Heat shock protein 60 regulates yolk sac erythropoiesis in mice

Abstract

The yolk sac is the first site of blood-cell production during embryonic development in both murine and human. Heat shock proteins (HSPs), including HSP70 and HSP27, have been shown to play regulatory roles during erythropoiesis. However, it remains unknown whether HSP60, a molecular chaperone that resides mainly in mitochondria, could also regulate early erythropoiesis. In this study, we used Tie2-Cre to deactivate the Hspd1 gene in both hematopoietic and vascular endothelial cells, and found that Tie2-Cre + Hspd1 f/f (HSP60 CKO) mice were embryonic lethal between the embryonic day 10.5 (E10.5) and E11.5, exhibiting growth retardation, anemia, and vascular defects. Of these, anemia was observed first, independently of vascular and growth phenotypes. Reduced numbers of erythrocytes, as well as an increase in cell apoptosis, were found in the HSP60 CKO yolk sac as early as E9.0, indicating that deletion of HSP60 led to abnormality in yolk sac erythropoiesis. Deletion of HSP60 was also able to reduce mitochondrial membrane potential and the expression of the voltage-dependent anion channel (VDAC) in yolk sac erythrocytes. Furthermore, cyclosporine A (CsA), which is a well-recognized modulator in regulating the opening of the mitochondrial permeability transition pore (mPTP) by interacting with Cyclophilin D (CypD), could significantly decrease cell apoptosis and partially restore VDAC expression in mutant yolk sac erythrocytes. Taken together, we demonstrated an essential role of HSP60 in regulating yolk sac cell survival partially via a mPTP-dependent mechanism.

Atypical protein kinase C is essential for embryonic vascular development in mice

“…Whole‐mount immunofluorescence staining in yolk sac was performed as previously described (Duan et al, 2019). In brief, embryo's yolk sac was collected and fixed with 4% PFA at 4°C overnight, washed and incubated in the blocking buffer (2% bovine serum albumin, 5% horse serum in PBS containing 0.2% Triton X‐100) for 1 hr at room temperature, followed by the incubation with anti‐CD31 antibody (BD Pharmingen, catalog no.…”

“…Whole-mount immunofluorescence staining in yolk sac was performed as previously described (Duan et al, 2019). In brief, embryo's yolk sac was collected and fixed with 4% PFA at 4 C overnight,…”

Inositol 1,4,5-trisphosphate receptors are essential for fetal-maternal connection and embryo viability

“…Mice were mated under the standard 12-hour light / dark cycle, and noon on the day of the appearance of the vaginal plug was defined as the embryonic day 0.5 (E0.5). Embryos and placentas were dissected and collected under a Leica MZ6 dissecting light microscope and photographed as previously described [28]. The tissues were then fixed in 4% paraformaldehyde (PFA) diluted in phosphate buffered saline (PBS).…”

“…Whole-mount PECAM staining was performed as previously described [28]. Briefly, the embryos dissected in ice cold PBS were fixed in 4% PFA overnight at 4˚C.…”

“…We first generated a series of conditional IP 3 R1 knockout mouse models in IP 3 R2 null background using cardiac muscle-specific expressing TnT-Cre [21], and endothelial / hematopoietic cell-specific expressing Tie2-Cre and Flk1-Cre [22,23]. It has been shown in our laboratory that TnT-Cre and Tie2-Cre are very efficient at deleting multiple floxed genes in mouse embryos [9,28,32,33]. We also performed quantitative real time PCR to examine expression of the Itpr1 gene, and found that Itpr1 mRNA levels were dramatically reduced in hearts of TnT-Cre + Itpr1 f/f Itpr2 -/embryos, and in blood cells of Tie2-Cre + Itpr1 f/f Itpr2 -/and Flk1-Cre + Itpr1 f/f Itpr2 -/embryos, when compared with those control embryos (S2A- S2C Fig).…”