Microinjection of miR-124-3p into fertilised mouse embryos at day 0.5 (miR-124*) results in mice with a ‘giant’ phenotype that is noticeable both prenatally and postnatally (Fig. 1A-B). Thus, an RNA fragment of 22 nucleotides in length can cause a phenotypically observable change. This finding explains how epigenetics, which we define as inherited changes in gene expression with no difference in the DNA sequence, acts as a mechanism. The prominent phenotype detected by Grandjean et al. (5) in miR-124* B6/D2 mice was also observed here in Balbc mice. This study was conducted to elucidate the molecular mechanism underlying the occurrence of this prominent phenotype in miR-124* mice.
Epigenetically modified transgenic mice with a microinjection of miR-124, known for its expression in the hippocampus, were tested behaviorally (15–23). All these behaviour test results, demonstrate alteration in expression levels of the transcripts may lead to the disappearance of the existing differences between males and females in terms of behaviour (stress and anxiety) in miR-124* mice; that is, similar behaviour in males and females may have been affected (Fig. 5). Prediction algorithms like miRDB and TargetScan are used to identify miRNA targets. Still, wet laboratory experiments are needed to confirm accuracy. For example, miRDB scores miR-124-3p's targeting of the doublesex and mab-3 related transcription factor like family A1 (Dmrta1) gene with 92 out of 100. Dmrta1 knockout mice showed no anatomical deficiency, were viable and productive, males exhibited mating behaviour with other males, and females had polypollicular ovaries (carrying more than one egg) (28). The majority of the embryos with miR-124* were found in twin formations within the uterus of the foster mothers (5). In this study, the Dmrta1 mRNA expression level decreased in miR-124* E7.5 compared to controls (Fig. 7A). This may explain frequent twin formations in miR-124* embryos.
The genetic and epigenetic mechanisms that control the size and weight of a mammal's body are regulated differently during the prenatal and postnatal periods. Grandjean et al. showed that B6/D2 miR-124* mice were approximately 30% larger (5). In this study, we observed that mice with miR-124* had a ‘giant' phenotype both prenatally (at E7.5 and E19.5) and postnatally. Compared to controls, miR-124* mice were heavier on postnatal day (PND) seven (Fig. 1C) and not heavier on PND21 (Fig. 2A) but rapidly gained weight and became heavier in the following weeks (Fig. 2A, B, and C). At PND7, miR-124* mice are probably heavier and larger (Fig. 1B) because they may behave more actively and consume more breast milk than the control. The presence of breast milk in the abdominal region of miR-124* at PND2 was observed before (5). At PND21, the mice stop breastfeeding and the litter starts feeding themselves. However, miR-124* mice become depressed and only feed on breast milk. So they are not heavier than the control group at PND21. However, after PND21, miR-124* mice recovered and were fed more actively than the control group. Behaviour test results showing that miR-124* mice moved faster than the control group support this hypothesis (Fig. 5A, B and E). This study found that Balb/c miR-124* male mice were 24–44% larger, and miR-124* female mice were 17–35% larger than their controls (calculated by considering the weighing data on the 28th day). Do miR-124* mice eat more and gain more weight? To find out, we weighed the mice and their food daily. Results showed that miR-124* male mice ate more than control males, while miR-124* females ate (Fig. 1E). This suggests that daily weighing may have caused stress in the female mice, preventing them from eating.
PHEMA-coated plates create a non-adhesive surface that encourages the growth of NS cells (9), unlike 2D culture, where neurons tend to become glial cells due to surface adhesion (29). NS provide a more natural three-dimensional environment for studying neural stem cells (30). Although they can differentiate into various cell types, they are less complex than early-stage embryos. While both models hold potential for studying neurodevelopment and embryogenesis, early embryos possess a greater cellular potential.
SoxE genes (Sox8, Sox9, and Sox10) play a crucial role in developing the nervous system and regulating the development of neurons, glial cells, and other necessary cell types (31, 32). They significantly impact neuronal development and function, with Sox8 being implicated in oligodendrocyte development and myelination (32–36). Homozygous Sox8 knockout mice were 30% smaller and 10% shorter than the control group after being weighed for 100 days (37). Sox8 mRNA expression levels were investigated in miR-124* embryos from early stages to adulthood. It was observed that Sox8 mRNA expression was undetectable in E2.5 embryos but increased towards adulthood. Sox8 may play a role in maintaining accelerated growth until adulthood. Our study found similarly lower Sox8 mRNA expression levels in miR-124* E7.5 embryos compared to the control. (Fig. 7A). However, a slightly increased Sox8 mRNA expression was observed in miR-124* E19.5 embryos (Fig. 8A). Sox8 mRNA expression level increased in NS with EGF (+), particularly at 12 and 21 days (p = 0.0028). No changes were observed in EGF (-) (Fig. 10A).
Sox9 mRNA expression levels were investigated in miR-124* B6/D2 mouse embryos from early embryonic to adulthood. Sox9 transcript levels decreased as embryogenesis progressed. High expression of Sox9 was seen in organs with high growth rates, such as the kidney and pancreas. Increased growth rates and cell proliferation continued to coordinate for the whole body (5). Subsequently, Grandjean et al. conducted more extensive investigations on Sox9. Since heterozygous Sox9 knockout mice died after birth (38), Grandjean et al. observed that when Sox9 expression was reduced by siRNA transfection at the zygote stage, embryos were small, development was abnormal, and embryos died at E10.5. Thus, they stated that Sox9 is required during early development but could not detect a specific effect on cell proliferation and growth control (5). Injecting Sox9 cDNA downstream of the CMV1 promoter resulted in larger miR-124* E7.5-day embryos with normal morphology, but development was arrested at E11.5 days. In our study, Sox9 mRNA expression level was lower in E7.5 compared to controls (Fig. 7A), while it was at the same level in E19.5-day-old embryos (Fig. 8B). However, no change was observed in Sox9 mRNA expression level in the EGF (-) in NS. Interestingly, in the EGF (+), Sox9 mRNA expression level was unchanged in miR124* 12-day-old NS compared to controls, whereas miR-124* was overexpressed in 21 day NS compared to both 12 (p < 0,001) and 21 (p < 0,0001) day control NS (Fig. 10B).
Sox10 is a crucial transcription factor in the regulatory network of Schwann cells and oligodendrocytes, required for lineage progression, final differentiation, developmental myelination, and myelin maintenance (39, 40). In this study, Sox10 mRNA expression was found to be decreased in miR-124* E7.5 (Fig. 7A) and miR-124* E19.5 hippocampus (Fig. 8C). Otherwise, Sox10 mRNA expression increased in miR-124* 21-day-old NS compared to controls (p = 0220) and miR-124* 12-day-old NS (p = 0315) (Fig. 10C).
Dcx is a neuron-expressed protein that regulates microtubules to direct neuron migration in the nervous system during development (41). Dcx knockout mice had normal growth until weaning but lower body weight at four months (42). Dcx knockout mice had slower growth rates and weighed 3–7% less than control mice in a larger cohort study (43). In this study, Dcx mRNA expression was at the same level as controls in miR-124* E7.5 and miR124* E19.5 hippocampus (Fig. 7A and Fig. 8D). Similarly, in the EGF (-), miR-124* Dcx mRNA expression was at the same levels as controls in 12 and 21-day-old NS. EGF (-), Dcx mRNA expression was deficient in miR-124* 21 day NS and at the same level as the control, but miR-124* was significantly increased in 12 day NS (p < 0,0001) (Fig. 10D). In a study, it was found that EGF infusion in ischaemic mice enhanced the proliferation of cells but inhibited their differentiation into neuroblasts (44). Similarly, in this study, Dcx mRNA expression in miR-124* mice increased without EGF but decreased to control levels in its presence. Prolonged neurosphere culture time could eliminate the neurogenesis capacity, and EGF may have negatively affected Dcx mRNA expression.
Neurod1, a neurogenic factor and proneuronal marker, was shown to be among the targets of miR124-3p by decreased gene expression after miR-124 microinjection into frog blastomeres (45). In this study, Neurod1 mRNA expression was increased in miR-124* E7.5 and E19.5 hippocampus (Fig. 7A and Fig. 8E). Neurod1 mRNA expression in miR-124* 21-day-culture NS was found to be at the same level as controls and significantly lower in miR-124* 12-day-culture NS compared to controls in the presence (p = 0.0014) and absence (p = 0.0002) of EGF (Fig. 10E). Following neurosphere formation, Neurod1 mRNA expression in miR-124* mice increases independently of the presence of EGF. However, it decreases to the same levels as controls as the culture time is prolonged. It indicates that prolonged neurosphere culture time almost eliminates the capacity of neurospheres for neuronal differentiation. When the culture period is prolonged till 21 days in miR-124* neurospheres, EGF overexpresses the transcription factors such as Sox8, Sox9 and Sox10; however, it decreases the mRNA expression of neurogenesis marker Dcx and neuronal differentiation marker Neurod1. These results demonstrate that EGF could have suppressed the neurogenesis and neuronal differentiation in miR-124* neurospheres by prolonged culture time. A similar mechanism could have been activated immediately after the microinjection of miR-124-3p into the one-cell embryo. Following microinjection of miR-124-3p, EGF could have been activated, and it suppressed miR-124-3p expression, leading to early neurogenesis and neuronal differentiation in the embryos, resulting in cognitively active and 'giant' phenotyped mice.
miR-124 is a microRNA recognised for its proneuronal role in developing and maturing the brain. The conservation of the sequence and expression pattern, miR-124 in the evolutionary process reveals that it is critically important in CNS development (46). Despite this, the functions of miR-124 in neuronal development are controversial. Neither inhibition nor overexpression of miR-124 significantly impacted neuronal differentiation (47). Overexpressing miR-124 through transfection in nerve progenitors of chick neuronal tubes resulted in decreased expression of small C-terminal domain phosphatase 1 (Ctdsp1). In the 3′UTR region of Ctdsp1 mRNA, regions compatible with the evolutionarily conserved miR-124 sequence were found. With this finding, they suggested that miR-124 facilitates neurogenesis at least partially by blocking the neuronal anti-REST/Ctdsp1 pathway (Visvanathan et al., 2007). The formation and development of neuritis increased in cell lines (HeLa and N2A) with decreased Polypyrimidine tract-binding protein (Ptbp) expression. Therefore, it is stated that some splicing events regulated by Ptbp may directly lead to the observation of neuronal phenotypes. They also reported that miR-124 targeted Ptbp1 by working as an antagonist to the REST pathway and initiated a neuron-specific splicing mechanism in support of Visvanathan et al (49). Mokabber et al. discovered that the mRNA levels of Sox9 and Ptbp1 increased when miR-124 expression decreased due to mimicking miR-124 transfection in hair follicle stem cells. It suggests that miR-124 suppresses Sox9 and Ptbp1, which in turn increases Ptbp2 and supports neuron-specific formation (25). Similarly, in this study, we observed that Ptbp1, Ptbp2 and Ctdsp1 mRNA expression levels decreased in miR-124* E7.5 (Fig. 8A). Ptbp1 and Ptbp2 genes, which are highly targeted by miR-124-3p, play a role in splicing mechanisms in miR-124* mice and contribute to significant phenotype and behavioural differences.
All these findings suggest that mice with a large phenotype in both prenatal and postnatal periods due to miR-124 microinjection are more sensitive to a growth factor such as EGF. It primarily affects epithelial cells, whereas Gh affects general growth and development in various tissues and organs. Nevertheless, they have similar properties, especially in the early embryonic period. Both EGF and Gh play important roles in regulating cellular processes and development during early embryogenesis (50, 51). While their functions are not entirely overlapping, there are some similarities in their effects during this stage. EGF and Gh are growth factors that promote cell division and proliferation in different cell types, contributing to overall embryonic growth. They also play crucial roles in the development and formation of organs during early embryogenesis(50). By regulating gene expression, they contribute to the complex orchestration of early embryonic processes and are involved in the development and function of the trophoblast (52).
The body growth pathway starts with Ghrh stimulating the release of Gh, which acts on target tissues through Ghr (53, 54). Gh also induces the production of Igf-1, which binds to Igf-1r and activates downstream signalling pathways. Igfbp1 and Igfbp5 regulate the availability and activity of Igf-1. The overall coordination of these factors and their signalling pathways play a vital role in regulating body growth, especially during the development and growth phases (54). Grandjean et al. showed that miR-124* embryos did not overexpress Gh, Igf1, Igf2 and their receptors at the early embryonic stage (5). In contrast, mRNA expressions of Ghrh, Gh, Ghr, Igf, Igf-1, Igfbp5, and Igfbp1 (p = 0.0132) increased, but Igf-1r decreased in miR-124* E7.5 day-old embryos in this study (Fig. 8B).