DOI: 10.1002/pd.4322
ORIGINAL ARTICLE
Differential changes in gene expression in human brain during late first trimester and early second trimester of pregnancy J. I. Iruretagoyena1*, W. Davis2, C. Bird1, J. Olsen2, R. Radue3, A. Teo Broman4, C. Kendziorski4, S. Splinter BonDurant2, T. Golos5, I. Bird6 and D. Shah1 1
Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI, USA Gene Expression Center, University of Wisconsin, Madison, WI, USA 3 University of Wisconsin, School of Medicine and Public Health, Madison, WI, USA 4 Department of Biostatistics and Medical Informatic, University of Wisconsin, Madison, WI, USA 5 National Primate Research Center and Department of Comparative Biosciences, University of Wisconsin, Madison, WI, USA 6 Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI, USA *Correspondence to: J. Igor Iruretagoyena. E-mail: [email protected] 2
ABSTRACT Objective This study aimed to describe brain development during the first (B1) and second trimester (B3) in human fetuses. Design Ten brains from 10 to 18 weeks of gestational age (GA) were collected, and the RNA was used for transcriptome analysis (Affymetrix 1.0 ST microarray chip). Differences in brain development within 10 to 18 GA were investigated by dividing the sample into 10 to 12 (B1), 13 to 15(B2) and 16 to 18(B3) weeks. A fold change of 2 or above, with a false discovery rate of 5%, was used as cut-off to determine differential gene expression for individual genes. Quantitative real-time PCR was used to confirm differences. Tests for enrichment procedures (using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) were then used to identify functional groups of mRNA. Results At 10 to 12 weeks, brains showed neuronal migration to be upregulated. From 10 to 18 weeks, brains showed genes coding for neuronal migration, differentiation and connectivity upregulated. ALDH1A1 and NPY genes, marker of spinal cord and striatum, were upregulated in B1 and B3 brains, respectively. Also, SLITRK6-HAS2 and CRYAB-PCDH18 genes for ear and eye sensory input were upregulated in B1.
Conclusions For the first time, brain global gene expression was described in human samples. Period B1 was dominated by genes coding for neuronal migration, differentiation, programmed cell death and sensory organs. B3 was dominated by neuronal proliferation, branching and myelination. Creating such a database will allow comparison with abnormals in future studies. © 2014 John Wiley & Sons, Ltd.
Funding sources: None Conflicts of interest: None declared
INTRODUCTION The central nervous system starts developing from the ectoderm in a sequential process around the third week of embryonic life. During this time, the neural tube and the neural crest become the first recognizable structures formed from the ectoderm. The neural tube then differentiates into the central nervous system and the neural crest into the peripheral nervous system including nerves and ganglia.1 The neural tube begins as an open structure with a cranial and caudal pole. By the end of the fourth week of gestation, the poles close and the cranial pole is divided into the prosencephalon, mesencephalon and rhombencephalon.1 Little is known about the active biological processes and cellular signaling leading to the central nervous system
Prenatal Diagnosis 2014, 34, 431–437
development. Most of the current data come from animal models. Few studies have been carried out using human fetal brain. Bystron2 evidenced the presence of a primordial neuronal-type cell in the fetal cortex by embryonic days 12–21. Mrzljak3 studied the neural development specifically in the dorsolateral and lateral prefrontal cortex of human fetal brain trying to elucidate the neuronal differentiation and development. NeuroD6, a neurogenic transcription factor important in neuronal differentiation, was interrogated in our cohort. De Graaf4 showed that neuronal migration and proliferation were among the earliest events in brain development and that synapse formation and glial cell proliferation, which begin in the second half of pregnancy, were among the later events. We studied genes coding for
© 2014 John Wiley & Sons, Ltd.
J. I. Iruretagoyena et al.
432
fibronectin, laminin, desmoplakin, fatty acid binding protein, integrin, Sparc and dexilin, among others, all involved in energy, microtubule construction system and cell to cell interaction. Letzen5 described the expression profile of the oligodendrocyte during the embryonic period in an effort to characterize glial cell maturational processes. In an effort to describe the contribution of the glial cell component in brain development, this study looked at the ratio between glial to neuronal gene ontology processes at different stages of fetal development. Mpzl2, a gene involved in myelination, is an important marker for glial cell component investigated in this research. These experiments tried to establish neuronal migration in early development. Trivedi6 have shown cellular migration during the embryonic period in human fetuses. Other authors, like Redmond et al.,7 have described the pathway of migration using a monkey model. In this microarray study, the aim is to show fetal human brain development, in a gestational age, further along than previous experiments have carried out, using human and animal models. The purpose of this study is to characterize pathways of normal brain development, during fetal rather than embryonic life. These findings would serve to generate hypotheses and confirm previous findings in animal models not only in cell migration but also in differentiation, growth and metabolism. Describing the active processes present during early fetal life would also create a template for comparing findings in fetal central nervous system pathology. This could not only aid in understanding the developmental stages of the brain but also potentially lead to new strategies of intervention.
MATERIAL AND METHODS Tissue collection and RNA isolation Human fetal brain tissue was collected after written informed consent following the University of Wisconsin Institutional Review Board (IRB) guidelines, from patients older than 18 years undergoing an elective dilation and curettage for termination of pregnancy due to unwanted pregnancy between 10 and 18 weeks of gestational age. Patients had already undergone the surgical consent process with their health team before they were approached to donate fetal tissue samples under an IRB approved protocol. Those who expressed willingness for such research tissue donation then underwent a research written informed consent process monitored and approved by the University of Wisconsin IRB. All pregnancies had a dating ultrasound carried out within a week of the procedure. The suction was generated by a vacuum with a standard suction unit providing variable controlled suction up to 550 mmHg and a suction flow rate of 30 lpm. Before the procedure, collection bottles were placed at 4 °C for 1 h. At the time of the procedure, 100 mL (at 4 °C), RNAsefree water solution (BP561-1 Water, Sterile for RNA work, Fisher Scientific, USA) mixed with 10× phosphate buffered saline (Ambion, USA) at a final concentration of 10%, was added to the collecting bottle. The procedure was performed within 5 min of adding the cold water. After the procedure, the brain was grasped with sterilized surgical instruments and carefully isolated to be Prenatal Diagnosis 2014, 34, 431–437
placed in RNAse-free plastic tubes. Fetal heart was also collected in the same fashion. No karyotype was carried out to confirm normal chromosomes. The heart tissue was used for comparison looking for major differences among the study group and the heart group as an internal validation. These tubes were immediately placed in liquid nitrogen for transport. The tissue was then stored in a 80 °C freezer. Tissue RNA was extracted using RNA STAT-60 reagents, as recommended by the manufacturer (Tel-Test, Inc., Friendswood, TX, USA). All procedures were run on ice. A starting ratio of 1 mL reagent/100 mg tissue was used to ensure efficient, intact RNA recovery. RNA concentration and purity were initially determined using spectrophotometric A260 and A260/280 ratios (NanoDrop Products, Thermo Fisher Scientific Inc., Wilmington, DE, USA). RNA quantification and quality control were performed using the Agilent Bioanalyzer (Agilent Technology, Santa Clara, CA, USA) 260/280 ratios and standard RIN-values (RNA integrity number). A value of 1.7 to 2.1 was considered workable for further testing.
Array preparation Ten brain RNA samples were divided into three Affymetrix ST 1.0 arrays according to gestational age. Array #1 analyzed four brain RNA samples from 10 to 12 weeks of gestation. Array #2 analyzed four brain RNA samples from 13 to 15 weeks of gestation, and Array #3 analyzed two brain RNA samples from 16 to 18 weeks of gestation. Ten heart samples from the same fetuses were used as internal controls. cDNA was synthesized from extracted RNA (300 ng) using the Ambion WT Expression Kit (Expression Kit Manual P/N 4425209 Rev. C). cDNA yield quantity and quality were assessed using NanoDrop Spectrophotometer (NanoDrop Products, Thermo Fisher Scientific Inc., Wilmington, DE, USA) and Agilent Bioanalyzer (Agilent Technology, Santa Clara, CA, USA). These samples were then further tested by cDNA generation for hybridization. cDNA was checked for yield and size distribution using a NanoDrop Spectrophotometer. A fragmentation check revealed a cDNA
ORIGINAL ARTICLE
Differential changes in gene expression in human brain during late first trimester and early second trimester of pregnancy J. I. Iruretagoyena1*, W. Davis2, C. Bird1, J. Olsen2, R. Radue3, A. Teo Broman4, C. Kendziorski4, S. Splinter BonDurant2, T. Golos5, I. Bird6 and D. Shah1 1
Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI, USA Gene Expression Center, University of Wisconsin, Madison, WI, USA 3 University of Wisconsin, School of Medicine and Public Health, Madison, WI, USA 4 Department of Biostatistics and Medical Informatic, University of Wisconsin, Madison, WI, USA 5 National Primate Research Center and Department of Comparative Biosciences, University of Wisconsin, Madison, WI, USA 6 Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI, USA *Correspondence to: J. Igor Iruretagoyena. E-mail: [email protected] 2
ABSTRACT Objective This study aimed to describe brain development during the first (B1) and second trimester (B3) in human fetuses. Design Ten brains from 10 to 18 weeks of gestational age (GA) were collected, and the RNA was used for transcriptome analysis (Affymetrix 1.0 ST microarray chip). Differences in brain development within 10 to 18 GA were investigated by dividing the sample into 10 to 12 (B1), 13 to 15(B2) and 16 to 18(B3) weeks. A fold change of 2 or above, with a false discovery rate of 5%, was used as cut-off to determine differential gene expression for individual genes. Quantitative real-time PCR was used to confirm differences. Tests for enrichment procedures (using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) were then used to identify functional groups of mRNA. Results At 10 to 12 weeks, brains showed neuronal migration to be upregulated. From 10 to 18 weeks, brains showed genes coding for neuronal migration, differentiation and connectivity upregulated. ALDH1A1 and NPY genes, marker of spinal cord and striatum, were upregulated in B1 and B3 brains, respectively. Also, SLITRK6-HAS2 and CRYAB-PCDH18 genes for ear and eye sensory input were upregulated in B1.
Conclusions For the first time, brain global gene expression was described in human samples. Period B1 was dominated by genes coding for neuronal migration, differentiation, programmed cell death and sensory organs. B3 was dominated by neuronal proliferation, branching and myelination. Creating such a database will allow comparison with abnormals in future studies. © 2014 John Wiley & Sons, Ltd.
Funding sources: None Conflicts of interest: None declared
INTRODUCTION The central nervous system starts developing from the ectoderm in a sequential process around the third week of embryonic life. During this time, the neural tube and the neural crest become the first recognizable structures formed from the ectoderm. The neural tube then differentiates into the central nervous system and the neural crest into the peripheral nervous system including nerves and ganglia.1 The neural tube begins as an open structure with a cranial and caudal pole. By the end of the fourth week of gestation, the poles close and the cranial pole is divided into the prosencephalon, mesencephalon and rhombencephalon.1 Little is known about the active biological processes and cellular signaling leading to the central nervous system
Prenatal Diagnosis 2014, 34, 431–437
development. Most of the current data come from animal models. Few studies have been carried out using human fetal brain. Bystron2 evidenced the presence of a primordial neuronal-type cell in the fetal cortex by embryonic days 12–21. Mrzljak3 studied the neural development specifically in the dorsolateral and lateral prefrontal cortex of human fetal brain trying to elucidate the neuronal differentiation and development. NeuroD6, a neurogenic transcription factor important in neuronal differentiation, was interrogated in our cohort. De Graaf4 showed that neuronal migration and proliferation were among the earliest events in brain development and that synapse formation and glial cell proliferation, which begin in the second half of pregnancy, were among the later events. We studied genes coding for
© 2014 John Wiley & Sons, Ltd.
J. I. Iruretagoyena et al.
432
fibronectin, laminin, desmoplakin, fatty acid binding protein, integrin, Sparc and dexilin, among others, all involved in energy, microtubule construction system and cell to cell interaction. Letzen5 described the expression profile of the oligodendrocyte during the embryonic period in an effort to characterize glial cell maturational processes. In an effort to describe the contribution of the glial cell component in brain development, this study looked at the ratio between glial to neuronal gene ontology processes at different stages of fetal development. Mpzl2, a gene involved in myelination, is an important marker for glial cell component investigated in this research. These experiments tried to establish neuronal migration in early development. Trivedi6 have shown cellular migration during the embryonic period in human fetuses. Other authors, like Redmond et al.,7 have described the pathway of migration using a monkey model. In this microarray study, the aim is to show fetal human brain development, in a gestational age, further along than previous experiments have carried out, using human and animal models. The purpose of this study is to characterize pathways of normal brain development, during fetal rather than embryonic life. These findings would serve to generate hypotheses and confirm previous findings in animal models not only in cell migration but also in differentiation, growth and metabolism. Describing the active processes present during early fetal life would also create a template for comparing findings in fetal central nervous system pathology. This could not only aid in understanding the developmental stages of the brain but also potentially lead to new strategies of intervention.
MATERIAL AND METHODS Tissue collection and RNA isolation Human fetal brain tissue was collected after written informed consent following the University of Wisconsin Institutional Review Board (IRB) guidelines, from patients older than 18 years undergoing an elective dilation and curettage for termination of pregnancy due to unwanted pregnancy between 10 and 18 weeks of gestational age. Patients had already undergone the surgical consent process with their health team before they were approached to donate fetal tissue samples under an IRB approved protocol. Those who expressed willingness for such research tissue donation then underwent a research written informed consent process monitored and approved by the University of Wisconsin IRB. All pregnancies had a dating ultrasound carried out within a week of the procedure. The suction was generated by a vacuum with a standard suction unit providing variable controlled suction up to 550 mmHg and a suction flow rate of 30 lpm. Before the procedure, collection bottles were placed at 4 °C for 1 h. At the time of the procedure, 100 mL (at 4 °C), RNAsefree water solution (BP561-1 Water, Sterile for RNA work, Fisher Scientific, USA) mixed with 10× phosphate buffered saline (Ambion, USA) at a final concentration of 10%, was added to the collecting bottle. The procedure was performed within 5 min of adding the cold water. After the procedure, the brain was grasped with sterilized surgical instruments and carefully isolated to be Prenatal Diagnosis 2014, 34, 431–437
placed in RNAse-free plastic tubes. Fetal heart was also collected in the same fashion. No karyotype was carried out to confirm normal chromosomes. The heart tissue was used for comparison looking for major differences among the study group and the heart group as an internal validation. These tubes were immediately placed in liquid nitrogen for transport. The tissue was then stored in a 80 °C freezer. Tissue RNA was extracted using RNA STAT-60 reagents, as recommended by the manufacturer (Tel-Test, Inc., Friendswood, TX, USA). All procedures were run on ice. A starting ratio of 1 mL reagent/100 mg tissue was used to ensure efficient, intact RNA recovery. RNA concentration and purity were initially determined using spectrophotometric A260 and A260/280 ratios (NanoDrop Products, Thermo Fisher Scientific Inc., Wilmington, DE, USA). RNA quantification and quality control were performed using the Agilent Bioanalyzer (Agilent Technology, Santa Clara, CA, USA) 260/280 ratios and standard RIN-values (RNA integrity number). A value of 1.7 to 2.1 was considered workable for further testing.
Array preparation Ten brain RNA samples were divided into three Affymetrix ST 1.0 arrays according to gestational age. Array #1 analyzed four brain RNA samples from 10 to 12 weeks of gestation. Array #2 analyzed four brain RNA samples from 13 to 15 weeks of gestation, and Array #3 analyzed two brain RNA samples from 16 to 18 weeks of gestation. Ten heart samples from the same fetuses were used as internal controls. cDNA was synthesized from extracted RNA (300 ng) using the Ambion WT Expression Kit (Expression Kit Manual P/N 4425209 Rev. C). cDNA yield quantity and quality were assessed using NanoDrop Spectrophotometer (NanoDrop Products, Thermo Fisher Scientific Inc., Wilmington, DE, USA) and Agilent Bioanalyzer (Agilent Technology, Santa Clara, CA, USA). These samples were then further tested by cDNA generation for hybridization. cDNA was checked for yield and size distribution using a NanoDrop Spectrophotometer. A fragmentation check revealed a cDNA
