletion of erythroid (CFU-E), Megakaryocytes (CFU-Mk) and total colony forming cells (Total CFCs) in E14.5 HdhneoQ50/Hdhex4/5 fetal liver. By E 18.5, the phenotype was completely rescued and no difference in erythroid (CFU-E and BFU-E (E)), megakaryocyte (CFU-Mk) myeloid (G, M, GM), erythroid-megakaryocyte (E/Mk) and multi-lineage mixed (Mix) colony forming cells was observed. Error bars represent standard deviations from the mean. d3, day3; d5, day5, d12, day12.
Fig 6.
Classes of genes implicated in organ development whose expression was altered by Httex4/5 null mutation.
A) Schematic describing the criteria applied to define the 4 classes of genes sensitive to both Hdhex4/5 null mutation and RA-induced differentiation. Up_up: genes up-regulated during differentiation and up-regulated by the Htt-null mutation; up_down: genes up-regulated during differentiation but down-regulated by Htt-null mutation; down_down: genes down-regulated during differentiation and further down-regulated by Htt-null mutation; down_up: genes down-regulated during differentiation, but upregulated in the context of Htt-null mutation. B) Expression trajectory plots describing variations in transcriptional levels across the two Htt genotypes (WT and dKO) and two developmental stages (ESC and RA-DIFF) of the genes belonging to the 4 categories described in A). a.u., arbitrary units. C) Bar blots displaying the number of genes (from RNA-seq analysis) for each of the 4 categories (up_up, up_down, down_down, down_up as described in A). Most of the genes were found in the up_up and up_down groupings. D) Enrichment analysis of genes belonging to the 4 categories (Up_up; up_down; down_up; down_down, as described in A), highlighting the most enriched GO-terms [BP, Biological Process; CC, Cellular Component; MF; Molecular Function; KEGGs pathways and Reactome pathways]. Significance of enrichment is based on FDR values [color code as in the legend].
Fig 7.
Httex4/5 null mutation, in the context of RA-differentiation, significantly perturbed genes implicated in ‘body weight’, ‘skin, skeleton, middle-ear development’ and ‘hematopoiesis’.
A) Venn diagram representation of the intersection of genes relevant to Htt hypmorph phenotypes (bodyweight, skin, skeleton, middle ear and hematopoiesis) and genes whose expression changes with Htt-genotype in stem cells (ESC) and during RA-EB differentiation (RA-DIFF). The genes in this intersection were analyzed further in B) and C). B) The number of intersection genes associated with each of the Htt hypomorph relevant phenotypes C) Expression trajectory plots (upper row) and enrichment analysis (lower row) of the four classes of genes (up-up; up-down; down-up; down-down; Fig 6) in the intersection with developmental genes associated with Htt hypmorph related phenotypes.
Fig 8.
Regulatory network analysis highlights Polycomb group protein and miRNAs as possible regulators.
A) Enrichment analysis of genes whose expression is changed by Hdhex4/5/ex4/5 null, that are involved in developmental phenotypes and regulated by Polycomb group proteins, based on ChEA ChIP-Seq annotation. Significance of enrichment is based on FDR values [color code as in the legend]. B) List of 22 miRNAs whose expression is altered by RA-differentiation and by Hdhex4/5 null genotype, but whose change in expression is significantly anti-correlated relative to expression change observed in the miRNA target genes. miRNAs identified by these criteria are by definition either down-down or down-up. miRNAs are ranked according to the p-value associated with the anti-correlation of their targets. C) Functional enrichment analysis of mRNA regulated by the 22 miRNAs displayed in Panel B. Only mRNAs affected by RA-Diff and the Htt null mutation, as well as anti-correlated with respect to their miRNA regulators were selected for the analysis. Enrichment FDR values are displayed in the bar chart.