Barrier Marianne, Dix David J, Mirkes Philip E
Birth Defects Research Laboratory, Division of Genetics and Development, Department of Pediatrics, University of Washington, Seattle, Washington, USA.
Birth Defects Res A Clin Mol Teratol. 2009 Aug;85(8):732-40. doi: 10.1002/bdra.20610.
It is well known that a variety of teratogens induce neural tube defects in animals; however, less is known about proteins that play a role in protecting embryos from teratogen-induced neural tube defects. Previously, our laboratory has shown that embryos overexpressing the 70-Da heat shock proteins (HSPs) Hspa1a and Hspa1b were partially protected from the deleterious effects of exposure to hyperthermia in vitro.
In the present studies, we have used a transgenic mouse in which both of the stress-inducible HSPs Hspa1a and Hspa1b were deleted by homologous recombination. Time-mated Hspa1a/a1b(-/-) (KO) and wildtype (WT) mice were exposed to hyperthermia in vivo on gestational day 8.5.
Results show that 52% of the gestational day 15 fetuses from KO litters were exencephalic, whereas only 20% of WT fetuses were affected. In addition, 6% of treated KO fetuses also exhibited eye defects (microphthalmia and anopthalmia), defects not observed in WT fetuses exposed to hyperthermia. Lysotracker red staining and caspase-3 enzyme activity were examined within 10 hours after exposure to hyperthermia, and significantly greater levels of apoptosis and enzyme activity were observed in the KO embryos compared with WT embryos.
These results show that embryos lacking the Hspa1a and Hspa1b genes are significantly more sensitive to hyperthermia-induced neural tube and eye defects, and this increased sensitivity is correlated with increased amounts of apoptosis. Thus, these results also suggest that Hspa1a and Hspa1b play an important role in protecting embryos from hyperthermia-induced congenital defects, possibly by reducing hyperthermia-induced apoptosis.
众所周知，多种致畸剂可在动物中诱发神经管缺陷；然而，对于在保护胚胎免受致畸剂诱导的神经管缺陷中发挥作用的蛋白质却知之甚少。此前，我们实验室已表明，过表达70 kDa热休克蛋白（HSPs）Hspa1a和Hspa1b的胚胎在体外可部分免受高温暴露的有害影响。
在本研究中，我们使用了一种转基因小鼠，其中应激诱导型HSPs Hspa1a和Hspa1b均通过同源重组被删除。将处于交配期的Hspa1a/a1b（-/-）（基因敲除，KO）小鼠和野生型（WT）小鼠在妊娠第8.5天进行体内高温暴露。
结果显示，基因敲除小鼠窝中15日龄胎儿中有52%为无脑儿，而野生型胎儿中只有20%受影响。此外，6%接受处理的基因敲除胎儿还表现出眼部缺陷（小眼症和无眼症），在暴露于高温的野生型胎儿中未观察到这些缺陷。在暴露于高温后10小时内检测了溶酶体示踪剂红色染色和半胱天冬酶-3酶活性，与野生型胚胎相比，在基因敲除胚胎中观察到明显更高水平的细胞凋亡和酶活性。
这些结果表明，缺乏Hspa1a和Hspa1b基因的胚胎对高温诱导的神经管和眼部缺陷明显更敏感，这种敏感性增加与细胞凋亡量增加相关。因此，这些结果还表明，Hspa1a和Hspa1b在保护胚胎免受高温诱导的先天性缺陷中发挥重要作用，可能是通过减少高温诱导的细胞凋亡来实现的。
