The Development And Asymmetry Of Fetal Frontal Lobe Structure And Protein Expression In Early And Middle Pregnancy

Background:The frontal lobe,located at the top and front of each cerebral hemisphere,is the largest part of the human brain.It is involved in executive control and planning,as well as various functions such as response inhibition,working memory,behavioral motivation,and high-level motor control.For hundreds of years,research has shown that there is a subtle structural asymmetry between the left and right hemispheres of the human brain.This inherent brain asymmetry is crucial for maintaining normal physiological functions of the nervous system.One of the significant features of structural brain asymmetry is the phenomenon of Yakovlevian torque,also referred to as brain torque.It is a geometric distortion resulting from hemispheric asymmetry,characterized by an overall counterclockwise twisting tendency of the brain around the dorsoventral axis.This can cause the broader right frontal lobe to shift leftward,while the left occipital pole bends to the right.Therefore,the frontal lobe is one of the earliest observed macroscopic brain structures with structural asymmetry,and this asymmetry can well reflect the structural and functional differences between the left and right hemispheres of the brain.Moreover,it is known that a series of neurological and psychiatric disorders are related to changes in frontal lobe asymmetry.The study of frontal lobe asymmetry can provide a more comprehensive and in-depth cognitive neuroscience theory,which is of great importance for the diagnosis and treatment of neurological and psychiatric disorders.The structural asymmetry of the frontal lobe is evident throughout the lifespan,and extensive research has investigated the asymmetry of this region after birth.However,limitations in specimen collection and ethical considerations have hampered studies on the structural asymmetry of the frontal lobe before birth,with existing research mainly focusing on the middle and third trimesters of pregnancy.A comprehensive understanding of the asymmetric development of the frontal lobe before birth can provide fundamental support for more in-depth research.The early and second trimesters of pregnancy,as the basis for subsequent development,are of particular importance for research in this area.Nevertheless,only a small number of studies have explored the development and asymmetry of the fetal frontal lobe in the early and middle stages of development,and they lack studies at the molecular level,with no consistent conclusion reached thus far.Due to the limited influence of habits and environmental factors on brain development before birth,the observed asymmetry in brain structure and motor behavior during fetal development is believed to be primarily regulated by asymmetric genetic developmental processes.Previous studies have shown that gene expression asymmetry already exists in the human spinal cord and hindbrain by the fourth week after fertilization,which may indicate a well-regulated genetic program to manage asymmetrical brain development before birth.Existing studies on fetal brain asymmetry mainly use transcriptomic techniques,but protein abundance differences between different brain regions are typically higher than RNA-level differences and cannot be reliably predicted by changes in mRNA encoding for proteins.Compared to transcriptomics,proteomics data can provide direct information on protein composition and functional status,and can directly address many biological questions by revealing the abundance of certain proteins in organisms.Therefore,exploring the development and asymmetry of the fetal frontal lobe at the protein level is necessary.However,there are currently very few studies on protein expression asymmetry in the human brain,with almost no studies focused on the frontal lobe.Little is known about the potential molecular mechanisms underlying frontal lobe structural asymmetry,and there is a lack of quantitative measurements and histological descriptions of frontal lobe asymmetry during early and mid-fetal development.There may be a series of proteins with expression-dependent asymmetric phenotype correlations during early development,which are key to understanding the formation of frontal lobe asymmetry in the future.To address the aforementioned issues,this study aims to investigate the changes and asymmetry of the fetal frontal lobe macrostructure and protein expression during early to middle development.The study collected fetal specimens and evaluated the expression of proteins related to the asymmetric development of the frontal lobe.The ultimate goal is to identify the asymmetric expression proteins associated with the macrostructural asymmetry of the human frontal lobe,laying the foundation for early diagnosis,treatment,and biomarker research of neuropsychiatric diseases related to asymmetric changes.The study is divided into three parts:1.Development and asymmetry of fetal neocortical laminar structure in early and middle pregnancy;2.Changes in and asymmetry of the proteome in the human fetal frontal lobe in early and middle pregnancy;3.Correlation between fetal frontal lobe volume asymmetry and protein expression in early and middle pregnancy.Objective:1.To explore the regional specific development pattern and asymmetry of fetal neocortical laminar structure from early to middle pregnancy,in order to enrich the early brain research of human life and provide reference for the development of normal fetal neocortex and disease diagnosis.2.To investigate the proteomic differences in bilateral frontal lobes of early to mid-term fetuses,focusing on protein expression changes and asymmetry in fetal development,and to explore the genetic mechanisms underlying typical frontal lobe development and inherent asymmetry.3.To explore the correlation between protein expression asymmetry and macroscopic structural asymmetry of human frontal lobes by analyzing the proteomics data and asymmetric volume index of bilateral frontal lobes in early and mid-term fetal specimens.Materials and Methods:Part Ⅰ:Twenty-four fetal specimens aged between 10 and 18 gestational weeks were collected,and fetal brain T2-weighted images were obtained by magnetic resonance imaging.The 3D Slicer software was used to manually segment the neocortex of the fetal brain,which was divided into two parts:marginal zone and cortical plate layer(MZ-CP layer),subcortical plate and intermediate zone layer(SP-IZ layer).The volume and surface area were measured,and the changes in the neocortical laminar structure with age and side differences were analyzed.The deformation vector values of each sampling point of the fetal neocortical MZ-CP and SPIZ were calculated using shape analysis,and the regional specificity and side differences of the morphology of the laminar structure during development were calculated by multiple regression analysis.Part Ⅱ:Four specimens of fetuses aged 9 to 17 weeks were collected,and their protein expression data were obtained using label-free quantitative proteomics technology.Differential expression proteins between gestational weeks and asymmetrically expressed proteins in each gestational week were screened,and their functional enrichment was analyzed.The protein interaction network was analyzed,and the developmental pattern of bilateral frontal lobe proteins was explored through short time-series expression analysis.Targeted proteomics technology was used to validate the expression of asymmetrically expressed proteins in another specimen of 13-week fetus.Part Ⅲ:Thirty fetal specimens aged 10 to 18 gestational weeks were collected and divided into three developmental stages:early,mid-early,and mid-term fetal stages.Twentyfour specimens were used for the first part of the study,and the remaining six specimens were excluded due to unclear boundaries of the laminar structure.Fetal brain T2WI was obtained by magnetic resonance imaging,and the frontal lobes were manually segmented using 3D Slicer software to calculate the volume and volume asymmetric index.Linear regression was performed to analyze the change pattern of the volume and volume asymmetric index with age.The protein expression asymmetry index of each protein in bilateral frontal lobes was calculated using the proteomics data of fetal specimens at 11,13,and 17 gestational weeks obtained in Chapter 2.Pearson correlation analysis was conducted between the protein expression asymmetry index and the volume asymmetric index in the three developmental stages.The significant proteins were further analyzed by protein interaction network analysis,GO functional enrichment,and KEGG pathway enrichment analysis.Results:Part Ⅰ:From 10 to 18 gestational weeks,the absolute surface area,relative surface area,and absolute volume of bilateral MZ-CP and SP-IZ showed linear growth with increasing gestational age,while the relative volume of MZ-CP showed linear reduction.Regional specificity of fetal neocortical development was found in the shape analysis,and the expansion rate of some areas in the parietal and temporal lobes was faster than in other brain lobes with increasing age.Meanwhile,similar to the postnatal brain torque phenomenon,fetal MZ-CP was deformed to the right near the frontal pole,and SP-IZ was deformed to the right near the frontal pole and to the left near the occipital pole.Part Ⅱ:A total of 1441 proteins with repeated expression were identified and quantified in the frontal lobe samples of fetuses aged 9 to 17 weeks.The expression patterns of these proteins varied between different hemispheres as gestational age changed.During this period,the number of differentially expressed proteins between gestational weeks increased,and the number of asymmetrically expressed proteins between hemispheres first decreased and then increased.Four proteins with 1.5-fold asymmetrical expression differences in all four gestational week samples were repeatedly identified.Part Ⅲ:The asymmetric volume index of the frontal lobes decreased linearly with age from 10 to 18 gestational weeks.Pearson correlation analysis revealed 66 proteins significantly correlated with the asymmetric volume index of the frontal lobes.These proteins had a significant protein interaction network and were significantly enriched in the splicing pathway.The top three significant GO biological processes were mRNA metabolism,mRNA splicing,via spliceosome,and RNA splicing.Conclusion and innovation:This study provides earlier and more detailed information on the developmental changes and asymmetry of the cortical plate structure in fetuses aged 10 to 18 gestational weeks,and provides a basis for identifying early and mid-term brain development abnormalities.This study obtained the youngest human frontal lobe proteomic dataset,which helps to determine the complex protein expression patterns in normal frontal lobe development and provides a reference for the study of diagnostic markers for brain asymmetry-related neurological and psychiatric disorders.This study identified a set of proteins related to the asymmetrical development of the frontal lobe in early to mid-term fetuses,further confirming that the inherent asymmetry of the frontal lobe is regulated by the expression of a large number of proteins.This lays the foundation for the early diagnosis,treatment,and biomarker research of certain asymmetry-related neurological and psychiatric disorders.