Heat shock proteins (Hsps), which have important biological functions, are a class of highly conserved genetic molecules with the capacity of protecting and promoting cells to repair themselves from damage caused by various stimuli. Our previous studies found that Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10, and Hsp40 may be related to all-trans retinoic acid (atRA)-induced phocomelic and other abnormalities, while HspA12B, HspA14, Trap1, and Hsp105 may be forelimb development-related genes; Grp78 may play an important role in forelimb development. In this study, the embryonic phocomelic, oligodactylic model of both forelimbs and hindlimbs was developed by atRA administered per os to the pregnant mice on gestational day 11, and the expression of 36 members of Hsps family in normal and abnormal development of embryonic hindlimbs was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). It is found that HspA1L, Hsp22, Hsp10, Hsp60, Hsp47, HspB2, HspB10, HspA12A, Apg1, HspB4, Grp78, and HspB9 probably performs a major function in limb development, and HspA13, Grp94 and Hsp110 may be hindlimb development-related genes.
Heat shock proteins (Hsps) are highly conservative protective proteins that are found in all living organisms. In mammalian cells, Hsps are classified into several families based on their apparent molecular masses and degrees of structural homology. The major Hsps in mammalian cells are small Hsps (sHsps) (15–40 kDa), HSP60, HSP70 (68–80 kDa), HSP90 (83–99 kDa), and high-molecular-weight Hsps (Hsp110).1,2 According to the literature by Kampinga et al.1 and the GenBank, there are 10 sHsps members in mice, namely Hsp27/p27/HspB1, HspB2/MKBP (myotonic dystrophy kinase-bind-protein), HspB3, α, A-crystallin/HspB4, α, B-crystallin/HspB5, Hsp20/HspB6, cvHsp/HspB7, Hsp22/H11/HspB8, HspB9 and HspB10 (the sperm outer dense fiber 1 protein).1,3,4 Hsp25 is a homolog of Hsp27.5,6 Ample evidence has demonstrated that HO-1, once transcribed and activated during enzyme degradation, has the function of 32 kDa Hsp and can participate in resisting oxidative stress and tissue damage.7,8 Hsp40 is preserved throughout evolution and important for protein translation, folding, unfolding, translocation and degradation, primarily by stimulating adenosine triphosphatase activity of chaperone protein Hsp70s.9 As Hsp10 is regarded as a chaperone of Hsp60,10 it is also one of the members of the Hsp60 family. There are 11 members in the Hsp70 family in mice, namely HspA1A (Hsp70-1, Hsp72), HspA1B (Hsp70-2), HspA1L, HspA2, Grp78 (Bip, HspA5), HspA8 (Hsc70, Hsc71, Hsp71, Hsp73), HspA9 (Grp75), HspA12A, HspA12B, HspA13, and HspA14 (Hsp70-4); there are four members in the Hsp90 family, namely Hsp90AA1 (HspC1, Hsp86), Hsp90AB1 (HspC3, Hsp84), Grp94 (HspC4, Hsp90B1), and Trap1 (HspC5, Hsp75); and there are four members in the Hsp110 family: Hsp105 (HspH1), Hsp110 (HspH2, Apg-2, HspA4), Apg-1 (HspH3, HspA4L), and Grp170 (HspH4, ORP150, Hsp12A).
In general, Hsps are induced by environmental stress conditions and various agents, including heat shock, anoxia, amino acid analogs, heavy metals, and certain inhibitors of mitochondrial respiration.11,12 The main biological function of them was known to maintain the necessary protein conformation in cell and protect cells’ life activities under cellular stress conditions. Hsps play an important role in protein folding, membrane transport, and stability of cytoskeletal, nuclear matrix, and so on, so they were called molecular chaperones.
In addition, Hsps are thought as chaperones of morphologic development of cells and organisms and believed to be closely related to normal and abnormal embryonic development. Hsp25 was related to development of the heart muscle tissue, and Hsp27 was associated with the stability of cytoskeleton in actin-containing cells.13 Structure type Hsc73, usually located in the cytoplasm and nucleus, participating neuronal differentiation, had high levels of expression in neural tube closure, neuroectodermal differentiation, and proliferation process.14 Hsp90β and Hsc70 may be involved in the occurrence of the nervous system and bone.15 Grp78 had a powerful function in the normal development and differentiation of muscle tissue.16 Hsp105, which located in the cytoplasm and nucleus of various tissue cells in mouse embryo, played a role in mouse embryonic organogenesis.17
In previous studies, we found that most members of sHsps, Hsp40, and Hsp60 families may play a stress-protective role in all-trans retinoic acid (atRA)-induced abnormal forelimb development, and abnormal expression of some genes (Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10, and Hsp40) may be related to atRA-induced phocomelic and other abnormalities.18HspA12B, HspA14, Trap1, and Hsp105 may be forelimb development-related genes; Grp78 may play an important role in forelimb development.19 As for the origin of embryonic development, the forelimbs and hindlimbs were all originated from the lateral plate mesoderm, but the hindlimbs develop later. So, in this study the embryonic phocomelic, oligodactylic model of both forelimbs and hindlimbs was developed by atRA administered per os to the pregnant mice at gestational day 11 (GD11),20 so that we could investigate the expression of Hsps family in normal and abnormal development of embryonic hindlimbs in an attempt to provide the basis for further study the role of Hsps during embryogenesis.
Materials and methods
Animals
Imprinting control region (ICR) mice were purchased from Sino-British SIPER B/K Lab Animal Co., Ltd. (Certificate No. SCXK Shanghai 2003-0002, Shanghai, China). Virgin female ICR mice were mated overnight in an environment maintained at 21 ± 2°C and a relative humidity of 55 ± 10% with a 12-h light/12-h dark cycle. Pregnancy was confirmed by the presence of vaginal plugs the following morning as GD0. Use of the animals was approved by the Committee on Ethics of Biomedicine Research of the Second Military Medical University (Shanghai, China).
Animal model construction and sample collection
Pregnant mice confirmed by the presence of vaginal plugs were equally randomized to a treatment group (n = 56) and a control group (n = 56). Using soybean oil as the solvent, atRA (Sigma, St Louis, Missouri, USA) was prepared to an 8 mg/ml suspension and administered per os to the treatment group on GD11 at a dose of 0.1 ml/10 g body weight. The same amount of soybean oil was administered per os to the control group. During the 7-day period from GD12 to GD18, eight mice from both groups were killed daily by cervical vertebral dislocation. The uterus was dissected to confirm the gestational status with naked eye, and then the fetuses were removed from the uterus. One fetal mouse was taken randomly from each pool of both groups for hsps messenger RNA (mRNA) test, and both hindlimbs of it were harvested under a stereomicroscope and immediately preserved in liquid nitrogen. Some fetuses of GD18 were photographed immediately after removing from the uterus, and then they were stained by alizarin red21 and photographed.
Total RNA extraction and real-time fluorescent quantitative PCR
The harvested hindlimbs from both groups were taken from the liquid nitrogen and lysed in Trizol (Invitrogen, Carlsbad, California, USA). Total RNA was extracted according to the protocol of the kit and dissolved in nuclease-free water, from which 1 μg total RNA was drawn and reverse transcribed to synthesize complementary DNA (cDNA) according to the reverse transcription kit (Promega, Madison, Wisconsin, USA).
cDNA obtained was amplified with real-time polymerase chain reaction (PCR) on ABI 7900 PRISM system using ABI SYBR buffer (Applied Biosystems, Foster City, California, USA). Primers were designed with ABI Primer Express 2.0 according to ABI design guidelines. All primers used in this study were synthesized by Bioasia (Shanghai, China) using the sequences as listed in Table 1. The real-time PCR reaction system is as follows: SYBR buffer 2.5 μl, cDNA 0.5 μl, upstream primer 0.3 μl, downstream primer 0.3 μl, Milli-Q water 6.4 μl. Reaction conditions were as: 95°C for 15 min, 95°C for 5 s, 60°C for 1 min in 40 cycles.
Ct value of the PCR amplification curve of the target gene (Hsps) was compared with the Ct value of the internal reference gene (β-actin) to obtain ΔCt, which was used to conduct relative quantitative analysis on the expression level of the Hsps. The HspsCt values normalized by subtracting the β-actinCt value, and the relative mRNA expression abundance was calculated by the formula .22
Statistical analysis
Data were expressed as , and at the same time, they were analyzed by multivariate analysis of variance (MANOVA) and repeated measures ANOVA using the SPSS v13.0 statistical software. Real-time PCR analysis was performed in duplicate and repeated a minimum of two times with independent RNA samples. The minimum level of significance was p < 0.05 for all tests.
Results
Morphologic comparison of normal and phocomelic limbs of fetal mice
Observation on GD18 showed that both the forelimbs and the hindlimbs of the fetuses of the treatment group whose mother was administered with atRA on GD11 were predominantly defected (Figure 1). The mean length of the left and right forelimbs as measured by a slide caliper was 10.96 ± 0.51 mm and 10.68 ± 0.48 mm, respectively, of the control group versus 8.12 ± 0.26 mm and 7.96 ± 0.31 mm of the treatment group (p < 0.05), and the mean length of the left and right hindlimbs was 9.60 ± 0.31 and 9.46 ± 0.33 mm respectively of the control group versus 6.04 ± 0.19 and 6.01 ± 0.23 mm of the treatment group (p < 0.05), indicating that both the forelimbs and the hindlimbs of the treatment group were significantly shorter than those of the control group. Staining showed that the humerus, radius, and ulna of both forelimbs of the treatment group were shorter than those of the control group, the femur and the tibia of both hindlimbs of the treatment group were shorter than those of the control group, and most of the fibula was missed (Figure 1).
Morphologic comparison between normal hindlimbs (R) and abnormal hindlimbs (L) at GD18. (a, b) An anterior view and a back view showed that the shorter forelimbs and hindlimbs of the fetal mouse of the treatment group, the forelimbs unable to embrace the thorax, and the hindlimbs was lacking strength and their joints were inconspicuous. (c, d) The humerus, radius, and ulna of both forelimbs of the treatment group were irregularly shorter than those of the control group, especially the radius which showed spotty. Of the treatment group, the femur of hindlimbs was significantly shorter than that of the control group, the tibia showed spotty, and most of the fetuses had no fibula. GD: gestational day.
Comparison of mRNA abundance of the Hsps families between the treatment and control groups
Here only the expression of embryonic hindlimbs was discussed in this study, since that of Hsps family in normal and abnormal development of embryonic forelimbs in atRA-induced phocomelic, oligodactylic was described in detail in our previous study.18,19 While that of embryonic hindlimbs was described in this study. As was shown in Figures 2to 6, there were 36 Hsps whose mRNA abundance was detected in the hindlimbs tissue of both treatment and control groups, all the other genes expressed differentially except HspA2.
Comparison of Hsp20, Grp170, HspA1A, HspB3, HspA14, HspA9, Hsp90AB1, Trap1, Hsp105, HspB5, Hsp25, HspA8, HspB7, and Hsp90AA1 mRNA abundance between the treatment and control groups. During GD12–GD18, the expression patterns of Hsp20, Grp170, HspA1A, HspB3, HspA14, HspA9, Hsp90AB1, Trap1, Hsp105, and HspB5 of the treatment group were the same as those of the control group. The mRNA abundance of Hsp20, Grp170, Hsp25, HspA1A, HspA8, HspB3, HspA14, HspA9, Hsp90AB1, Trap1, and Hsp105 of the treatment group was higher than that of the control group during GD12–GD18, and the mRNA abundance of HspB5, HspB7 and Hsp90AA1 was higher than that of the control group during GD12–GD17, while there was no difference between the two groups at GD18. — —: the control group; : the treatment group; *: higher than that of the control group, p < 0.05, MANOVA. MANOVA: multivariate analysis of variance; GD: gestational day; mRNA: messenger RNA.
Comparison of Hsp27, Hsp40, Hsp65, HO-1, and HspA1B mRNA abundance between the treatment and control groups. The mRNA abundance of Hsp27, Hsp40, and Hsp65 of the treatment group was higher than that of the control group during GD12–GD17, and lower than that of the control group before birth (at GD18). The mRNA abundance of HO-1 and HspA1B of the treatment group was higher than that of the control group during GD12–GD15, and lower than that of the control group during GD16–GD18. — —: the control group; : the treatment group; *: higher than that of the control group; #: lower than that of the control group, p < 0.05, MANOVA. MANOVA: multivariate analysis of variance; GD: gestational day; mRNA: messenger RNA.
Comparison of HspB2, HspB10, HspA12A, HspB9, HspA1L, Apg1, HspB4, Hsp22, Hsp10, Hsp60, and Grp78 mRNA abundance between the treatment and control groups. The expression patterns of HspB2, HspB10, HspA12A, HspB9, HspA1L, Apg1, HspB4, and Hsp22 of the treatment group were the same as or similar to those of the control group during GD12–GD18. During GD12–GD14, the mRNA abundance of HspB9 of the treatment group was found lower than that of the control group, but when we focus on the other genes, the lower mRNA abundance came out during GD16/GD17–GD18. As for Hsp60, the mRNA abundance was higher than that of the control group at GD17, with that of the other genes higher during GD16/GD17–GD18. — —: the control group; : the treatment group; *: higher than that of the control group; #: lower than that of the control group, p < 0.05, MANOVA. MANOVA: multivariate analysis of variance; GD: gestational day; mRNA: messenger RNA.
Comparison of HspA13, Grp94, and Hsp110 mRNA abundance between the treatment and control groups. The expression patterns of HspA13, Grp94, and Hsp110 of the control group were the same as those of the treatment group, and the mRNA abundance of the treatment group was higher than that of the control group during GD12–GD18. — —: the control group; : the treatment group; #: lower than that of the control group, p < 0.05, MANOVA. MANOVA: multivariate analysis of variance; GD: gestational day; mRNA: messenger RNA.
Comparison of Hsp47, HspA12B, and HspA2 mRNA abundance between the treatment and control groups. The expression patterns of Hsp47, HspA12B, and HspA2 of the control group were the same as those of the treatment group. The mRNA abundance of Hsp47 was lower than that of the control group during GD12–GD15, and had no difference between the two groups during GD16–GD18. The mRNA abundance of HspA12B of the treatment group was lower than that of the control group at GD15, higher during GD16–GD18, and had no difference with the control group during GD12–GD14. The mRNA abundance of HspA2 of the treatment group had no difference with that of the control group during GD12–GD18. — —: the control group; : the treatment group; *: higher than that of the control group; #: lower than that of the control group, p < 0.05, MANOVA. MANOVA: multivariate analysis of variance; GD: gestational day; mRNA: messenger RNA.
In the control group, the mRNA abundance of Hsp20, Hspb7, HspA8 (Figure 2), Hsp27, Hsp65 (Figure 3), Hspb4 (Figure 4), and HspA13 (Figure 5) tended to increase with the embryonic aging, and the mRNA abundance of Hsp20, Hspb7, HspA8, Hsp27, Hsp65 and Hspb4 during GD15/GD16–GD18 was higher than that during GD12–GD14, whereas that of HspA13 during GD17–GD18 was higher than that during GD12–GD14. During GD12–GD18, the mRNA abundance of HspB5, HspA1A, Hsp90AA1 (Figure 2), HO-1, Hsp40 (Figure 3), Hsp10, HspA12A (Figure 4), and HspA2 (Figure 6) was fluctuated and reached their highest range before birth (GD18). The expression peaks of Hsp105 (Figure 2), Hsp60, Grp78 (Figure 4), Grp94, Hsp110 (Figure 5), and HspA12B (Figure 6) were observed at GD14, while those of Hspb2 and Hspb10 were observed at GD15 (Figure 4); those of Hsp90AB1 and Trap1 appeared at GD16 (Figure 2), and HspA1B at GD17 (Figure 3). The mRNA abundance of HspA14 (Figure 2) and Apg1 (Figure 4) was lowest separately at GD14 and GD15 and that of HspA1L (Figure 4) showed a downward tendency with the growth of gestational days.
In the treatment group, as well as the control group, the mRNA abundance of Hsp20, Grp170 (Figure 2), Hsp65 (Figure 3), HspB4 (Figure 4), and HspA13 (Figure 5) tended to increase with the embryonic aging and that of HspA1L tended downward with the growth of gestational days. The expression peaks of Grp94, Hsp110 (Figure 5), and Hsp105 (Figure 2) were observed at GD14, those of Hsp90AB1 and Trap1 emerged at GD16 (Figure 2), HspA1B at GD17 (Figure 3). However, the expression was lower for HspA14 (Figure 2) and Apg1 (Figure 4) and appeared at GD14 and GD 15, respectively. HspB2 had an expression peak at GD17 and 2 days later than that of the control group (Figure 4), while HspB10 reached its peak at GD16 and one day later than that of the control group (Figure 4).
Compared with the hindlimbs of the same gestational age of the control group, gene expression in the hindlimbs of the treatment group was significantly higher (p < 0.05) for Hsp20, Grp170, HspA1A, HspB3, HspA14, HspA9, Hsp90AB1, Trap1, Hsp105, Hsp25, and HspA8 during GD12–GD18 (Figure 2), for HspB5, HspB7, and Hsp90AA1 (Figure 2), Hsp27, Hsp40, and Hsp65 (Figure 3) during GD12–GD17, for HO-1 and HspA1B during GD12–GD15 (Figure 3), for HspB2, HspB10, HspA12A, HspB9, Apg1, HspB4, and Grp78 (Figure 4), and HspA12B (Figure 6) during GD16–GD18, for HspA1L, Hsp22, Hsp10 and Hsp60 during GD17–GD18 (Figure 4). In contrast, gene expression was significantly lower (p <0.05) for HspA13, Grp94 and Hsp110 during GD12–18 (Figure 5), for HspA1L, Hsp22, Hsp10 and Hsp60 during GD12–GD16 (Figure 4), for HspB2, HspB10, HspA12A, Apg1, HspB4, Grp78 (Figure 4) and Hsp47 (Figure 6) during GD12–GD15, for HspB9 at GD12-GD14 (Figure 4), for HspA12B at GD15 (Figure 6), for HspA1B at GD16 (Figure 3), and for Hsp27, Hsp40 and Hsp65 at GD18 (Figure 3).
Discussion
Previous studies showed that Hsp32, Hsp30, Hsp27, Hsp26, Hsp25, Hsp23, and Hsp22 of the sHsps family,14,23–27 Hsp40 of the Hsp40 family,28 Hsp60, Hsp62 and Hsp56 of the Hsp60 family,29–31 HspA1A, HspA1B, HspA2, Grp78, HspA8, HspA9, HspA12B and HspA14 of the Hsp70 family,32–39 Hsp90AA1, Hsp90AB1, Grp94, and Trap1 of the Hsp90 family,40–43 and Hsp105 and Hsp110 of the Hsp110 family17,44 were expressed in embryonic tissues of different animals.
It is generally believed that Hsps can protect embryonic development and on the other hand interferes with it.45 In the process of embryonic formation, it is necessary to regulate the balance between cell cycle and apoptosis so as to maintain the appropriate number of cells. Hsps play an important role as molecular chaperones in regulating cell cycle,46 and they also have the function of anti cell apoptosis.47–49
Based on our previous studies,18,19 the present research aimed at investigating the expression of Hsps family in normal and abnormal development of embryonic hindlimbs to explore the effect of the Hsps family on the development of mouse embryos’ hindlimbs. Except for HspA2, it was found in this study that there were significant differences in the expression of the other 35 Hsps in most part of the developmental period between the treatment group and the control group. The development time of the mouse embryonic hindlimbs was usually known during the GD12–GD14,50 while of the treatment group, the mRNA abundance of Hsp20, Grp170, HspA1A, HspB3, HspA14, HspA9, Hsp90AB1, Trap1, Hsp105, Hsp25 and HspA8 during GD12–GD18 (Figure 2), that of HspB5, HspB7, Hsp90AA1 (Figure 2), Hsp27, Hsp40 and Hsp65 (Figure 3) during GD12–GD17 and that of HO-1 and HspA1B during GD12–GD15 (Figure 3) was observed to be higher than that of the control group. Furthermore, the expression patterns of many of these genes of the treatment group were the same as or similar to those of the control group. So we could draw a conclusion that these genes might be involved in stress-induced protective responses.
In the treatment group, the mRNA abundance of HspA1L, Hsp22, Hsp10, and Hsp60 during GD12–GD16 (Figure 4), that of Hsp47 (Figure 6), HspB2, HspB10, HspA12A, Apg1, HspB4 and Grp78 (Figure 4) during GD12–GD15, and that of HspB9 (Figure 4) during GD12–GD14 were all lower than the mRNA abundance of the control group; however, in the subsequent development period, the mRNA abundance of these genes of the treatment group at most time points was higher than that of the control group. It suggested that these genes probably played an important role in normal and abnormal development of embryonic hindlimbs. The expression patterns of HspA13, Grp94, and Hsp110 of the control group was the same as those of the treatment group during GD12–GD18 (Figure 5), and the mRNA abundance of these genes of the treatment group was lower than that of the control group, and all these suggested that the abnormal expression of the three genes, perhaps hindlimb development-related ones, was closely related to the occurrence and progression of atRA-induced short hindlimbs malformations, and they may be hindlimb development-related genes.
The mRNA abundance of HspA12B of the treatment group had no difference with that of the control group during GD12–GD14, and that of the treatment group at GD15 was lower than the control group, while higher during GD16–GD18, which suggested that this gene maybe played some role in normal and abnormal development of embryonic hindlimbs. The mRNA abundance of HspA2 of the treatment group had no difference with that of the control group during GD12–GD18, and this indicated that this gene hadn’t been affected by the atRA-induced short hindlimbs malformations.
Our previous studies found that Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10, and Hsp40 may be related to atRA-induced phocomelic and other abnormalities18 and HspA12B, HspA14, Trap1 and Hsp105 may be forelimb development-related genes; Grp78 may be play an important role in limb development.19 Summarizing the results of this study and our previous studies, we are convinced that HspB2, HspB9, HspB10, and Grp78 probably performed an important role in normal and abnormal development of embryonic forelimbs and hindlimbs, HspA12B, HspA14, Trap1, and Hsp105 might be forelimb development-related genes, while Grp94 and Hsp110 were probably hindlimb development-related genes.
Footnotes
Conflict of interest
The authors declared no conflicts of interest.
Funding
This work was supported by Scientific Foundation for the Doctors of Hunan Normal University (53112-2323) and Grant of the National Natural Science Foundation of China (81273104).
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