Indian Journal of Animal Research

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Research Article

Impact of Vitamin A on the Expression Profile of Development-Controlling Genes in Chick Embryos

Abdalla A. Sayed1,2,*, Gamal M. Bekhet3
1Department of Biological Science, College of Science, King Faisal University, Saudi Arabia.
2Department of Zoology, Faculty of Science, Minia University, Minia-Egypt.
3Department of Zoology, Faculty of Science, Alexandria University, Alexandria-Egypt.

ABSTRACT

Background: Vitamin A has potentially harmful effects on the development of chick embryos even at very low dose concentration. Vitamin A different doses cause embryotoxic and teratogenic effects on the developing of the chick embryo. Hence, due to teratogenicity of this vitamin it can be used with utmost caution during pregnancy.      

Methods: Fertile chicken eggs (obtained from local hatchery) were injected with three doses of Vitamin A (15 IU/ml, 30 IU/ml, 75 IU/ml) into the center of the egg yolk. Morphometric parameters as mortality rate, body weight (g), crown rump length (mm) of embryo and Frequency % of different anomalies gross malformations were measured. Whole chicken samples were taken by 4th, 8th and 18th incubation day. Daam, Hox and Tbx genes expression profile were analyzed.

Result: Dose-dependent defects and embryo lethality in the chick embryos developed when exposed to higher concentrations. Our study record for the first time the duplication in the heart, brain and eye. Also showed duplication of the neck forming S-shaped showed in some malformed chick embryos. The three investigated genes showed different fluctuations confirming the malformations detected.

INTRODUCTION

Retinoic acid signaling and receptors in gene regulation have an essential  role in the vertebrate development including the formation of the heart (El Robrini et al., 2016)., brain (Uribe et al., 2018) and congenital defects (Shukrun et al., 2019); gut embryonic development (Shimozono et al., 2013); human fetal gonad and Spermatogenesis (Griswold,  2016), pluripotent embryonic stem cells promoted neural lineage entry through multiple pathways. (Lu et al., 2009), fate and differentiation of olfactory progenitor cell (Paschaki et al., 2013), craniofacial development (Dubey et al., 2018). Retinoids bind to   retinoid X receptors (RARs and RXRs) to regulate tissue-specific genes.

Dose-dependent defects and embryo lethality in the humans embryos developed when exposed to higher concentrations of retenoic acid, rat embryos (Kessel and Gruss, 1991), chick embryos and Xenopus tadpoles (Sive et al., 1990). severe malformation including : central nervous system defects, skeletal defects, cleft palate, ear and other craniofacial malformations, defects of the heart, thymus and urogenital system and limb and digit reduction or duplication. (Dora, et al., 2008). The planar cell polarity (PCP) control the fate of different cells and leads finally to organization of different functional organs (Ma et al., 2003) signals of PCP transported throughout a group of regulators of disheveled associated activator of morphogenesis (Daam) which is a member of Wnt signaling  in different pathways (Kida et al., 2004). For normal development some complexes between genes should be formed (MacDonald et al., 2009). Daam proteins coordinate to amplify Wnt signaling where it potentiate the activity of Wnt ligand. Genome-Wide Linkage Analysis Identifies Loci for Physical Appearance Traits in Chickens (Sun et al., 2015). T-box proteins have an essential role in the normal development of chicken (Angenee et al., 2013).where it control the specification of each one of the germ layers and the organ forming cells (Abrahams et al., 2010). T-box gene expression are controlled by cell cycle regulators such as E2F4 and PML, nuclear factor Y (NF-Y) (Martin et al., 2012). T-box protein manage and control the pharyngeal muscles and specification of the fate and differentiation of HSN and PHB neurons those derived from Aba blastomere (Lupski, 2015).

The hox genes specify regional differences along the anterior-posterior (A/P) axis of the vertebrate embryo. This function appears to reflect an ancestral role of the hox gene complex and is conserved across phyla. During the evolution of vertebrates, this gene complex has been recruited to perform other functions as well, many of which occur later in development. (Gaunt SJ, 2015).

MATERIALS AND METHODS

Four hundreds of fertile chicken eggs (obtained from local hatchery) were incubated at 38°C with a relative humidity of 70%. Three doses of Vitamin A palmitate were directly injected (single dose for each) in the broader side into the center of the egg yolk using an established protocol (Drake et al., 2006).  Eggs were divided into four groups 100 for each. The control group received sterile physiologic 70% ethanol, the second group injected by 15 I.U./ml of Vitamin A (Retinol palmitate ) diluted in 0.1 ml sterile 70% ethanol, the third group received 30 I.U./ml and the 4th group injected with 75 I.U./ml. After the fourth day of incubation till hatching, all eggs were opened and surviving and dead embryos were carefully separated from the yolk sac, cleaned and examined for gross abnormalities and classified according to Hamburger and Hamilton (1951). The potential  teratogenic effect of Vitamin A were detected through the following  morphometric parameters: (1) mortality rate of embryos in which development was arrested; (2)  Body weight (g) and Crown rump length(mm) of embryo (3) Frequency % of different anomalies gross malformations; were examined using dissecting light microscopy.

Whole chicken samples were taken by 4th, 8th and 18th day of incubation and total RNA was extracted. mRNA was purified from the total RNA using. 2 μg of mRNA was used for cDNA synthesis for each sample by oligo-dt12-28 primer (GIBCO-Invitrogen, USA). The obtained cDNA then purified. The purified cDNA was stored at - 200 C until using for PCR. Primers were designed for the target genes Daam, Hox and Tbx genes.

RESULTS AND DISCUSSION

Vitamin A palmitate have teratogenic effects on the development of neural tube, heart, limbs, genitourinary system, eye, respiratory tract, abdominal wall and skin. In the present study, the injection of developing chick embryos with Vitamin A decreased their survivability compared to control (Fig 1). This is in accordance with previous studies (Drake et al., 2006, Brand et al., 2008 and Grant et al., 2018). The chick embryos showed increased mortality when examined 24-72 hours later. Embryos were recovered at days 4, 8 of incubation and newly hatched chicks. Effects of Vitamin A palmitate on embryonic mortality with different doses are summarized in Fig 1.

Fig 1: Effect of vitamin A (15 IU/ml, 30 IU/ml, 75 IU/ml) on embryonic mortality (X±SE): Values are Mean±SEM of 10 replicates. a ,b, c and d Means within each column for each item with different superscripts are significantly different (P<0.05) with each other [One way ANOVA-Tukey’s multiple comparison test].



The percentage of embryonic mortality was observed in egg injected with 15, 30 and 75 IU/ml Vitamin A palmitate decreased significantly with increasing dose respectively compared to the control group. This finding is in accordance with other studies which reveals that there is a significant in the treatment with 75 IU/ml (Dubey et al., 2018 and Shukrun et al., 2019). The wet body weight and, crown rump length were recorded and compared with control group and summarized Fig (2 and 3). There were a significant (p>0.05) reduction in both parameters length in all treated groups compared to control group. Our results are coincide with that reported by (Abbas s et al., 2016; and Bekhet 2019).

Fig 2: Effect of Vitamin A (15 IU/ml, 30 IU/ml, 75 IU/ml) on Body weight of chick embryos.



Fig 3: Effect of Vitamin A (15 IU/ml, 30 IU/ml, 75 IU/ml) on crown rump length of chick embryos.



As regards to the qualitative malformations that observed in the treated groups as compared to control groups were summarized in Table 1,2. Such as microcephaly, hydrocephaly, hematoma formation, Omphalocele, microphthalmia, anomalous legs, everted viscera and ectopia cardis and heart and neural defects. Moreover many malformations were appeared in the developing chick embryo as  shown in Fig 4 were brain decreased in size and changed in shape (Fig 4 b-f) compared to control group (Fig 4a). In addition to the newly hatching  chick ,with haematoma observed in (Fig 4g) ,there were failure to retraction of their yolk sac (Fig 5,YS) , dermatitis (Fig 6,D) accompanied with skin devoid of feathers, (Fig 6NS ) and everted viscera (Fig 7, EV). Nearly all of these malformation obtained by this study results were confirmed by other researchers such as Farzaneh et al., 2017 and  Reda et al.,  2019. Our study record the duplication in the heart (Fig 4e), brain and eye (Fig 4f). Also showed duplication of the neck forming S-shaped (Fig 7, DN) for first time.

Table 1: Primers sequence used in PCR amplification.



Table 2: Frequency % of different anomalies observed in vitamin A treated chick embryos.



Fig 4: showing chick embryo after 5 days of incubation (a) Control. (b) Treated with 15 IU/ml. (c) Treated with 30 IU/ml.(d) Treated with 30 IU/ml. (e) Treated with 75 IU/ml. (f) Treated with 75 IU/ml. (g) Treated with 15 IU/ml. Abbreviations: FB (Fore brain),MB (mid brain), HB (Hind brain), E (Eye), H (Heart).



Fig 5: Showing newly hatching chick, (a) Treated with 15 IU/ml. (b) Treated with 30 IU/ml. (c) Treated with 75 IU/ml. Abbreviations : (YS): Yolk Sac. (EV): Everted Viscera and (NK): naked skin trunk.



Fig 6: Showing chicks treated case with 30 IU/ml showing variety of malformed legs. a) Twisted hind leg (b) Direction of hind legs backward (c) Creeping hind limb. (d) Crocked toes and pointing of hind legs inside. (e) Deformed spraddel leg and crocked toes.



Fig 7: Showing newly hatched treated case with 75 IU/ml with duplication of the neck (DN) in the form of S - shaped and also accompanied with malformed leg.



Regarding development control genes Daam1, Daam2, Tbx1, Tbx2, Tbx3, Tbx4,Tbx5, Hox3, Hox5, Hox7 and Hox10 expression  were analyzed in 5 days , 8 days and 18 days embryo. The expression frame is showed in Fig (8-10). Daam1 and Daam2 expression analysis showed in Fig 8. Daam1 showed a very high expression by the 5th day and the same expression rate continuing up to 8th day by examining the rate of expression by 18th day of incubation the expression rate was decreased. The dose concentration effects is appear in the two high doses. Daam1 and Daam2 control the myocardial growth and differentiation (Singhvi et al., 2008, Li et al., 2011), so its expression was affected in the early development days and this may explain why vitamin A high doses increase the expression rate in the 5th and 8th days embryo (Fig) Vitamin A also affect the expression profile of Daam2 which usually compensate the role of Daam1 one when its expression rate is affected (Ajima et al., 2015 and Kannaki et al., 2018).  This may explain as it is shown in figure the expression rate of Daam2 in three treatment doses was nearly same Fig 8. Also Daam2 is a modulator for spinal cord development through the increasing of Dvl3/Axin2 clustering inside cells by this way it can promotes Wnt signaling by stabilization of such clusters (Lee and Deneen 2012). Results of this work showed that Daam1 is more affected than Daam2 and this may be due to the effect of vitamin A on GTP-bound Rho (Goode and Eck 2007- Li and Higgs 2005).

Fig 8: Expression analysis of Daam1 (a) and Daam2 (b) in chicken eggs treated with vitamin A for different doses throughout 5, 8, 18 days of incubation. The expression rate of both genes showed a different flocculation and disturbance in groups treated with vitamin A.



In addition, study of five members of Tbx gene (Tbx1, Tbx2, Tbx3, Tbx4 and Tbx5) were examined in this work and results showed a different pattern for each member as shown in Fig 9. Tbx1 expression is affected by high dose of vitamin A. Tbx1 and Tbx5 down regulated by time of incubation and the other three members are fluctuated between upregulation and down regulation. The expression results are in agree with results of (Esteban et al., 1999) which controlled by the FGF signaling.

​

Fig 9: Expression analysis of Tbx1 (a) and Tbx2 (b), Tbx3 (c), Tbx4 (d) and Tbx5 (e) in chicken eggs treated with vitamin A for different doses. The expression rate in Tbx1 and Tbx5 was affected and disturbed more than the other genes of this family.

CONCLUSION

Our results concluded that vitamin A has potentially harmful effects on the development of chick embryos even at very low dose concentration. New record was reported with the duplication of organs on the malformed chick embryo including heart, brain and neck. All malformations were confirmed by molecular expression analysis for genes those control chick development such as Daam, Hox and Tbx genes. Hence, due to teratogenicity of this vitamin it can be used with utmost caution.

ACKNOWLEDGEMENT

The author acknowledge the deanship of scientific Research at King Faisal University for the financial support under Nasher Track (Grant No. 186396).

Conflict of Interest

The author(s) declare(s) that there is no conflict of interests regarding the publication of this article.

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