Genetic Analysis of Anterior–Posterior Expression Gradients in the Developing Mammalian Forebrain

Cerebral Cortex September 2007;17:2108--2122 doi:10.1093/cercor/bhl118 Advance Access publication December 05, 2006 Genetic Analysis of Anterior--Posterior Expression Gradients in the Developing Mammalian Forebrain Keywords: cortical development, Fhl1, Fhl2, gene expression, microarray, protomap Introduction There has been significant progress in our understanding of the processes of regional patterning in the cerebral cortex during embryogenesis. The now classic and enduring model for cortical regionalization is the ‘‘protomap’’ model (Rakic 1988) in which the cells derived from neural progenitors have distinct intrinsic identity that drives regionalization. A growing body of data demonstrates that intrinsic gradients of homeobox transcription factors, such as Emx2 and Pax6, and other signaling molecules play a critical role in cortical regionalization (Miyashita-Lin et al. 1999; Bishop et al. 2000, 2002; Mallamaci et al. 2000; Muzio et al. 2002a,b; Muzio and Mallamaci 2003; Sansom et al. 2005). Otx1 and Otx2 provide further examples of homeobox transcription factors with restricted expression patterns that are involved in proper cortical organization and development (Acampora, Avantaggiato, et al. 1999; Acampora, Barone, et al. 1999). The identification of genes, many of which are transcription factors, with distinct temporal and spatial gradients in the developing telencephalon show that there are intrinsic mechanisms controlling early cortical regionalization  The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 1 Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA, 2 Institute of Molecular Medicine, Department of Medicine, University of California at San Diego, School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0613, USA, 3Vanderbilt Kennedy Center for Research on Human Development and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37203, USA and 4Department of Human Genetics, David Geffen School of Medicine, UCLA, Los Angeles, CA and Program in Neurobehavioral Genetics, and Center for Autism Research, Semel Institute for Neuroscience and Behavior, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA and that the pattern of gene expression is functionally relevant (Levitt et al. 1997; Miyashita-Lin et al. 1999). To further define other factors that show spatial gradients during the period of cortical regionalization, we used a genomic screening approach based on custom complementary DNA (cDNA) microarrays to assess gene expression gradients during corticogenesis. We chose to study gene expression at E12.5, a time during which time intrinsic signals are known to direct early telencephalic patterning (Barbe and Levitt 1991; Arimatsu et al. 1992; De Carlos and O’Leary 1992; Cohen-Tannoudji et al. 1994). We identified 106 genes with consistent and significant (P < 0.01) anteroposterior (A-P) gene gradients. We analyzed the majority of identified gene expression patterns (n = 88) using in situ hybridization and reverse transcription--polymerase chain reaction (RT-PCR) and confirmed a gradient of telencephalic expression for a subset of genes. We also investigated the role of one of these genes, Fhl1, a transcription factor without a previous known role in cortical development, in vivo (Morgan et al. 1995; Morgan and Madgwick 1996). We were unable to demonstrate a clear anterior--posterior patterning phenotype in Fhl1 knockouts, suggesting either redundancy with other close family members or a role in later neuronal maturation. This gene and other transcription factors, or signaling molecules identified here clearly provide an important group of genes for future study of cortical development. Materials and Methods Dissections and RNA Preparation Anterior, middle, and posterior regions of the dorsal pallium, the forerunners of frontal parietal and occipital cortical areas, were dissected (A, M, P) from E12.5 C57Bl6 J mice from Jackson Laboratory. Anatomical limits were chosen to increase reproducible dissection of samples. The cut between the anterior and the middle regions was made about 1--2 mm behind the middle of the ganglionic eminence because there are no surface landmarks. The posterior section is a tissue slice of approximately 2 mm above the rhinal sulcus identifiable at E12.5. Four litters were dissected resulting in 4 sets of pooled, independent replicate samples. The microdissected regions were flash frozen on dry ice and stored at –80
C. Total RNA was extracted from each of the pooled samples using TRIzol reagent (GibcoBRL, Carlsbad, CA) according to the manufacturer’s recommendations. RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA) and Agilent 2100 Bioanalyzer software were used to analyze and quantify the RNA samples. Only high-quality RNA samples were used, for example 28S:18S ratio > 1.6 and A260/280 ~2.0 (Ultrospec 2000, Pharmacia, Piscataway, NJ). Intrinsic regulatory factors play critical roles in early cortical patterning, including the development of the anteroposterior (A-P) axis. To identify genes that are differentially expressed along the A-P axis of the developing cerebral cortex, we analyzed gene expression in presumptive frontal, parietal, and occipital cerebral walls of E12.5 mouse using complementary DNA microarrays. We identified 106 genes, including expressed sequence tags (ESTs), expressed in an A-P gradient in the embryonic brain and screened 88 by in situ hybridization for confirmation. Central nervous system (CNS) expression patterns of many of these genes were previously unknown. Others, such as Sfrp1, CoupTF1, and FABP7, were expressed in a manner consistent with previous studies, providing independent confirmation. Two related transcription factors, previously not implicated in CNS development, Fhl1 and Fhl2, were observed to be enriched in posterior and anterior telencephalon, respectively. We studied patterning gradients in Fhl1 knockout mice but observed no changes in gene expression related to A-P regionalization in the Fhl1 knockout mice. These data provide an important set of new candidates for studies of cortical patterning and maturation. Lili C. Kudo1, Stanislav L. Karsten1, Ju Chen2, Pat Levitt3 and Daniel H. Geschwind1,4 Fhl1 Knockout Animals Fhl1 knockouts had been generated on a black Swiss background by inserting a LacZ sequence into the Fhl1 locus (Chu, Ruiz-Lozano, et al. 2000). Because Fhl1 is located on the X chromosome, heterozygous females were mated to wild-type males to generate Fhl1 knockout males and wild-type littermates. Genotyping of the animals was performed by using 2 sets of primers for wild-type and the knockout, for which the primers incorporate a portion of the LacZ gene. Microarrays Transcripts were analyzed using a mouse 10 000-element custom cDNA microarray printed at University of California at Los Angele (...truncated)