Developmental Expression of Mouse Kruppel-Like Transcription Factor

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

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.

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.

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). Northern blot hybridiza- (Sassoon and Rosenthal, 1993). Riboprobes included those corre- tions have documented the widespread distribution of KLF sponding to the transactivation domain and 5Ј UTR of Klf7 (probes 6 and 7 transcripts in adult human tissues, with predomi- 1 and 2), and to the NeuroD and SCG10 genes (generous gifts from nance of the former in placenta and of the latter in brain and Drs. J. Lee and D. J. Anderson, respectively) (Lee et al., 1995; Stein spinal cord (Koritschoner et al., 1997; Matsumoto et al., et al., 1988). Hybridizations were performed with both Klf7 probes, 1998). Based on these data and pending additional develop- but only representative examples obtained with probe 1 are shown mental studies, we preliminarily assigned the name here. Sense and antisense riboprobes were transcribed in vitro on UKLF—for ubiquitously expressed KLF—to what was sub- appropriately linearized DNA templates using RNA polymerases as previously described (Ohnishi et al., 2000; Sumiyoshi et al., sequently called KLF7 (Matsumoto et al., 1998; Turner and 2001). For whole-mount in situ hybridizations, staged mouse Crossley, 1999). embryos were fixed overnight in 4% paraformaldehyde, dehy- Here we report the isolation of the mouse Klf7 gene and drated, and stored in ethanol at Ϫ20°C for up to 6 months. the establishment of its expression pattern during embryo- DIG-labeled riboprobes were synthesized using a DIG-labeling kit genesis. The results indicate that Klf7 is predominantly according to the manufacturer’s recommendations (Roche, India- expressed in the central and peripheral nervous systems napolis, IN). Prior to hybridization, embryos were hydrated and (CNS and PNS) and according to three broadly identified proteinase-K treated for 10 to 25 min, depending on the develop- phases; they coincide with neuronal cell fate restriction in mental stage. After fixation in 4% paraformaldehyde/0.1% glutar- the embryonic spinal cord, with the establishment of the aldehyde, embryos were washed first in PBS/Tween 20 (PTW) and ϫ neuronal phenotype in the postnatal brain, with synapto- then in 1:1 PTW:hybridization buffer (50% formamide; 1.3 SSC, pH 5.0; EDTA, pH 8.0; 50 mg/ml yeast tRNA; 0.2% Tween 20; genesis and maintenance of the mature synaptic circuitry in 0.5% Chaps; 100 mg/ml heparin). This treatment was followed by the adult cerebellum, and survival and/or phenotypic main- another wash and by 1 h prehybridization at 65°C, both carried out tenance of dorsal root ganglia. Furthermore, we show that in hybridization buffer. Probes were denatured at 95°C for 5 min Klf7 overexpression induces growth arrest of transfected and then added to an appropriate amount of prewarmed hybridiza- cells. Based on these results, we propose that transcription tion solution at a final concentration of 0.1 to 1 ␮g/ml. Embryos factor KLF7 regulates neuroblast differentiation and neuro- were hybridized overnight at 65°C. After hybridization, embryos

were washed several times at 65°C in the hybridization buffer, then combination of the Ad5 derivative sub360 adenovirus (Aoki et al., in 1:1 hybridization solution:MABT (100 mM maleic acid; 150 mM 1999). Infection and propagation of the neuroblastoma cell line NaCl, pH 7.5; 0.1% Tween 20), and ﬁnally in MABT alone. Prior to NB-OK1 with AdKLF7 or control AdEGFP adenovirus construct incubation with the AP-anti-DIG antibody, embryos were blocked were according to the published protocol (Aoki et al., 1999). in MABT/2% Boehringer Blocking Reagent (Roche)/20% heat- Expression of mKLF7-FLAG was monitored by Western blot inactivated sheep serum. Antibody binding was performed over- analysis with polyclonal antibodies raised against the bacterially night at 4°C in fresh blocking solution and at 1:2000 antibody expressed transactivation domain of the protein. To this end, dilution. The antibody was subsequently removed by extensive cellular extracts were separated by SDS–PAGE on 10% gels and washes in MABT. Embryos were then equilibrated in NTMT transferred onto an Immobilon-P membrane (Millipore Corp., Bed-

staining buffer [100 mM NaCl, 50 mM MgCl2, 100 mM Tris–HCl ford, MA) and probed with the corresponding primary antibody. (pH 9.5), 1% Tween 20] and then covered with BMP-purple sub- Anti-Flag monoclonal M5 antibody (Sigma) was used at a 1:1000 strate (Roche) and incubated at 4°C until staining became visible. dilution; anti-p21 goat and anti-cyclin D1 rabbit antibodies were In situ hybridizations on tissue sections were performed as de- purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and scribed previously (Ohnishi et al., 2000; Sumiyoshi et al., 2000). used at 1:100 and 1:400 dilutions, respectively; anti-mKLF7 anti- Briefly, embryos and tissues were fixed in paraformaldehyde, body was used at a 1:1000 dilution; anti-p53 antibody (Pharmingen, embedded in paraffin, and sectioned at 8 ␮m. Deparaffinized San Diego, CA) was diluted 1:100; anti-actin antibody was used at sections were proteinase-K treated and incubated with [35S]UTP- a 1:1000 dilution (Sigma). Horseradish peroxidase-conjugated anti- labeled riboprobes. Slides were dipped in emulsion and exposed at goat (Santa Cruz), anti-rabbit, and anti-mouse secondary antibodies 4°C for 5 to 8 days and counterstained with hematoxylin–eosin (Amersham Pharmacia, Piscataway, NJ) were each used at 1:1000 (H&E) for bright-field views. Photographs were taken with a Zeiss dilution, followed by enhanced chemiluminescence detection Axioscope or Nikon dissecting microscope and a digital camera (Amersham Pharmacia). system. For postprocessing of the data, PhotoShop 5.0 imaging To monitor DNA replication, cells were exposed to 10 ␮M software (Adobe) was used. bromodeoxyuridine (BrdU) for 30 min, washed several times with PBS, and fixed in 70% ethanol (50 mM glycine buffer, pH 2.0) for 30 min at Ϫ20°C. Positive cells were detected using a 5-bromo-2Ј- Immunohistochemistry deoxyuridine detection kit (Kit I; Roche) according to the manu- Embryos and tissues were fixed, embedded, and sectioned as facturer’s instructions. Cells were examined in a fluorescence described above. After deparaffinization and rehydration, sections microscope and counted. For flow cytometry, cells were harvested, ␮ were treated with hydrogen peroxide to reduce background. Block- resuspended in 200 l of cold citrate buffer (0.25 M sucrose, 40 mM ␮ ing of the sections and antibody binding were performed in 10% sodium citrate, pH 7.6), added to 800 l of cold DNA staining/lysis ␮ FBS in PBS. Detection of primary antibodies was done with solution (20 g/ml propidium iodide, 0.5% NP-40, 0.5 mM EDTA biotinylated secondary antibodies (1:500 dilution) and ABC reagent in PBS calcium-, magnesium-free, pH 7.2), and analyzed in a (Vector Laboratories, Burlingame, CA). Binding of primary and FACScan analytical flow cytometer with CellQuest software in the secondary antibodies and ABC reagent were for 3 h, 60 min, and 30 Shared Resource Facility of the Mount Sinai School of Medicine min, respectively. Unbound antibodies were removed by several (Director, Dr. H. Snoeck). washes in PBS. HRP activity was visualized by a DAB/metal concentrate (Pierce, Rockford, IL). The following primary antibodies were used at the indicated dilutions: PCNA (Sigma, St. Louis, MO; 1:100), ␤-tubulin isotype III (aka TuJ; Sigma; 1:100), nestin/rat RESULTS 401 (Developmental Studies Hybridoma Bank, Iowa City, IA; undiluted), NeuN (Chemicon, Temecula, CA; 1:100), tyrosine KLF7 is a member of a group of mammalian zinc finger hydroxylase (Roche; 1:5). Photographs of the resulting sections transcription factors that have been implicated in the were taken and processed as described above. control of several developmental programs by regulating the process of cell differentiation (Turner and Crossley, Cell Transfections 1999). Based on preliminary Northern analyses, we origi- The full-size Klf7 cDNA was reconstructed in the pBS vector nally called this zinc finger protein UKLF, for ubiquitously (Stratagene, La Jolla, CA) from appropriately derived fragments of expressed KLF (Matsumoto et al., 1998). In so doing, we the overlapping cDNA clones and resubcloned into the pCMV-tag1 were aware of two intrinsic limitations of the approach vector (Stratagene) to yield clone mKLF7-FLAG. To generate stable upon which the name assignment was based. First, the transfectants in the Tet-on/Tet-off inducible system, mKLF7- Northern analysis was performed using RNA from tissues FLAG was subcloned into the Tet-repressible expression vector that include many different cell types; consequently, it did p-TRE2 (Clontech) and stably transfected into NIH3T3 fibroblasts not address possible restriction of expression to a limited (a kind gift from Dr. S. Friedman) that harbor plasmids pTet-Tk number of cell types. Second, the RNA was purified from (Life Technologies, Rockville, MD) and pPUR (Clontech). Cells tissues of 22-week-old human fetuses; consequently, the were selected using histidine-deficient DMEM and 2 ␮g/ml puro- analysis did not monitor KLF7 expression during most of mycin with and without addition of 2 ␮g/ml tetracycline (Shockett et al., 1995). Individual clones were picked and monitored for organogenesis. We therefore decided to isolate the mouse mKLF7 expression by Western blot analysis with anti-FLAG anti- gene (Klf7) in order to define the cellular source(s) of Klf7 body (Sigma); approximately 90% of the clones tested were positive expression during embryogenesis and, thus, provide the after 48 h induction. The recombinant adenovirus vector encoding conceptual framework for assessing the putative function(s) mKLF7-FLAG (AdKLF7) was prepared by Cre/loxP-mediated re- of this transcription factor.

FIG. 4. Klf7 is expressed in postmitotic neurons of neural tube and hindbrain. Klf7 expression in the E11.5 neural tube was compared to the expression of selected markers for cell proliferation (PCNA) and for glial (Nestin) and neuronal (NeuN, TuJ, SCG10) differentiation. With the exception of Klf7 and SCG10, expression of other marker genes was assessed by immunohistochemistry.

Characterization of Mouse KLF7 et al., 1998). Based on these experimental criteria, we concluded that mKLF7 is the homolog of hKLF7. Mouse Klf7 cDNAs were isolated from an E11.5 embryonic library and found to theoretically code for a 33-kDa protein (mKLF7) virtually identical to human KLF7 (hKLF7 Pattern of Klf7 Gene Expression in Fig. 1). Identity between the two proteins includes three In situ hybridizations to whole mounts and tissue sec- polypeptide sequences previously shown to be required for tions demonstrated that during development Klf7 expres- reporter gene transactivation, nuclear localization, and in sion is predominantly confined to the nervous system. vitro binding to the consensus sequence 5Ј GGNGNGGGN They also revealed that the temporal profile of Klf7 expres- 3Ј (Fig. 1) (Matsumoto et al., 1998). An additional conserved sion occurs in three broadly defined phases during embryo- feature of the two proteins is the presence of a potential genesis and postnatal life. phosphorylation site by Erk kinases in the hydrophobic Embryonic phase. The embryonic phase of Klf7 mRNA segment (Fig. 1). Like human KLF7, Northern analysis accumulation commences at about E9.5, is maximal at documented ubiquitous expression of mouse Klf7 in adult about E11.5, and declines later. Very low Klf7 expression tissues (Fig. 2a) (Matsumoto et al., 1998). Furthermore, the was first detected in the rostralmost portion of the head and FISH technique localized the Klf7 gene to chromosome in the trigeminal ganglia of the E9.0 embryo (data not 1C1–3 (Fig. 2b); this region of the mouse genome is a shown). Appreciable Klf7 mRNA accumulation was clearly segment of conserved synteny with human chromosome and reproducibly observed in the CNS and PNS of the E9.5 2q32 where KLF7 has been previously mapped (Matsumoto embryo (Fig. 3a). Moreover, whole-mount in situ hybridiza-

FIG. 5. Temporal profile of Klf7 expression. (a) Localization of Klf7 transcripts on cross sections at the thoracic level of E11.5 to E18.5 embryos (top, dark field; bottom, bright field). Sustained expression is seen in the dorsal root ganglia (drg), whereas expression in the neural tube (nt) decreases over time. (b) Localization of Klf7 transcripts on E11.5 to E16.5 sagittal sections. During earlier stages (E11.5), Klf7 expression is strictly confined to the nervous system. At later stages (E16.5), weak diffuse expression can also be detected outside the nervous system. Additional abbreviations: fb, forebrain; mb, midbrain; hb, hindbrain; pc, plexus coeliacus; scg, superior cervical ganglia; tg, trigeminal ganglia.

tions of E9.5–E10.5 embryos showed that Klf7 activity vestibulocochlear, petrosal, superior, jugular, nodose, acces- follows the same proﬁle as the neurospeciﬁc gene NeuroD sory, and dorsal root ganglia (Fig. 3b, left). Expression along (Fig. 3a) (Lee et al., 2000); as such, the temporal expression the dorsal root ganglia and neural tube could also be seen of Klf7 mirrors the rostral-to-caudal emergence of cranial from a dorsal view of the embryo (Fig. 3b, middle), whereas sensory ganglia (Ma et al., 1998; Fode et al., 1998). By E11.5, a cross section of the embryo revealed Klf7 signals in the Klf7 is intensively expressed in the fore-, mid-, and hind- dorsal root ganglia and in the ventral horn and roof of the brain; the forming eye; and the trigeminal, geniculate, neural tube (Fig. 3b, right).

In the E11.5 neural tube, Klf7 is expressed in the mantle zone, where postmitotic neuroblasts are located, but not in the proliferative ventricular zone. Thus, Klf7 expression colocalizes with markers for neuronal differentiation (NeuN, TuJ, SCG10), but not with markers for progenitors and cell proliferation (Nestin, PCNA) (Fig. 4). Cross sections at the thoracic level from E11.5 to E18.5 embryos documented sustained Klf7 expression in dorsal root ganglia and gradual decrease in the neural tube (Fig. 5a). Temporal progression of the Klf7 profile from strong expression predominantly in the nervous system to weak expression diffusely distributed throughout the whole embryo can be best appreciated by comparing the sagittal sections of E11.5–E.16.5 embryos (Fig. 5b). Additional Klf7-positive sites noted at these late developmental stages include the neural retina of E17.5 embryos and the olfactory epithelium of the E16.5 embryo (data not shown). There were also neural tissues never found to express Klf7 throughout development; they include the neural crest-derived chro- maffin cells of the adrenal medulla and the enteric nervous system (data not shown). Postnatal and adult stages. Postembryonic expression of Klf7 is observed in a few sites of the postnatal brain and, later, is almost exclusively confined to the adult cerebellum and dorsal root ganglia. An illustrative example of postnatal Klf7 expression in the brain can be seen in a sagittal section of the day 2 (P2) mouse head (Fig. 6a). Here, Klf7 transcripts accumulated most strongly in the cerebral neocortex and hippocampus, nasal epithelium, and trigeminal ganglion (Fig. 6a). Expression was also noted in the rostral migratory stream of the forebrain where, similar to the other sites of the brain, Klf7 activity is downregulated in the adult animal (data not shown). Klf7 accumulation in the developing cerebellum follows the spatiotemporal profile of neuronal differentiation. Dur- FIG. 6. Postembryonic Klf7 expression. (a) At early postnatal stages (P2), Klf7 expression is particularly strong in the developing the first 3 weeks of postnatal life, progenitors in the ing cerebral cortex (cn), the hippocampus (hc), the nasal epithe- external granule cell layer (EGL) cease to proliferate, begin lium (ne), and the trigeminal ganglia (tg); no expression is de- axonal growth, and descend into the internal granule cell tected in the developing cerebellum. (b) In the 6-month-old layer (IGL) (Hatten et al., 1997). Analysis of P5, P14, and brain, strong Klf7 expression is restricted to the IGL of the P21 cerebellum specimens documented the gradual transi- cerebellum (c); weak expression can be detected in the hip- tion of Klf7 expression from the inner portion of the EGL to pocampus (hc), the olfactory bulb (ob), and some brain-stem differentiating neuroblasts in the IGL (Fig. 7). The pattern nuclei (bn). (c) In the 6-month-old mouse, Klf7 is strongly ex- closely parallels that of NeuroD and is clearly distinct from pressed in the neurons of the dorsal root ganglia (arrow) but not the accumulation of Sonic hedgehog (Shh) transcripts in in the surrounding Schwann cells. Purkinje cells (Fig. 7). By 6 months, Klf7 continues to be strongly expressed in the IGL and, to a lesser extent, in other brain structures, such as the olfactory bulb, the hippocampal formation, and some brain-stem nuclei (Fig. 6b). Expression also remains high in neurons of the adult cord or brain, but only in the developing mantle zone and dorsal root ganglia (Fig. 6c). Consistent with the diffusely cortical plate, which contain differentiating postmitotic low expression in late embryos (Fig. 5b, right), other organ neuroblasts. Klf7 expression in the PNS was exclusively systems of the adult mouse (such as skin, lung, muscle, and seen at times when neuronal precursors reach the desig- kidney) were found to exhibit weakly widespread Klf7 nated ganglia, exit the cell cycle, and differentiate into signal without preferential accumulation in any particular mature neurons. Expression in the postnatal cerebellum, tissue or cell type (data not shown). on the other hand, is more dynamic, in that it initially In summary, in situ hybridizations did not detect Klf7 involves the EGL and, later, the mature granule neurons transcripts in the proliferative ventricular zone of spinal of the IGL.

FIG. 7. Klf7 expression in the neonatal cerebellum. Accumulation of Klf7 transcripts in the P5, P14, and P21 cerebellum (a, d, and g) is shown in comparison to NeuroD (b and e) and Shh (c, f, and i). (h) Part of the P5 image (a) at higher original magniﬁcation (200ϫ); it highlights restricted Klf7 expression in the inner (i) portion as opposed to the outer (o) portion of the external granule layer (EGL). Other abbreviations indicate the internal granule layer (IGL), Purkinje cell layer (PCL), and molecular layer (ML).

KLF7 Overexpression Induces Growth Arrest or without addition of tetracycline documented G1 arrest only in those in which KLF7 is overexpressed (Fig. 8e). In order to gain additional insight into mKLF7 function, In order to strengthen the correlation of the above and we overexpressed this transcription factor in transfected the gene expression data, the analysis was repeated within a cell cultures. To this end, we first employed the Tet-on/ “neural” context by overexpressing mKLF7 in the human Tet-off inducible gene system to monitor the effect of stably overproducing mKLF7 in NIH3T3 fibroblasts (Shockett et neuroblastoma line NB-OK1 (Nishimura et al., 1996). Western al., 1995). The analysis was carried out on two randomly analysis of extracts from cells infected with AdKLF7 or chosen clones (c3 and c21) which produce comparable AdEGFP construct specifically correlated mKLF7 produc- amounts of the KLF7-FLAG fusion protein, as assessed by tion by the former with stimulation of p21 and inhibition of Western blot with anti-FLAG antibody (Fig. 8a). DNA cyclin D1 (Fig. 9, left). Consonant with the changing levels replication was measured by BrdU incorporation and found of cell cycle regulators, a substantial decrease in BrdU to be 2- to 4-fold less than control when clones were incorporation was seen in AdKLF7- compared to AdEGFP- cultured in the absence of tetracycline (Fig. 8b). The de- infected cells or uninfected cells (Fig. 9, right). These in crease parallels the 8- to 14-fold increase of the p21 protein vitro experiments therefore demonstrated the antiprolifera- in the induced clones (Fig. 8c). In contrast, tetracycline tive potential of mKLF7, thus raising the possibility that withdrawal had no effect on the levels of the p53 protein this transcription factor may participate in maintenance of (Fig. 8d). Flow cytometry analysis of c21 cells cultured with the postmitotic state.

FIG. 8. Growth arrest after mKLF7 induction in NIH3T3 ﬁbroblasts. Two different clones (c3 and c21) and untransfected ﬁbroblasts (3T3) were incubated with or without tetracycline for 48 h. Cell lysates were analyzed by Western blot against KLF7-FLAG (a), p21 (c), and p53 (d). (b) DNA replication was analyzed by measuring the percentage of cells that incorporate BrdU after 0, 24, and 48 h of growth in the absence of tetracycline. (e) FACS analysis of control and c21 cells cultured with or without addition of tetracycline.

DISCUSSION postembryonic gene expression and by assessing the conse- quences of ectopically producing the protein in different The main goal of the present study was to gather correla- cell lines. Albeit correlative, the results nevertheless impli- tive insights into the function(s) of transcription factor cate mKLF7 in neuroblast differentiation and function and mKLF7 by establishing the profiles of embryonic and in cell cycle progression. Development of the vertebrate nervous system can be broadly identified by the overlapping phases of neuroecto- dermal induction, neuraxial patterning, and neuronal differentiation (Tanabe and Jessel, 1996). Differentiation of distinct neuronal subtypes begins with the patterning of the neural plate and culminates with the activation of genetic programs that define neuronal connectivity and function. Superimposed onto neuronal diversification is the pathway that governs acquisition of generic neuronal properties by progenitor cells. Generation of mature neuronal circuits is therefore the end-product of a dynamic interplay between extrinsic signals (secreted or transmembrane molecules) and intrinsic determinants (transcription factors) that occurs at precise stages of development (Lemon and Tjian, 2000). Our in situ hybridizations indicate that Klf7 is predominantly, but not exclusively, expressed in the CNS and PNS; FIG. 9. Growth inhibition of human neuroblastoma NB-OK1 cell expression is according to three dynamic phases (embry- line overexpressing mKLF7. NB-OK1 cells were infected with the onic, postnatal, and adult) which in turn correspond to construct AdKLF7 or AdEGFP (m.o.i. 50) and cell lysates were analyzed by Western blot with antibodies against KLF7 (␣KLF7), specific neurogenic stages. Expression in embryonic spinal cyclin D1, p21, and actin (left). The percentage of cells that incorpo- cord, which occurs in the ventral horn outside the prolif- rated BrdU was analyzed after infecting the NB-OK1 cells with the erative ventricular zone, coincides with the establishment constructs AdKLF7 or AdEGFP at a m.o.i. of 50 for 24 h (right). of neuronal phenotype in motor neurons, including process

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 316 Laub et al. outgrowth and initial synapse formation. Expression in time of withdrawal from it (Edlund and Jessel, 1999). The postnatal cerebral cortex occurs well after neuronal deter- notion of mKLF7 involvement in cell entry into the post- mination and during a period characterized by dramatic mitotic state is indirectly supported by the finding that the synaptogenesis and synaptic remodeling. Sustained expres- protein has an antiproliferative effect on different cell lines. sion in adult cerebellum and dorsal root ganglia suggests The effect includes the selective modulation of the cell maintenance of phenotype and/or involvement in neuronal cycle regulators p21 and cyclin D1 with consequential survival. arrest in G1. The idea is also consistent with genetic and in Examination of the embryonic profile of Klf7 expression vitro data overwhelmingly implicating KLFs in the regula- detected substantial mRNA accumulation in ventral horn tion of cell differentiation (Nuez et al., 1995; Perkins et al., neuroblasts at about the time patterning is completed and 1995; Shields et al., 1996; Kuo et al., 1997a,b; Wani et al., irrespective of their position along the anterior–posterior 1998; Cook et al., 1998; Segre et al., 1999; Shie et al., 2000; and dorsoventral axes (Pfaff and Kintner, 1998; Jessell, Zhang et al., 2000). 2000). Hence, Klf7 is expressed too late and too widely to be Our data also suggest that mKLF7 may be implicated in involved in patterning of the nervous system. Klf7 tran- maintaining the neuronal phenotype of cerebellar granule scripts were never detected in the forming enteric nervous cells and dorsal root ganglia. Although largely unknown, system nor in a particular cell lineage; they were also found the mechanisms responsible for phenotype maintenance in to accumulate in the derivatives of neural and surface postmitotic neurons are believed to be probably different ectoderm, as well as of some neural crest cells. Hence, Klf7 from those operating in proliferating cells (Edlund and is neither a panneural marker nor specific for a given Jessel, 1999). One possibility is that long-term stabilization lineage or a neuron subtype. The only discernable feature of of gene expression may involve the participation of activa- the embryonic profile of Klf7 is expression in postmitotic tors in chromatin remodeling (Edlund and Jessel, 1999; neuroblasts undergoing terminal differentiation. It is there- Lemon and Tjian, 2000). Along these lines, KLF1 has been fore tempting to speculate that mKLF7 may be part of the shown to be required for the formation of an SWI/SNF- network that drives or enables postmitotic differentiation related chromatin remodeling complex (Armstrong et al., and maturation of several neuronal subtypes. This conclu- 1998). Furthermore, a recent study has correlated KLF1 sion is supported by the confined accumulation of Klf7 binding to the Locus Control Region of the ␤-globin gene mRNA in the postnatal and adult brain and in the embry- cluster with regulation of chromatin accessibility through- onic and postnatal dorsal root ganglia. out the whole locus (McMorrow et al., 2000). While this Neocortical neurons are formed and make synapses be- work was under review, we became aware of another study fore birth. However, the first postnatal weeks are a period of suggesting that mKLF7 may regulate expression of the Trk extraordinary organization and reorganization, including gene family (Lei et al., 2001). Ongoing generation of Klf7 growth and retraction of axons and dendrites and synapto- null mice will be instrumental in establishing the contri- genesis (Yuste and Sur, 1999). The strong expression of Klf7 bution of this transcription factor to organismal develop- in the cerebral cortex during this period suggests that it may ment and function. play a special role in cerebral cortical development. Like- wise, Klf7 is strongly and specifically expressed in adult cerebellar granule cells that are formed in the mouse during ACKNOWLEDGMENTS the first weeks of postnatal life. They proliferate from progenitor cells in the EGL and with trophic support by The authors thank Drs. Anderson, Friedman, and Lee for provid- Purkinje cells in the form of secreted Shh (Hatten et al., ing critical reagents; Dr. L. Parada for sharing data prior to publi- 1997; Weschler-Rey and Scott, 1999; Wallace, 1999; Dah- cation; and Ms. K. Johnson for typing the manuscript. The work mane and Ruiz I Altaba, 1999). Our data indicate that Klf7 was supported by grants from the National Institutes of Health expression gradually increases during the first weeks of (AR45581), German Academic Exchange Service, Gottlieb Daimler postnatal life and becomes restricted to postmitotic granule and Karl Benz Foundation, and Ministry of Health and Welfare of cells that ultimately remain the sole defined source of high Japan. Klf7 activity in the adult animal. Thus, in the cerebellum (as in the embryonic spinal cord) Klf7 probably lies some- where downstream of the Shh-induced cascade and is con- REFERENCES nected with granule cell differentiation and phenotype maintenance. 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