Developmental Biology 233, 305–318 (2001) doi:10.1006/dbio.2001.0243, available online at http://www.idealibrary.com on Developmental Expression of Mouse Kru¨ppel-like Transcription Factor KLF7 Suggests a Potential Role in Neurogenesis Friedrich Laub,*,1 Rafael Aldabe,*,1 Victor Friedrich, Jr.,* Shunsuke Ohnishi,† Teruhiko Yoshida,† and Francesco Ramirez* *Brookdale Center, Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine–New York University, One Gustave L. Levy Place, New York, New York 10029; and †Genetics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan To identify potential functions for the Kru¨ppel-like transcription factor KLF7, we have determined the spatiotemporal pattern of gene expression during embryogenesis and in the adult organism. We show that the profile of Klf7 expression predominantly involves the central and peripheral nervous systems and is broadly identified by three separate phases. The first phase occurs early in embryogenesis with increasingly strong expression in the spinal cord, notably in motor neurons of the ventral horn, in dorsal root ganglia, and in sympathetic ganglia. The second robust phase of Klf7 expression is confined to the early postnatal cerebral cortex and is downregulated thereafter. The third phase is characterized by high and sustained expression in the adult cerebellum and dorsal root ganglia. Functionally, these three phases coincide with establishment of neuronal phenotype in embryonic spinal cord, with synaptogenesis and development of mature synaptic circuitry in the postnatal cerebral cortex, and with survival and/or maintenance of function of adult sensory neurons and cerebellar granule cells. Consistent with Klf7 expression in newly formed neuroblasts, overexpression of the gene in cultured fibroblasts and neuroblastoma cells repressed cyclin D1, activated p21, and led to G1 growth arrest. Based on these data, we argue for multiple potential functions for KLF7 in the developing and adult nervous system; they include participating in differentiation and maturation of several neuronal subtypes and in phenotypic maintenance of mature cerebellar granule cells and dorsal root ganglia. © 2001 Academic Press Key Words: cerebellum; Kru¨ppel-like factor; motor neurons; mouse nervous system; neocortex; neurogenesis; neuronal differentiation; sensory ganglia; zinc finger protein. INTRODUCTION several peptide motifs that transcription factors use to contact DNA; the composition, number, and organization The organization and differentiation of vertebrate organs of the zinc fingers segregate zinc finger proteins into dis- are generally defined as the process whereby cells progres- tinct groups (Klug and Schwabe, 1995). One of them is sively restrict developmental potential while acquiring characterized by three highly conserved zinc fingers of the lineage-specific patterns of gene expression in response to C2H2 type and additional sequence homology to the Dro- environmental signals. Implicit in this definition is the sophila segmentation gene product Kru¨ppel (Turner and concept that, irrespective of the underlying mechanism, Crossley, 1999). This particular group of Kru¨ppel-like fac- transcriptional regulation is central to the control of the tors (aka KLFs) includes 12 different members (KLFs 1–12), whole process. Transcriptional regulation of eukaryotic in addition to Sp1 and the related proteins Sp2–Sp4 (Turner genes often involves combinatorial interactions among and Crossley, 1999). ubiquitous, restricted, and/or cell type-specific nuclear pro- The founding member of the KLF group is EKLF (aka teins (Lemon and Tjian, 2000). The zinc finger is one of the KLF1), so named because it was originally isolated as an erythroid-specific product (Miller and Bieker, 1993). Ho- 1 These authors contributed equally to the work. mologous gene targeting in the mouse later corroborated 0012-1606/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 305 306 Laub et al. FIG. 1. Alignment of the deduced amino acid sequences of human and mouse KLF7 (hKLF7 and mKLF7). Identical residues are boxed in dark gray and conservative changes in light gray. Below is a schematic representation of the protein domains with the amino acid reference at the points of transition. Abbreviation: NLS, nuclear localization signal. Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. KLF7 Expression in Neuronal Tissues 307 FIG. 2. Characterization of the Klf7 gene. (a) Expression of Klf7 in adult mouse tissues. (b) Chromosomal localization of the mouse Klf7 gene by fluorescence in situ hybridization (FISH) analysis. In the top, two bright signals are visible at corresponding positions on the sister chromatids of chromosome 1; below are the same mitotic figures stained with DAPI and at the bottom, a diagram that summarizes the mapping results with each dot representing a double FISH signal on chromosome 1. Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 308 Laub et al. FIG. 3. Klf7 expression during E9.5–E10.5 mouse embryogenesis. (a) Klf7 expression can be seen in the head, trigeminal ganglia (t), and geniculate ganglia (g) and then progresses in a rostral–caudal direction to include the emerging vestibulocochlear (v), petrosal (p), and nodose ganglia (n) and to the forming dorsal root ganglia (drg) and the neural tube (nt). (b) (Left) Klf7 expression at E11.5 includes the emerging superior (s), jugular (j), and accessory (a) ganglia; additionally, there is intense expression in the entire fore- (fb), mid- (mb), and hindbrain (hb) regions. (Middle) dorsal view of embryo shown on the left. (Right) Cross section of the embryo shown in the other views. Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. KLF7 Expression in Neuronal Tissues 309 this notion by documenting the absolute requirement of nal function by participating in maintenance of postmitotic KLF1 for onset of adult ␤-globin production and liver phenotype. erythropoiesis (Nuez et al., 1995; Perkins et al., 1995). The former activity is mediated through binding to a G/C-rich promoter element, also known as the CACCC box (Miller MATERIALS AND METHODS and Bieker, 1993). The same genetic approach has provided insights into the function of KLFs 2, 3, and 4 as well. Loss Cloning and Mapping of the Mouse Klf7 Gene of the Klf2 gene is associated with abnormalities in the A mouse 11.5-day embryonic (E11.5) cDNA library in the ␭gt11 program of naı¨ve T cell quiescence and survival, as well as vector (Clontech, Palo Alto, CA) was screened with the 680-bp impaired development of the lung and vascular system (Kuo human KLF7 cDNA fragment that mostly corresponds to the 5Ј et al., 1997a,b; Wani et al., 1999); ablation of Klf3 leads to a untranslated region (UTR) of the message under conditions of myeloproliferative disease (Perkins et al., 1998), and mice reduced stringency (Ohnishi et al., 2000). The resulting positive without the Klf4 gene have altered skin integrity, due to clone was used for subsequent rounds of screening that ultimately late-stage lesion(s) in keratinocyte differentiation (Segre et yielded overlapping cDNAs that cover the entire length of the al., 1999). In vitro studies, on the other hand, have associ- coding sequence and portions of the UTRs. The coding sequence of ated serum stimulation of cultured fibroblasts with higher the mouse Klf7 gene has been deposited with GenBank under Klf4 expression in growth-arrested compared to exponen- Accession No. AF327568. Northern analysis of adult mouse tissues tially growing cells (Shields et al., 1996). Consonant with was performed using the cDNA fragments that codes for the transactivation domain of the protein and a poly(A)ϩ RNA- this finding, KLF4 overexpression represses cyclin D1 and containing filter (Mouse Multiple Tissue Northern; Clontech). The induces the cyclin-dependent kinase inhibitor p21 in a mouse cDNAs were employed to isolate two nonoverlapping p53-dependent manner; in both cases, the effect is mediated clones from a 129/sv genomic library constructed in the Lambda by KLF4 binding to G/C-rich promoter elements (Shie et al., DASH phage vector (Andrikopoulos et al., 1995). Selected sequenc- 2000; Zhang et al., 2000). A similar antiproliferative effect ing of the clones identified the exon encoding the 5Ј UTR and has been reported for KLF11 overexpression in CHO cells transactivation domain (exon 1) in one of them and the exon (Cook et al., 1998). encoding the remaining non-zinc finger portion of the protein (exon Structural features have segregated the KLF family of 2) in the other. The exon 1-containing clone was chosen to map the proteins into evolutionarily related subgroups, one of which gene using the fluorescence in situ hybridization (FISH) procedure includes KLFs 6 and 7 (Matsumoto et al., 1998; Turner and performed by a commercial firm (SeeDNA, North York, Ontario, Canada) according to the published protocol (Heng et al., 1992). Crossley, 1999). The amino acid sequences of the zinc fingers of KLFs 6 and 7 exhibit 84% homology, whereas the amino-termini are nearly identical (only four conservative In Situ Hybridizations substitutions in 47 residues) and similarly function as In situ hybridizations were carried out on whole-mount embryos et transactivators in cell transfection assays (Koritschoner and on tissue sections from staged embryos and adult animals al., 1997; Matsumoto et al., 1998).