Figure 1.
FoxA3-Cre mediated Yy1 cKO deletion results in prominent yolk sac defects at 9.5 dpc.
A-L) Bright field images of WT (A–F) and cKO (G–L) embryos (EM) alone or embryos within their yolk sacs (YS) at the indicated stages. A, G) 8.5 dpc mutant embryos (inset in G) are sometimes slightly delayed compared with WT (inset in A) but display no noticeable yolk sac defects. B–C, H–I) 9.0 dpc mutants display relatively normal yolk sac blood vessel development. D–E, J–K) 9.5 dpc mutant yolk sacs have dilated vessels (asterisk in J) and poor vessel organization (compare D to J). cKO embryos (K) are smaller than WT embryos (E) from the same litter. F, L) While prominent large blood vessels are easily detected in 10.5 dpc WT yolk sacs (F), the yolk sacs of mutants are uniformly pale (L). M–P) A comparison of WT and mutant H&E stained yolk sac sections demonstrates that while no differences are found at 8.5 dpc (M, O), the 9.5 dpc cKO yolk sac (P) has fewer and larger vessels compared with WT (N). Q) Investigation of the same sized area at 9.5 dpc revealed significantly fewer vessels in mutant compared with WT yolk sacs (*** = p<0.001; error bar = standard error). R) A size distribution chart at 9.5 dpc reveals that mutants contain fewer of the small vessels (<100 µm) and more of the larger vessels (>100 µm) compared with WT yolk sacs.
Figure 2.
Yy1 cKO visceral endoderm displays a reduction of apical lysosome size and other epithelial characteristics.
A–B, E–F) Transmission electron microscopy (TEM) of WT (A–B) and cKO (E–F) visceral endoderm (VE) sections reveals large apical lysosomes in 8.5 and 9.0 dpc WT visceral endoderm (asterisks, A–B) and in 8.5 dpc cKO visceral endoderm (asterisks, E). At 9.0 dpc the size of the apical lysosomes are greatly reduced in the cKO (compare asterisks, in F to B). C–D, G–H) IgG localization (green) at the apical surface is readily noted in 9.0–9.5 dpc WT yolk sac sections (YS, C–D) while IgG distribution is reduced in the mutant at the same stages (G–H). Inset is a higher magnification view of a portion of the visceral endoderm. I–J, M–N). Whole-mount LysoTracker Red staining reveals large filled lysosomes from 9.0–9.5 dpc (I–J) in WT tissue while the LysoTracker-filled areas are reduced in the mutant samples (M–N). K–L, O–P) Immunolocalization of E-Cadherin (CDH1; green) reveals epithelial cell-cell adhesions in the visceral endoderm of WT yolk sac sections from 9.0–9.5 dpc (K–L). CDH1 expression is slightly reduced at 9.0 dpc and more profoundly reduced at 9.5 dpc in cKO visceral endoderm (O–P). ME = mesoderm; N = nucleus.
Figure 3.
Efficient excision of YY1 in definitive and visceral endoderm is accompanied by reduced HNF4α.
Immunofluorescence analysis of sectioned WT (A–D, I–K) and cKO tissue (E–H, L–N) at the stages indicated. A–C, I) YY1 (green) is ubiquitous in WT embryonic and extraembryonic tissues. A–B, D, K) HNF4α (red, orange when co-expressed with YY1) labels the visceral endoderm (A–B, D) and the developing liver bud (K). E–G, L) In cKO embryos, YY1 is downregulated in the extraembryonic visceral endoderm (VE) at 7.5 dpc (E) and is completely lost in the embryonic visceral endoderm by 8.75 dpc (F), when YY1 is also depleted in the definitive endoderm (DE) of the foregut. By 9.25 dpc YY1 is lost in most cells of the liver bud (L). E–F, H, N) Although HNF4α is present in the YY1-deficient visceral endoderm until 8.75 dpc (E, F) it is greatly reduced in both the visceral endoderm and in the nascent liver bud by 9.5 dpc. J, M) Despite the loss of YY1 in the nascent liver bud, the liver bud differentiation marker PROX1 is maintained in the cKO liver bud (M) at levels comparable to that observed in WT (J). The dotted line in C–D and G–H represent the division between the visceral endoderm and mesoderm derivative of the yolk sac, while in I–N the dashed line outlines the liver bud (LB).
Figure 4.
cKO embryos display a variety of defects in the yolk sac mesoderm.
A–B, D–E) Whole mount immunofluorescence of 9.5 dpc WT (A–B) or mutant (D–E) yolk sacs (YS) using the endothelial marker PECAM (green) and the vascular smooth muscle marker (αSMA) demonstrates that the large disorganized vessels in the cKO (D) are not surrounded by αSMA (E). C, F) Section immunofluorescence of WT (C) and cKO (F) 9.5 dpc yolk sacs demonstrates loss of αSMA in the cKO. G–L) Section immunofluorescence of VEGFA (green) demonstrates relatively uniform VEGF levels in the 9.0, 9.25 and 9.5 dpc WT yolk sac (G–I) while VEGF distribution in the visceral endoderm of the mutant is progressively diminished at each stage (J–L). M) A Western blot of whole yolk sacs at the indicated stages. The ratio of VEGFA to GAPDH signal intensities for the cKO relative to each stage-matched WT control is displayed under each band. N) Cleaved Caspase-3 staining was used to assess the percentage of cell death in the yolk sacs layers of WT and cKO sections at 8.5 and 9.0 dpc. A significant increase in apoptosis was observed in the cKO mesoderm (ME) at 9.0 dpc. O) Phosphohistone-H3 (PH-3) staining was similarly used to assess proliferation and a significant decrease in proliferation was found in the cKO yolk sac mesoderm at 9.0 dpc. *** = p<0.001, ** = p<0.01; error bars = standard error; dotted line is drawn between the visceral endoderm (VE) and mesoderm derivatives (ME) on yolk sac sections.
Figure 5.
Changes in yolk sac gene expression in cKO embryos.
A–E) RT-PCR and qPCR performed with cDNA prepared from whole 9.0 and 9.5 dpc cKO and WT yolk sacs. A) As expected, Yy1 is significantly downregulated in whole cKO yolk sacs. β-actin and Hprt expression are used as loading controls. B) No expression differences between WT and cKO samples are noted for VegfA using primers that recognize all (Exon 2–3) or the alternative VegfA isoforms (Exons 3–8) nor in the Vegf transcriptional regulator Hif1α. C) While many visceral endoderm-specific genes show no expression differences, expression of vHnf1, Hnf4α and Pgc1α were all downregulated in cKO samples when compared to WT at 9.0 and 9.5 dpc. D) An examination of genes involved in lysosome biogenesis reveals no expression differences between WT and cKO yolk sacs with the exception of Enpp-2, which is upregulated in mutant samples at both stages examined. E) qPCR reveals that Yy1 is expressed at ∼30% of WT levels in whole yolk sacs, where mesoderm derivatives maintain Yy1. qPCR was used to confirm that VegfA expression is not significantly altered between cKO and WT and that expression of the visceral endoderm gene, Hnf4α is significantly downregulated in cKO yolk sacs. *** = p value<0.001; error bars = standard error.
Figure 6.
Exogenous VEGF rescues Yy1 cKO yolk sac defects.
A–Y) WT and cKO embryos cultured from 8.5–9.5 dpc in the presence (+VEGF) or absence (−VEGF) of VEGF. A–E) WT cultured embryos display normal yolk sac vasculature (A–A’), typical embryonic size (inset in A) and the presence of YY1 (brown) in the visceral endoderm (VE) and in yolk sac mesoderm (ME; B). In WT yolk sac sections, αSMA (red) surrounds mature vessels (C), HNF4α (red) is expressed in the visceral endoderm (D) and IgG is localized to the apical visceral endoderm (E). F–J) WT embryos cultured with exogenous VEGF display robust yolk sac vasculature (F, F’), YY1 expression in both YS layers (G), normal αSMA in mature vessels (H), typical HNF4α in the visceral endoderm (I) and high levels of apical IgG (J). K–O) Cultured cKO embryos demonstrate poor vascular development, including pooled blood in the proximal yolk sac (K–K’), no YY1 in the visceral endoderm (L), reduced αSMA (M), reduced HNF4α (N) and decreased apical IgG (O) when compared with WT cultured embryos (A–E). P–T) cKO embryos cultured with exogenous VEGF display normal yolk sac vasculature (P–P’) and increased embryo size (inset in P) when compared to cKO embryos cultured without exogenous VEGF (K–K’). cKO embryos cultured with VEGF lack visceral endoderm YY1 (Q) but have increased αSMA in the yolk sac mesoderm (R) and increased levels of HNF4α (S) and apical IgG (T) in the visceral endoderm when compared to untreated cKO embryos (M–O). U–Y) Immunofluorescence against cleaved Caspase-3 (CASP3, green) and CDH1 (red) of sectioned yolk sacs revealed that typical CDH1 expression found in WT (U) and WT cultured with VEGF (V), was downregulated in cultured cKO embryos but more normal visceral endoderm expression restored when cKO embryos were cultured with VEGF (X). Y) Quantification of cleaved Caspase-3 positive cells demonstrates that the addition of VEGF to cKO embryos restores WT levels of apoptosis. *** = p<0.001; error bars = standard error; dotted line is drawn between the visceral endoderm (VE) and mesoderm derivatives (ME) on yolk sac sections.
Figure 7.
Inhibition of FLK1 in WT embryos results in yolk sac defects similar to Yy1 cKO.
A–J) WT 8.5 dpc embryos cultured until they reached 9.5 dpc in the absence (A–E; −SU1498) or presence of the small molecule SU1498 (F–J; +SU1498). Compared with control embryos (A–E), SU1498 treated embryos displayed clear yolk sac abnormalities, including pooled blood in the proximal yolk sac (F), a small embryo (G), reduced HNF4α (H), reduced apical IgG localization (I) and higher amounts of cleaved Caspase-3 (CASP3) staining (J). Asterisks in J indicate cleaved Caspase-3 positive cells; dotted line represents the division between the yolk sac mesoderm (ME) and visceral endoderm (VE).
Figure 8.
Loss of YY1 leads to defects in paracrine signals necessary for angiogenesis and visceral endoderm integrity.
A, B) A summary of the signaling events downstream of YY1 in WT and mutant yolk sacs. A) In the presence of YY1, normal VEGF levels produced by the visceral endoderm (VE) allow the underlying mesoderm derivatives (ME) to undergo events associated with vascular remodeling. The underlying vascular tissue is the source of a VEGF-dependant paracrine signal(s) that is required by the visceral endoderm to maintain characteristics such as epithelial polarity, large apical lysosomes and HNF4α expression. B) In the absence of YY1 in the visceral endoderm, decreased levels of paracrine VEGF result in defective angiogenesis, increased apoptosis and decreased proliferation in the adjacent mesoderm. Because of reduced VEGF signaling, the yolk sac mesoderm does not generate the paracrine signal(s) needed to maintain epithelial characteristics in the visceral endoderm, resulting in decreased HNF4α, a loss of large lysosomes and reduced CDH1 levels.