Neuroscience 162 (2009) 1150–1162

CHARACTERIZATION OF Foxp2-EXPRESSING CELLS IN THE DEVELOPING SPINAL CORD

Y. MORIKAWA,* T. HISAOKA AND E. SENBA neurons process incoming sensory information from the Department of Anatomy and Neurobiology, Wakayama Medical Uni- trunk and limbs and relay this information to higher brain versity, 811-1, Kimiidera, Wakayama 641–8509, Japan centers. In contrast, ventral horn neurons modulate motor output and transmit it to the target muscles. To process the Abstract—Two members of winged-helix/forkhead transcrip- signals from sensory input to motor output efficiently, the tion factors, Foxp1 and Foxp2, are expressed in the develop- arrangement of neurons is organized along the dorsoven- ing and adult CNS, including the striatum, cerebral cortex, tral axis of the spinal cord. In this pathway of signal pro- and thalamus. In a previous study, we have demonstrated cessing, functionally distinct classes of interneurons mod- that Foxp1 is expressed in a subpopulation of V1 interneu- ulate and integrate information from the periphery and rons in addition to motor neurons of the spinal cord during higher brain center (Rexed, 1952). mouse embryogenesis. However, the detailed expression pattern of Foxp2 and its relationship with Foxp1 in the devel- In the ventral spinal cord of adult mice, motor behavior oping spinal cord remains to be elucidated. To shed light on is controlled by the coordinated activity of motor neurons the potential roles of Foxp1 and Foxp2 in the developing (MNs) and local circuit interneurons. During the develop- spinal cord, we characterized Foxp2-expressing cells during ment of the embryonic spinal cord, MNs and four classes mouse embryogenesis. At embryonic day (E) 11.0, Foxp2- of ventral interneurons (V0, V1, V2, and V3) are generated expressing cells were first observed in the ventral spinal from five distinct populations of progenitor cells. These cord, which were Pax6؊, p27؉, and neuron-specific class III -tubulin؉ postmitotic neurons. Between E13.5 and E15.5, progenitor cells express unique combinations of homeodo-␤ high expression of Foxp2 was observed in both medial and main transcription factors through exposure to a gradient lateral parts of the ventral spinal cord. Double-immunofluo- of sonic hedgehog; MNs are marked by their expression of rescence staining for Foxp2 with some homeodomain tran- Islet1/2 (Isl1/2), V0 cells by Evx1/2, V1 cells by En1, V2 scription factors revealed that Foxp2-expressing neurons ,.؉ ؉ ؊ ؊ ؊ ؊ cells by Chx10/Gata3, and V3 cells by Sim1 (Briscoe et al were Pax2 , En1 , Evx1 , Chx10 , Gata3 , and Lhx3 V1 2000; Jessell, 2000; Lee and Pfaff, 2001). Each class of interneurons in the intermediate zone throughout the ventral spinal cord, indicating that Foxp2-expressing neurons were the postmitotic neurons comprises some distinct subtypes. also V1 interneurons with the same phenotypes as Foxp1- For example, V2 interneurons are subdivided to excitatory expressing interneurons. In addition, neither Foxp1 nor V2a and inhibitory V2b, which are characterized by Chx10 -Foxp2 was expressed in ventral calbindin؉ Renshaw cells. and Gata3, respectively (Li et al., 2005). In the V1 inter However, Foxp2 did not colocalize with Foxp1 in interneu- neurons, two functional subtypes of neurons, Renshaw rons of the ventral spinal cord. These findings suggest that cells and non-Renshaw cells including Ia inhibitory inter- Foxp1 and Foxp2 are expressed in the distinct subsets of V1 interneurons that belong to non-Renshaw cells in the ventral neurons, are defined, and the formers strongly expressed spinal cord during embryogenesis. Thus, Foxp1 and Foxp2 calbindin (Carr et al., 1998; Alvarez et al., 2005). may be involved in the determination of the cell type identi- The forkhead-box (Fox) family of transcription factors ties during late embryogenesis: the classes of neurotrans- is characterized by a highly conserved, winged-helix DNA- mitters and the functional subtypes of non-Renshaw cells, binding domain, functioning as both transcriptional activa- such as Ia and Ib inhibitory interneurons. © 2009 IBRO. Pub- tors and repressors (Lai et al., 1990; Weigel and Jäckle, lished by Elsevier Ltd. All rights reserved. 1990; Carlsson and Mahlapuu, 2002). These proteins have Key words: transcription factor, development, immunohisto- been shown to play critical roles in the regulation of embry- chemistry. onic development of various organs, including the control of cell differentiation, cell cycle regulation, and pattern formation (Lai et al., 1993; Kaufmann and Knöchel, 1996; Carlsson and The dorsal and ventral horn of the spinal cord in adult Mahlapuu, 2002). In the CNS, Foxg1 (formerly BF-1) has vertebrates is populated by neurons that process sensory been shown to be a critical regulator of the developing telen- and motor information, respectively (Brown, 1981; Pear- cephalon by the control of the proliferation and differentiation son, 1993; Caspary and Anderson, 2003). Dorsal horn of progenitor cells (Tao and Lai, 1992; Xuan et al., 1995; *Corresponding author. Tel: ϩ81-73-441-0617; fax: ϩ81-73-441-0617. Hanashima et al., 2002), and Foxb1 is essential for normal E-mail address: [email protected] (Y. Morikawa). Abbreviations: BSA, bovine serum albumin; DAPI, 4=,6-diamidino-2- development of the diencephalon and midbrain (Labosky et phenylindole dihydrochloride; E, embryonic day; Fox, forkhead-box; al., 1997; Wehr et al., 1997; Alvarez-Bolado et al., 2000). In IgG, immunoglobulin G; Isl, Islet; LMC, lateral motor column; MNs, addition, Foxn4 plays important roles in the diversification of motor neurons; P, postnatal day; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RT, room temperature; TuJ1, neuron-specific the V2 interneuron lineage into the V2a and V2b subtypes in class III ␤-tubulin. the developing spinal cord (Li et al., 2005). 0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.05.022

1150 Y. Morikawa et al. / Neuroscience 162 (2009) 1150–1162 1151

The Foxp subfamily of transcription factors is charac- protected in 0.1 M PBS containing sucrose (15% and then 30%) at terized by the zinc-finger domain and the leucine-zipper 4 °C for 16 h, embedded in OCT medium (Miles, Elkhart, IN, USA), Ϫ motif as well as the most divergent winged-helix domain frozen rapidly in cold n-hexane on dry ice, and stored at 80 °C. At all times, the experiments were carried out under the control within the Fox family (Shu et al., 2001; Lu et al., 2002). of the Animal Research Control Committee in accordance with the Among the Foxp subfamily, Foxp1 and Foxp2, initially Guidelines for Animal Experiments of Wakayama Medical University identified as the genes that repress the mouse CC10 and and Japanese Government Notification on Feeding and Safekeeping human surfactant protein C promoters, have been reported of Animals (No. 6) and the National Institutes of Health Guide for the to be expressed in various tissues, including the brain, Care and Use of Laboratory Animals (NIH Publications No. 80–23) lung, heart, and spleen (Shu et al., 2001; Ferland et al., revised 1996. All efforts were made to minimize the number of 2003; Takahashi et al., 2003, 2008). Previously, we have animals used and their suffering. also demonstrated that Foxp1 is expressed in the devel- Immunohistochemistry oping and adult CNS, such as the striatum, cerebral cortex, and spinal cord (Tamura et al., 2003, 2004). Recently, two Immunohistochemical staining was performed with some modifi- groups have reported that coordinated actions of Foxp1 cations as described previously (Morikawa et al., 2004). Briefly, and Hox proteins determine the fate of lateral motor col- 6-␮m-thick frozen sections were preincubated in 0.1 M PBS con- umn (LMC) MNs (Dasen et al., 2008; Rousso et al., 2008), taining 5% normal donkey serum and 0.1% Triton X-100 at room temperature (RT) for1htoblock non-specific binding of second- suggesting that Foxp1 plays important roles in the devel- ary antibodies, and then incubated with primary antibodies con- opment of the spinal cord. On the other hand, human taining 1% bovine serum albumin (BSA) and 0.1% Triton X-100 at FOXP2 has been identified as the gene mutated in patients 4 °C for 16 h. Primary antibodies were used at the following with a severe speech and language disorder (Lai et al., dilutions: rabbit anti-Foxp1 (1:1000; Abcam, Cambridge, UK), 2001). Mice carrying an identical point mutation to that of mouse anti-Foxp1 (JC12; 1:100; Abcam), rabbit anti-Foxp2 (1: the patient display impaired motor-skill learning and abnor- 2000; Abcam), goat anti-Foxp2 (1:200; Santa Cruz Biotechnology, mal synaptic plasticity in the striatum and cerebellum (Fu- Santa Cruz, San Jose, CA, USA), rabbit anti-Pax6 (1:50; Mizugu- chi et al., 2001), sheep anti-Chx10 (1:100; Abcam), rabbit anti- jita et al., 2008; Groszer et al., 2008). Thus, Foxp2 Pax2 (1:400; Zymed, Carlsbad, CA, USA), mouse anti-Evx1 (3A2; (FOXP2) plays important roles in the development of mo- 1:100; Developmental Studies Hybridoma Bank, University of tor-skill learning in mice and in the development of speech Iowa, Iowa City, IA, USA), mouse anti-En1 (4G11; 1:800; Devel- and language in humans. Although it has been reported opmental Studies Hybridoma Bank), mouse anti-Lhx3/Lim3 that Foxp2 is expressed in the developing spinal cord (Shu (4E12; 1:400; Developmental Studies Hybridoma Bank), mouse et al., 2001; Dasen et al., 2008), the characterization of anti-Gata3 (HG3-31; 1:50; Santa Cruz Biotechnology), mouse Foxp2-expressing cells remains to be elucidated. Re- anti-Lhx1/5 (4F2; 1:4000; Developmental Studies Hybridoma Bank), guinea-pig anti-Isl2 (1:4000; Abcam), mouse anti-p27 (1: cently, we have reported that Foxp1 is expressed in some 800, BD Biosciences Pharmingen, San Jose, CA, USA), mouse interneurons in addition to LMC MNs of the ventral spinal anti-neuron-specific class III ␤-tubulin (TuJ1; 1:2000; Covance, cord during mid- to late embryogenesis (Morikawa et al., Richmond, CA, USA), mouse anti-NeuN (1:400, Chemicon, Te- submitted for publication). To understand the potential mecula, CA, USA), rabbit anti-calbindin (1:200; Cell Signaling roles of Foxp1 and Foxp2 in the development of spinal Technology, Beverly, MA, USA). After washing with 0.1 M PBS cord interneurons, we examined the detailed expression containing 0.1% Triton X-100, the sections were incubated in the Cy2/Cy3-conjugated secondary antibodies (Jackson ImmunoRe- pattern of Foxp2 and its relationship to Foxp1 in the de- search Laboratories, West Grove, PA, USA) containing 1% BSA veloping spinal cord. and 0.1% Triton X-100 at RT for 1 h. All sections were counter- stained with 4=,6-diamidino-2-phenylindole dihydrochloride (DAPI, EXPERIMENTAL PROCEDURES Molecular Probes, Eugene, OR, USA). The following controls were performed: (i) incubation with Tissue preparation protein A–purified goat or rabbit immunoglobulin G (IgG) instead of the primary antibody; (ii) incubation with isotype-matched con-

Eight-week-old C57BL/6J mice were purchased from CLEA Japan trol antibody (mouse IgG1 for anti-En1, anti-Lhx3, anti-Gata3, (Tokyo, Japan). To obtain the embryos at the exact developmental anti-Lhx1/5, anti-p27, and anti-NeuN antibodies and mouse IgG2a stages, we performed the limited mating of the mice. Briefly, a for anti-Foxp1, anti-Evx1, and anti-TuJ1 antibodies) instead of the couple of mice were mated only for 1 h. The stage of mouse primary antibody; (iii) incubation without the primary antibody or embryos was designated as embryonic day (E) 0 when the cop- without primary and secondary antibodies. All controls revealed ulation plug was confirmed at the end point of 1 h mating. Preg- no labeling (data not shown). nant mice were deeply anesthetized with diethyl ether and the embryos were obtained by cesarean section at E10.5, E11.0, Image analysis and quantification E11.5, E12.0, E12.5, E13.5, E15.5, and E17.5. The embryos were immersed in ice cold 0.1 M phosphate-buffered saline (PBS, pH Fluorescently labeled cells were quantified in the developing spi- 7.4) containing 4% paraformaldehyde (PFA) for 1–4 h. Neonatal nal cords with some modifications as described previously mice were deeply anesthetized with diethyl ether at postnatal day (Tamura et al., 2005). Briefly, brachial parts of the specimens from (P) 0 and perfused transcardially with ice cold 0.85% NaCl fol- three embryos in each stage (E11.0, E11.5, E12.0, E12.5, E13.5, lowed by 4% PFA in 0.1 M PBS. The spinal cords were quickly E15.5, and E17.5) and three neonatal mice (P0) were pooled and removed and immersed in the same fixative at 4 °C for 4 h. Three processed for sectioning. The spinal parts were serially sec- parts were isolated from each specimen: brachial (C4–C8), tho- tioned at 6 ␮m and four sections that were 120 ␮m apart were racic (T4–T8), and lumbar (L1–L5). These three parts were cryo- selected for immunostaining. After staining, color digitized im- Download English Version: https://daneshyari.com/en/article/4340235

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