The Chicken Embryonic Life Regulators: miRNAs ?
Tembhurne P. A. *Bawaskar M.S. and Bhojne G.R
Nagpur Veterinary College, Nagpur
Maharashtra Animal and Fishery Sciences University, Nagpur
High Land Drive Seminary Hills, Nagpur 440006.
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Key words: Chicken, Embryo, life regulators, miRNAs, HeLa cells, Somites, C.elegans, miR-125b, miR-142-3p and miR-21.
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The developmental sciences have delivered the in-depth knowledge from embryogenesis to the new born. The advent in understanding the gene expression opens ups the new era of understanding the signals and pathways how the entire cell universe works. The early development of life is always been a plethora of functional process which has the effects of different gene controlled processes at several time points of the embryonic development. Deciphering such unsolved puzzles intrigue the scientist whether the coding genes alone regulates these cellular processes.
The recent advances established that these genes are controlled by a large class of small non-coding RNAs, which are functioning as important regulators of a wide range of cellular processes by modulating gene expression. Three classes of small RNAs have been identified to play major role in biological system, including Micro RNAs (miRNAs), siRNAs and Piwi-interacting RNAs (piRNAs) (Farazi et al., 2008) The small RNA sequence is able to regulate the expression of multiple genes because it can bind to target genes as either an imperfect or perfect complement (Ha et al., 2008).MicroRNAs are small non-coding endogenous RNA (21 to 23 nt in length) which are generated from endogenous hairpin-shaped transcripts. MicroRNAs are one of the largest gene families, constitute approximately 1 to 3% of the genome and are predicted to regulate 30% of human genes (Sandhu et al., 2011). Micro RNAs act as the delicate regulatory switches and fine tuners of gene expression by binding mainly to 3’ UTR sites as well as to the ORF and 5’ UTR sites occasionally in sequence specific manner to regulate stability and translation of the target mRNAs. Micro RNAs interact with mRNAs and either block protein translation or lead to protein degradation (Bartel, 2004). miRNA found to play a major role in normal biological pathways such as cell cycle, survival, differentiation, proliferation and migration of cells.
The microRNAs regulating gene expression has wide impact on the various biological processes, such as cell cycle control, apoptosis, haematopoiesis, adipocyte differentiation, insulin secretion, tumorigenesis, virus-host interactions, immunity and immune response.
The microRNAs miR–124and miR–1, which are preferentially expressed in brain and muscle respectively, were introduced into HeLa cells, they caused the gene expression profiles to shift toward those of brain and muscle respectively, suggesting that, these miRNAs assist in controlling differentiation during development. These studies indicate that miRNAs serve an important regulatory role during vertebrate development.
Regulation of gene expression in developmental processes is an important aspect of miRNA function. The first identified miRNA, termed lin-4 and let-7, were found to regulate larval stage transition in C.elegans development (Lee et al., 1993). Studies of Dicer null embryos in several vertebrate species, including zebra fish and mice, have revealed that miRNA are essential for proper embryonic development (Giraldez et al., 2005; Harris et al., 2006). Dicer knockout in both studies was lethal, with embryos failing to develop past the earliest stages of development.
The highly expressing organ specific miRNA’s suggested their importance in establishing or maintaining the cells fate in that organ. MicroRNAs plays vital role in regulation of immunity, including the development and differentiation of B and T cells, proliferation of monocytes and neutrophils. They are critical regulators of the migration of cancer cells; for example, miR-23b, miR-146b and miR-34a found to be involved in cancer cell migration. Regulation of gene expression during embryonic development is one of the most important functions of miRNA.,
Role of microRNA in chicken developments
The chicken orthologue of the C. elegans gene lin-41 is expressed in chick limb development. This indicates that they control over the expression of chicken lin-41.miR-196 is also involved in specifying hind limb development.
During the somite development in the chick embryo revealed that miR-124 and miR–206, are involved in the regulation of the nervous system. They are expressed in the early developmental stage, because the nervous system is one of the earliest developing systems during embryonic development. The miR-124is specifically involved in the development of the central nervous system, whereas miR-206is specifically expressed in developing somites.
microRNA expression in the early chick embryo ranging from day 0.5 to 5 days reveals that 135 different microRNAs were found to be expressed and out of 75 showed differential expression over the different developmental time points. This study demonstrates that microRNAs are a very important gene regulatory mechanism in the chick development, similar to what has been found in other organisms (Darnell et al., 2006).To evaluate further the role miRNA in development of livestock species, miRNA transcriptome profiles have been created from tissues at specific stages of development in chick embryo and adult chicken. Large proportion of miRNA are ubiquitously expressed at all stages of the developing embryo and adult, a pattern of increased abundance of miRNA during progression of development is observed, with the greatest expression of most miRNA in the adult tissues compared with the embryo (Darnell et al., 2006).
Similar to other vertebrate species, tissue specific miRNA were identified including miR-122 in the liver, miR-132 in the cerebrum, and miR-338 in the cerebrum and cerebellum. Together, these data discussed to this point suggest a potential role for both tissue-specific and ubiquitously expressed miRNA in skeletal muscle, adipose tissue, and embryo development of multiple livestock species. One of our research have shown some of the miRNA (miR-125b, miR-142-3p and miR-21) are highly expressed in at embryonic developmental stages of (11 day) embryo and in immune organs i.e. Bursa of Fabricius and Spleen embryonic day 15 and day 20 which involve a variety of cell proliferation, differentiation and migration of T and B cells (Hicks et al., 2008, 2009).
The study by Hicks and Liu (2021), where transcriptome and miRNome analyses revealed that a dynamic and diverse metabolic program exists in the adipose tissue during the embryonic-hatch transition in chicks. Several important developmental and metabolic systems have distinct expression patterns between adipose tissue of embryonic and post-hatch chicks. The adipocytic expression of miR-26a increases from E18 to D3 and MiR-26a is known to target a number of metabolic and adipogenic genes. It is possible that the increased adipocytic expression of miR-26a during the embryonic to hatch transition serves, in part, to relieve Activin B inhibition of PNPLA2 expression to facilitate the lipolysis of adipose tissue triglyceride stores to supply the energy needed for hatching and the rapid growth which follows. The dwarfing in cheek as shown to be regulated by the some of the miRNA in one study showed as let-7b, miR-128 and the MAPK pathway might play key roles in the GHR-deficient induced muscle mass loss, and that the reduced cell division and growth are potential cellular processes during the SLD chicken skeletal muscle development. It provides new insights into the molecular mechanism underlying muscle mass loss in the SLD chickens, and some regulatory networks that are crucial for chicken skeletal muscle development. In another study it is observed that the gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens ( Weihua et al., 2022)
The another study have shown a expression of miR-203 is transitory during chicken skeletal muscle development but also showed a novel role of miR-203 in inhibiting skeletal muscle cell proliferation and differentiation by repressing c-JUN and MEF2C, respectively as miR-203 exhibited downregulated expression during myoblast differentiation into myotubes. miR-203 overexpression inhibited myoblast proliferation and differentiation, whereas its loss-of-function increased myoblast proliferation and differentiation. During myogenesis, miR-203 can target and inhibit the expression of c-JUN and MEF2C, which were important for cell proliferation and muscle development (Luo et al 2014, 2016)
It is been observed that over 800 mRNAs and 30 miRNAs was altered in the embryonic liver between embryonic day 18 and posthatch day 3, and many of these differentially expressed mRNAs and miRNAs are associated with metabolic processes, indicating miRNAs in controlling the metabolic switch that occurs between embryonic and posthatch development in the chicken ( Hicks et al., 2017 ). The adipocytic expression of the GH receptor (GHR) significantly increases during the embryonic-hatch transition. It could be due to the reduced expression of miR-15a (a GHR regulator) during this time. The BCL6 is down-regulated during the peri-hatching period while, its miRNA regulator, miR-10a is up-regulated, GFBP5 is a member of the insulin-like growth factor family, which is stimulated by GH signaling. The adipocytic expression of IGFBP5 was up-regulated from E18 to D3 (and conversely the IGFBP5 regulator, miR-140, was down-regulated during this time.In summary we could see that miRNAs play a vital role as an important component of the BCL6/GH feedback loop and this regulatory system may contribute to maintaining lipolysis of fat stores at a sufficient level to meet the energy expenditures associated with hatching ( Hicks and Liu ., 2021 ).
The further research on sequencing based identification and characterization of more ncRNAs (mainly microRNAs (miRNAs), long non-coding RNAs (LncRNAs), and circular RNAs (CircRNAs)) involved in the development of domestic chicken especially the skeletal muscle, could decipher the different regulatory functions action with cellular proliferation, differentiation, fusion, and apoptosis of myoblasts and satellite cells, and the specification of muscle fiber type.
Similarly it is observed that the some miR expression viz expression level of miR-15a in hypoxic Tibet chicken embryos increased and remained relatively high at embryonic day (E)16–20, whereas in normal chickens, expression increased and peaked at E19–20, at which time the cross-current gas exchange system (CCGS) is developing. This indicates the epigenietic controls on during the developmental stages also contributes to the regulating the cellular processes especially as mR-15 in lung hypoxia management. Indicating the miR-15 could be better solution or a novel therapeutic strategy for hypoxia insults and altitude adaptation ( Hao 2014).
There are differential expression of several miRNAs in the Chick Embryonic days E 11 , E15-E-20 days showing differential expression of miRNA, especially some of the miRNS were found transiently expressed differentially at day E15 and day E20 in spleen and Bursa ( Hicks et a al., 2008, 2009 ) In our laboratory we have deciphered the developmental role of miRNAs , where in study, we decipher the miRNA-142-3p which is highly expressed during the developmental stages, whether this miRNA needed for maintaining the functional activity of immune organ by regulating the gene expression in organs. We have knockdown of this miRNA-142-3p at developmental and we found its role in functional stages of these immune organ and in other organs. ( Manesh et al., 2017) The elucidated importance of miRNAs in respect to the target organs, importance in structural integrity of immunological important organs / or other target organs, involvement in maturation and migration of B and T cells provides a miRNAs as futuristic tools.
The miRNA shown their regulatory role in the developmental stages of chicken emberyo . The chicken embryo as a suitable model to decipher the exact role of each miRNA, miRNA therapeutics or the epigenetic control owing to several advantages. The understanding the miRNA in developmental biology through the chicken embryos enhancing the current knowledge of avian biology. The mRNA shown potential for futuristic tools was bio-markers, therapeutic agents (currently under clinical trials for miR-122). The understanding of fine tuning of gene expression by the miRNA in developmental stages shown the post hatch implication.
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