PATTERNS & PHENOTYPES

Calmin Expression in Embryos and the AdultBrain, and Its Regulation by all-transRetinoic AcidMark A. Marzinke,1 Elizabeth M. Henderson,2 Katherine S. Yang,1

Angela Wai-Man See,1,3 Danielle C. Knutson,1,3 and Margaret Clagett-Dame1,2,3*

The vitamin A metabolite, all-trans retinoic acid (atRA), is a regulator of nervous system development.Using a subtracted cDNA library constructed from neuroblastoma cells, the atRA-responsive gene calmin(Clmn) was identified (Merrill et al. [2004] Biol Chem 385:605-614). The Clmn transcript is detected veryearly in rat embryonic development and is sensitive to retinoid status. In vitamin A-deficient embryos,Clmn mRNA is dramatically down-regulated in the neuroepithelium adjacent to the somites, and thisexpression can be rescued with the addition of atRA. In embryonic day 18.5 embryos, CLMN is detected inregions where newly differentiated neurons are found, including the neural retina and the cortical plate;and in the adult brain, CLMN is most highly expressed in the neuron cell bodies of the hippocampus, cere-bellum, and olfactory bulb. Thus, Clmn is sensitive to retinoid status during early gestational stages, andits expression is relegated to postmitotic neuronal cells in the adult rat brain. Developmental Dynamics239:610–619, 2010. VC 2009 Wiley-Liss, Inc.

Key words: calmin; differentiation; embryogenesis; hippocampus; regulation; vitamin A

Accepted 18 October 2009

INTRODUCTION

Vitamin A plays an essential role invertebrate embryogenesis, includingdevelopment of the nervous system(McCaffery and Drager, 2000; Clagett-Dame and DeLuca, 2002; Duester,2008). The vitamin A metabolite, all-trans retinoic acid (atRA), regulatesthe transcription of target genes bybinding to the retinoic acid receptor(RAR) (Chambon, 1996). The RARheterodimerizes with the retinoid Xreceptor, and this ligand-bound com-plex interacts with specific enhancer

regions of DNA, called retinoic acid

response elements (RARE), to modu-

late the expression of atRA-regulated

genes (Clagett-Dame and Plum, 1997;

Balmer and Blomhoff, 2002). In an

effort to identify atRA-responsive

genes that play a role in neuronal

development, a subtractive cDNA

library prepared from atRA-treatedand untreated human SH-SY5Y cells

was screened, and calmin (Clmn or

calponin-like, transmembrane; also

known as retinoic acid induced in neu-

roblastoma 12 or Rainb12) was identi-

fied (Merrill et al., 2004). Our group

has shown that Clmn is widely distrib-

uted in cancer cell lines, and is up-

regulated in human neuroblastoma,

breast cancer, and myeloid leukemia

cells within 4 to 24 hr after exposure to

atRA.

CLMN is a carboxy-terminal trans-

membrane-containing protein of un-known function. Based on sequence

homology databases, CLMN is pro-

posed to contain two N-terminal calpo-

nin homology domains, which are

known actin-binding interfaces found

Additional Supporting Information may be found in the online version of this article.1Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin2Pharmaceutical Science Division, University of Wisconsin-Madison, Madison, Wisconsin3Interdepartmental Graduate Program in Nutritional Sciences; University of Wisconsin-Madison, Madison, Wisconsin*Correspondence to: Margaret Clagett-Dame, Department of Biochemistry, University of Wisconsin-Madison, 433 BabcockDrive, Madison, WI 53706-1544. E-mail: dame@biochem.wisc.edu

DOI 10.1002/dvdy.22171Published online 11 December 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:610–619, 2010

VC 2009 Wiley-Liss, Inc.

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in several scaffolding proteins, includ-

ing spectrin and dystrophin (Gimona

et al., 2002). Additionally, studies

reveal sequence homology with en-

aptin as well as NUANCE, a protein

shown to connect the nucleus to the

actin cytoskeleton (Zhen et al., 2002).

In addition to an ORF encoding for a

1021 amino acid (aa) murine protein

(NCBI accession no. AB047978), sev-eral Clmn splice variants have been

described, including two isoforms pre-

dicted to result in truncated proteins

both lacking the transmembrane do-

main, and a third with a 31 aa inser-

tion that retains the transmembrane

domain (Ishisaki et al., 2001).Analysis of adult mouse tissues

shows Clmn is expressed in the brain,liver, kidney, large intestine, and thetestis (Ishisaki et al., 2001; Takaishiet al., 2003). Clmn expression in-creases in themouse testis duringmat-uration, and the CLMN protein isfound specifically in thematuring sper-matids. Other than skin, the expres-sion of calminmRNAor protein hasnotbeenstudied indevelopingembryos.

In the present work, we have char-

acterized the spatio-temporal expres-

sion of calmin using immunohisto-

chemistry and in situ hybridization in

rat embryos. We show that Clmn

mRNA expression in the neural tube

of early embryos is sensitive to reti-

noid status. Additionally, we report

that the expression of the full-length

CLMN protein is confined to neurons

in the adult hippocampus, cerebel-

lum, and olfactory bulb.

RESULTS

Immunoblot Analysis of

CLMN Protein

To study the CLMN protein, an anti-body was generated in rabbit against aC-terminal peptide (aa 947-961, Fig.1A). This antibody was designed todetect the full-length protein. To testantibody specificity, the full-lengthCLMN open reading frame wasexpressed using both a mammalianand a bacterial expression system.Murine CLMN containing a C-termi-nal 3x-flag tag was detected at 160kDa using the anti-flag or the anti-CLMN antibody, and was not detectedwhen nonimmune serum was used

(Fig. 1B). Similar results wereobtained when the full-length humanprotein was overexpressed in mamma-lian cells (data not shown). A proteinof approximately 160 kDa was alsospecifically detected by the anti-CLMN antibody in rat and mousebrain (Fig. 1C), and agrees with themolecular size reported by Takaishiet al. (2003) in mouse cerebrum andcerebellum using a polyclonal anti-body generated using bacteriallyexpressed CLMN. CLMN expressed asa N-terminal fusion protein with GSTin bacteria appeared at approximately190 kDa (27 kDa GST þ 160 kDaCLMN) using both a GST monoclonalantibody and the anti-CLMN antibody(data not shown). Thus, the anti-pep-tide CLMN antibody generated herespecifically detects human, mouse,and rat CLMN.

Distribution of CLMN Protein

in the Adult Rat Brain

CLMN immunostaining was observedin the hippocampus, olfactory bulb,cerebellum, cerebral cortex, piriformcortex of the rhinencephalon, medialhabenular nucleus of the dorsal thala-mus, and hypothalamic nuclei (Fig. 2,and data not shown). It was also

observed in a subset of large cells lin-ing the border of the periaqueductalgray; these cells may represent neu-rons of the mesencephalic nucleus ofthe trigeminal nerve (Supp. Fig. S1,which is available online). In the hip-pocampus, CLMN was found in thedentate gyrus and all CA regions aswell as the hilus (Fig. 2A and inset).The CLMN staining was specific, asshown by the lack of staining in thecontrol section in which nonimmuneserum was used (Fig. 2B). Within thecortex, staining was most notable inlayers II–V (Fig. 2C) and staining inthe piriform cortex was also observed(Fig. 2D). In the olfactory bulb, CLMNwas specifically found within cellbodies of neurons in the granule celllayer, as evidenced by expressionin neuron-specific NeuN-positive cells(Fig. 2E,F). CLMN was highly ex-pressed inPurkinje cells in the cerebel-lum (Fig. 2G,H) as confirmed by coloc-alization with calbindin D28K (Fig.2I,J). While calbindin D28Kwas foundboth in the cell bodies and neuronalprocesses (red) of the Purkinje cells,CLMN staining (green) was confinedto the Purkinje cell body (Fig. 2J). Verylight CLMN staining was also noted inthe internal granule cell layer, but wasabsent from the both the molecular

Fig. 1. Analysis of CLMN protein by immunoblotting. A: Schematic of the full-length mouseCLMN protein and the putative functional domains including two calponin homology (CH)domains (hatched) and a transmembrane domain (solid); the location of the CLMN peptide usedto generate the CLMN antibody is also shown (black bar). B: Immunodetection of overexpressedmurine CLMN (arrowhead) containing a 3X flag tag (CLMN-3xFl) using either the CLMN antibodyor a monoclonal antibody to the flag sequence. Immunoblotting with nonimmune serum isshown as a control. Lysates from N2A cells were prepared after transfection with CLMN-3xFl orempty vector (pIRES; negative control). C: Immunoblotting of endogenous CLMN protein in adultrat and mouse brain lysates using the CLMN antibody or nonimmune serum. Immunoblottting oflysates from cells transfected with CLMN-3xFl was performed as a positive control. aTubulin (55kDA) was analyzed in B and C by immunoblotting as a control for protein loading.

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layer and the white matter of the cere-bellum. The expression of CLMN wasverified in several regions of the adultbrain by immunoblotting. A 160 kDaprotein was specifically detected in thecortex, cerebellum, hippocampus, andolfactory bulb (Supp. Fig. S2).

Because CLMN protein was highlyexpressed in the adult rodent hippo-campus, we next examined whetherstaining was confined to neurons,or whether the protein was also ex-pressed in non-neuronal cells. CLMN(green) staining was found in the samecells as the neuronal marker, NeuN(red) in theCA1–3 regions (Fig. 3A, anddata not shown), as well as in the den-tate gyrus (Fig. 3B) and hilus (data notshown). CLMN staining did not over-lap that of the glial cell marker, glialfibrillary acidic protein (GFAP; red;Fig. 3C,D). CLMN staining was foundin the cell bodies of neurons that alsostained positive for MAP2, a brain spe-cific microtubular marker, but did notcolocalize with MAP2 in the dendriticcompartment of cells (Fig. 3F,G). Thus,in the adult brain, CLMN immuno-staining was found exclusively in post-mitoticneuronal cell bodies.

CLMN Expression in the

Embryo

Because little is known about CLMNexpression in developing embryos,antibody staining was examined infrontal sections of the head of anembryo at 18.5 days (E18.5). In theforebrain region, light CLMN immu-nostaining was seen in the corticalplate, whereas staining was absent inthe intermediate zone (Fig. 4A,A0).There was also very faint staining inregions corresponding to the ventricu-lar and subventricular zones, identi-fied by staining an adjacent sectionwith Ki67 (Fig. 4B), which marks cellsin interphase and undergoing mitosis(Scholzen and Gerdes, 2000). Therewas little CLMN expression in the

midbrain, except in a small subset ofcells that appeared to define the bor-der of the periaqueductal gray (Fig.4C,C0), and in the inferior tectal neu-roepithelium (Fig. 4D). In the cerebel-lum, CLMN protein was most abun-dant in the external granule celllayer, a transient cell population thatwill ultimately give rise to the inter-nal granule cell layer (Fig. 4D,D0). Inall of these regions, the specificity ofCLMN antibody staining was con-firmed by the absence of staining inthe nonimmune control sections (Fig.4A00,C00,D00). Staining of the hypoglos-sal nerve (CN12) was also evident inthe medulla (data not shown). In theeye, CLMN was prominently express-ed in the differentiating cell layer, aswell as in a few cells at the outer edgeof the proliferating retina (Fig. 4E,G),and the staining was specific asshown by the nonimmune control sec-tion (Fig. 4F). The differentiatingregion of the retina was identifiedwith ISL1 (Islet-1; Fig. 4H), a proteinexpressed in the early differentiatingretinal ganglion cells (Gong et al.,1995; Galli-Resta et al., 1997) andthe proliferating layer was observedby Ki67 staining (Fig. 4I). Finally,CLMN was expressed in several otherhead structures, including the epithe-lium of the nasal cavity, dorsal surfaceof the tongue, and the ocular muscles(Fig. 4G, and data not shown).

At very early stages of embryonic de-velopment, Clmn expression wasexamined by in situ hybridization.This approach was used because theClmn signal to background stainingratio was better when using the ribop-robe as compared to antibody stainingin early embryos. Clmn mRNA wasfirst observed in presomitic (late headfold stage) embryos, with strong stain-ing observed in the caudal mesoderm(Fig. 5A). At the 1- to 2-somite (s)stage, light staining was also noted inthe head region (data not shown), andat 3–4s, staining was found in associa-

tion with the presumptive fore- andmid-brain regions of the embryo (Fig.5B). At 3–4s, transcript was also pres-ent in the neuroepithelium adjacent tothe developing somites (Fig. 5C) aswell as in the tail region in the meso-derm but not the neuroepithelium ofthe tail region (Fig. 5D). At 7s, stain-ing in the forebrain and midbrain neu-ral plate was evident, and stainingappeared in the neuroepithelium justcaudal to the otic sulcus, with thestrongest staining immediately adja-cent to the entire somitic mesoderm(Fig. 5E). With development, the cau-dal domain of Clmn expression in theneuroepithelium/neural tube followedthat of somitic development (Fig. 5F–H). By E12.5, Clmn mRNA wasdetected in the ependymal layer of thedeveloping spinal cord, dorsal rootganglion and emerging motor axons,sympathetic ganglion, as well as thedeveloping lung bud (Fig. 5I).

Clmn Expression Is Down-

regulated in Early Vitamin

A-Deficient Embryos

Because Clmn mRNA is regulated byatRA in some cell lines, we next deter-mined whether Clmn expression wasaltered in early embryos madeseverely deficient in vitamin A.Embryos (4–5s) from vitamin A-suffi-cient (VAS) mothers showed theexpected pattern of Clmn expressionin the neuroepithelium in the headregion and adjacent to the somites, aswell as in the tail mesoderm. In con-trast, as shown in transverse vibra-tome sections, vitamin A-deficient(VAD) embryos showed a dramatic lossof staining in the neuroepithelium ad-jacent to the developing somites (com-pare Fig. 6E to 6B), whereas expres-sion in the head region was lessaffected and that in the tail region wasunchanged (compare Fig. 6D to 6A;Fig. 6F to 6C). At 6–7s, VAS embryos

Fig. 2 CLMN expression in coronal sections of the adult rat brain. A: Specific CLMN immunostaining in the dentate gyrus (dg) and cornu ammonis(CA) regions of the hippocampus. A higher magnification showing CLMN labeling of cells in the hilus (h) region is shown in the inset. B: Note the ab-sence of specific staining in the hippocampus when nonimmune serum is used (compare B with A). C,D: CLMN immunoreactivity is detected in theadult cortical laminae (II–V; C), and the piriform cortex (pc; D) in the rhinencephalon. E: CLMN immunostaining in the olfactory bulb. F: dotted box inE is the region of the granule cell layer (gcl) shown in higher magnification in F, where CLMN (green immunofluorescence) is found in the same cellsstained with nuclear neuronal marker NeuN (red). G: CLMN is detected at high levels in the cell soma of Purkinje neurons (pur) as well as in the gran-ule cell layer (gcl) in the cerebellum. H: The region shown in the dotted box in G is shown in higher magnification. I: Calbindin D28K is expressed inthe cell soma and dendritic processes of the Purkinje neurons in the molecular layer (ml) while CLMN expression is evident in the cell soma of thePurkinje neurons and in the internal granule cell layer (compare H with I). J: Overlay of CLMN (green) and calbindin D28K (red) immunofluorescenceshows colocalization (yellow) in the cell soma of Purkinje neurons. wm, white matter.

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continued to express Clmn in the neu-roepithelium adjacent to the somites(Fig. 6G), whereas expression in thisregion was lost in VAD embryos (Fig.6H). However, VAD embryos frommothers supplemented with atRA (250mg/g diet) from the onset of pregnancyshowed a rescue of Clmn expression in

Fig. 3.

Fig. 2.

Fig. 3. CLMN immunostaining is confined tothe cell body of hippocampal neurons.A,B: CLMN (green) and NeuN (red) staining isfound in the same cells in the CA3 region anddentate gyrus (dg). C,D: Non-neuronal cellsthat stain positive for glial fibrillary acidic pro-tein (GFAP; red) are not immunoreactive forCLMN (green). F,G: Within neurons, CLMNand MAP2 staining do not overlap; CLMN im-munostaining is confined to the cell bodywhereas MAP2, a microtubular marker, specif-ically stains the neuronal processes. E: Aschematic of the adult mammalian hippocam-pus. Regions within the CA3 and dg that wereshown in A–G are indicated by the dottedboxes. CA, cornu ammonis; h, hilus.

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Fig. 4.

Fig. 5. Clmn mRNA is expressed in early rat embryos. A: Detection of Clmn by whole-mount in situ hybridization in the caudal mesoderm (cm) at thelate head fold (presomite) stage. B–D: At the 3- to 4-somite (s) stage, Clmn staining is seen in the presumptive forebrain (fb) and midbrain (mb) (B), theneuroepithelium adjacent to the developing somites (C, arrow), and in the caudal mesodermal layers of the tail region (D). E: At 7s, Clmn mRNA isexpressed in the neuroepithelium caudal to the otic sulcus (os) and in the forebrain and midbrain regions. F–H: Clmn is found in the neuroepithelium ofthe developing neural tube at 12–19s. Note, Clmn expression in the neuroepithelium ends coincident with the most recently formed somite (G). I: ClmnmRNA is also expressed in the developing nervous system at E12.5, with expression in the dorsal spinal cord (sc), floor plate (fp), dorsal root ganglion(drg), motor neurons (mn) and sympathetic ganglion (sg);Clmn is also found at high levels in the developing lung bud (lb). LHF, late head fold; ov, otic ves-icle; pos, preotic sulcus.

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the trunk region (Fig. 6I). Thus, atRAacts to regulate Clmn expression inthe neuroepithelium adjacent to thedeveloping somites in the earlyembryo.

VAD embryos contain undetectablelevels of atRA as assessed using a sen-sitive HPLC-F9-RARE-lacZ reporterassay, whereas VAS embryos at E10.5from mothers supplemented with reti-nol contain approximately 60 fmolatRA (Supp. Table S1). The presenceof Clmn expression in the head andtail regions in embryos devoid of

measurable atRA suggests that ex-pression in these regions may be in-dependent of atRA. Embryos frommothers fed a high level of atRA(250 mg/g diet) have detectable levelsof atRA and also show rescued Clmnexpression in the neuroepitheliumadjacent to the somites supportingthat, at least in this region, mainte-nance of Clmn expression requiresatRA.

To determine whether the effect ofatRA on Clmn expression is direct,SH-SY5Y cells were treated with ve-

hicle or 10�6 M atRA in the absenceand presence of the transcriptional in-hibitor, actinomycin-D, or cyclohexi-mide, an inhibitor of protein transla-tion. The induction of Clmn mRNA byatRA at 4 hr was eliminated with theaddition of actinomycin-D, but wasnot reduced by cycloheximide (Fig. 7).In fact, Clmn mRNA was increased7.6-fold above vehicle when proteinsynthesis was inhibited, suggesting astabilization of the mRNA. The rapidinduction of Clmn mRNA and its inhi-bition by actinomycin-D, suggest thatClmn is directly regulated by atRAand its receptors.

DISCUSSION

In the present study, we show thatcalmin is widely distributed in boththe embryonic and adult nervoussystem. In early embryos, at least asubset of Clmn expression is respon-sive to retinoid status. The vitamin

Fig. 6. Clmn expression in the neuroepithelium of the developing neural tube is sensitive to ret-inoid status. A: At the 4- to 7-somite (s) stage, Clmn transcript is expressed in the neuroepithe-lium adjacent to the somites, as well as in the developing forebrain/midbrain and tail mesodermin vitamin A-sufficient (VAS) embryos. D: Vitamin A-deficient (VAD) embryos show a specific lossof Clmn expression in the neuroepithelium adjacent to the somites. Transverse sections reveal aloss of Clmn due to VAD in the neuroepithelium (compare E with the VAS section, B) but not inthe mesodermal layer of the developing tail (compare F with C). G–I: At 6–7s, Clmn expressionin the neuroepithelium adjacent to the developing somites (s) is present in VAS (G) but not VAD(H) embryos, and is rescued by the addition of atRA (I). pos, preotic sulcus.

Fig. 7. The induction of Clmn mRNA byatRA does not require new protein synthesis,but is blocked by an inhibitor of transcription.Clmn is up-regulated by exposure of SH-SY5Y cells to atRA for 4 hr. Co-treatment withthe transcriptional inhibitor actinomycin-Dresults in a total loss of Clmn induction by all-trans retinoic acid (atRA), whereas exposureof atRA-treated cells to the protein translationinhibitor cycloheximide does not block theinduction of Clmn mRNA. Values are mean 6SEM.

Fig. 4. CLMN immunostaining in frontal (coronal) sections of the head at embryonic day (E) 18.5. A: CLMN immunostaining is present in the fore-brain cortical plate (cp). A higher magnification of the cortical plate region stained with the CLMN antibody is shown in (A0), whereas specific stain-ing is absent in the adjacent panel (A00) in which nonimmune serum was used. B: Adjacent section to (A) probed with the proliferating marker Ki67show prominent staining in the ventricular (vz) and subventricular zones (svz) of the forebrain where faint CLMN staining is also detected (compareA with B). C,D: In the midbrain, CLMN is localized in a small subset of cells at the border of the dorsal and ventral periaqueductal gray (C, arrow)and is also found in the inferior tectal neuroepithelium (D, itn). In the cerebellum (cb), CLMN is localized to the external granule cell layer (D, egl).A higher magnification of specific CLMN staining in the cells lining the periaqueductal gray and in the cerebellum is shown in (C0) and (D0), respec-tively; and the lack of staining in these regions with nonimmune serum is shown in (C00) and (D00). E,F: CLMN is expressed in the differentiating celllayer of the neural retina (nr), and in a smaller subset of cells lining the outer edge of the proliferating retina (pr; E); F shows the lack of staining inthe retina when nonimmune serum is used. G: A higher magnification of specific CLMN immunostaining in the retina and ocular muscle (om).H,I: ISL1, a protein expressed in the early differentiating retinal ganglion cells (H), and Ki67 (I) staining are shown for reference. iz, intermediatezone.

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A metabolite all-trans retinoic acid(atRA) is required for early embryo-genesis, including the developingnervous system (Clagett-Dame andDeLuca, 2002; Duester, 2008). Em-bryos made deficient in retinoid, ei-ther through nutritional or geneticmethods, die early in development(White et al., 1998, 2000; Nieder-reither et al., 1999; Mic et al., 2002).Provision of atRA in amounts suffi-cient for early embryogenesis, butlimiting at later gestational times,yields fetuses with a host of develop-mental abnormalities (See et al.,2008). The first report of Clmn regula-tion by retinoid was in human neuro-blastoma cells, where it is up-regu-lated by atRA (Merrill et al., 2004).Here, we report that Clmn expressionis lost in the neuroepithelium adja-cent to the developing somites inembryos that are severely deficient invitamin A. Furthermore, the expres-sion of Clmn can be rescued by theaddition of atRA to these embryos. Inthe early embryo, atRA is produced inthe paraxial mesoderm with a rostralboundary at the level of the firstsomite, and later it is generated bythe somites. Several groups haveshown that the atRA-synthesizingenzyme, Raldh2, is expressed in theseregions, whereas it is not expressedby the neuroepithelium proper (Nie-derreither et al., 1997; Berggrenet al., 1999; Swindell et al., 1999;Molotkova et al., 2005). Taken to-gether, this supports the idea thatClmn expression in the neuroepi-theum is maintained by retinoid sig-naling from the underlying somiticmesoderm. Because atRA induction ofClmn mRNA is eliminated by thetranscriptional inhibitor, actinomy-cin-D, but is not altered by a proteinsynthesis inhibitor, Clmn appears tobe directly regulated by atRA and itsreceptors. Thus, Clmn is found indeveloping embryos before the timethat neurons differentiate, and vita-min A is required to maintain expres-sion specifically in the neuroepithiumadjacent to the developing somites.

Several regions in which CLMN im-munostaining is found at a later stage(E18.5) in the rat embryo correspondto sites in which neuronal progenitorshave recently exited the cell cycle andundergone differentiation and migra-tion. In the developing eye, retinal

neurogenesis occurs in a fixed histo-genetic order during late embryonicand early postnatal life, and at E18.5in the rat, both retinal progenitorcells and differentiated neurons arepresent. The nuclei of the retinal pro-genitor cells move within the outerneuroblastic layer according to thephase of the cell cycle, whereas celldifferentiation occurs in the innerlayer of the retina. CLMN expressionis strong in the differentiating regionof the developing retina, and is foundin the same region that stains posi-tively for ISL1, a homeodomain pro-tein associated with several specificpopulations of early differentiatingneurons in the central nervous sys-tem (CNS), including postmitotic reti-nal neurons (Galli-Resta et al., 1997).CLMN immunostaining is also foundat the outermost edge of the retina,the site where cells would be complet-ing mitosis and generating both cellsthat will continue to divide and post-mitotic daughter cells.

At E18.5, CLMN is also expressedin the nondividing cell layer of thecortex (cortical plate). Neuronal dif-ferentiation in the mammalian neo-cortex peaks at about this time in ges-tation, whereas non-neuronal cellssuch as astrocytes and oligodendro-cytes develop later (Ross et al., 2003).Neurogenesis in the cortex occurs in aninside out manner. Nuclear migrationand division of cells in the ventricularzone is followed by cell cycle exit, gen-erating a neuron that then migratesthrough the intermediate zone into thecortical plate (Guillemot, 2005). Theformation of the CNS involves preciseregulation of neural cell progenitor cellproliferation, cell fate specification,and migration and synaptogenesis(Donovan and Dyer, 2005). It will beimportant to determine whetherCLMN plays a role in the regulation ofcell cycle exit or cell fate specification,as it is most abundantly expressed inthe late embryonic CNS in regions ofthe retina and cortex where these proc-esses are actively taking place.

The strongest CLMN immunostain-ing in the adult brain is observed inthe pyramidal neurons of the hippo-campus (CA1–CA3) and the dentategyrus, as well as the olfactory bulband the cerebellum, where the Pur-kinje cell body and cells in the granu-lar layer, but not the molecular layer,

are stained. Co-staining experimentswith glial and neuronal markers inregions of the adult brain in whichCLMN is expressed show specific im-munostaining that is restricted topostmitotic neurons, and more specifi-cally to the cell bodies of these neu-rons. The detection of CLMN in thehippocampus agrees with a previousreport in which Clmn mRNA wasfound in these same regions using ariboprobe designed to the 30 end of thefull-length transcript (Takaishi et al.,2003). Of interest, vitamin A defi-ciency in mice affects adult rodentsynaptic plasticity between pyramidalneurons in the CA1 and CA3 neuronsof the Schaffer Collateral pathway(Misner et al., 2001). Impairment ofsynaptic plasticity and a deficiency inspatial learning is also observed inRARb knockout mice (Chiang et al.,1998). Furthermore, retinoid activityis present in the adult hippocampusas evidenced by several in vivo RA re-porter systems (Misner et al., 2001;Luo et al., 2004). Whether CLMNplays a role in any of these eventsremains to be determined.In summary, Clmn is expressed in

embryos from the late headfold stage(presomite stage) onward, and is mostabundant in tissues of the central andperipheral nervous systems. At theearly somite stage, vitamin A isrequired to maintain the normalexpression of Clmn in the neuroepi-thelium adjacent to the somitic meso-derm. In E18.5 embryos, CLMN isdetected in regions where newly dif-ferentiated neurons are found, includ-ing the neural retina and the corticalplate; and in the adult brain, CLMNis most highly expressed in the neu-ron cell bodies of the hippocampus,cerebellum, and olfactory bulb.

EXPERIMENTAL

PROCEDURES

Retinoids

All-trans retinoic acid (atRA) wasobtained from Spectrum Chemical Co.(New Brunswick, NJ) and wasdeemed greater than 99% pure byreverse-phase high-performance liq-uid chromatography (Motto et al.,1989).

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Cell Culture and Transfection

The murine neuroblastoma cell lineNeuro2A (N2A, ATCC, Manassas, VA)was grown at 37�C with 5% CO2 inMEM with 2 mM L-glutamine, 1.5 g/Lsodium bicarbonate, 0.1 mM nones-sential amino acids, 1 mM sodium py-ruvate, and 10% fetal bovine serum.N2A cells were transfected withFugene 6 (Roche, USA) according tothe manufacturer’s guidelines. SH-SY5Y human neuroblastoma cellswere cultured as previously described(Clagett-Dame et al., 1993).

Actinomycin-D/Cycloheximide

Experiments

SH-SY5Y cells were exposed to atRA(10�6 M) or vehicle (0.2% ethanol) for 4h in the absence or presence of actino-mycin-D (5 mg/ml) or cycloheximide(10 mg/ml) after which time poly(A)þ

RNA was isolated and reverse tran-scribed using AMV enzyme (Promega,Madison, WI) and random hexamers.Quantitative PCR was performedusing the LightCycler faststart DNAmaster SYBR green 1 kit (Roche, Indi-anapolis, IN) using primers for humanClmn (NCBI accession no. AB047979;upstream 50-GTGAAAGACCAGAGGAAGGCTA-30 and downstream 50-TGATGCGAACAAAAGTGGAT-30) as wellas human GAPDH (NCBI accessionno. AB062273; upstream 50-GCTTAGCACCCCTGGCCAAGGTCA-30 anddownstream 50-CATGTGGGCCATGAGGTCCACCAC-30) to normalize forinput cDNA. The identity of polymer-ase chain reaction (PCR) products wasverified by sequence analysis.

Generation of Embryo and

Brain Samples

Female rats (Harlan Sprague-Dawley,Madison, WI) were maintained onlaboratory chow diet (Harlan Teklad,USA) and mated with male rats of thesame strain. Vitamin A-deficient(VAD) embryos were generated andatRA rescue studies were performedas described previously (White et al.,1998, 2000). Briefly, the VAD groupwas fed the vitamin A-free purifieddiet supplemented with 1.5 mg atRA/gdiet starting at embryonic day (E) 0.5;this amount of atRA is close to theminimum needed to support embry-

onic survival (White et al., 2000). ForatRA rescue studies, the 250 mg atRA/g diet was fed. The VAS groupreceived a daily oral bolus of retinylpalmitate (500 IU/day) starting atE0.5. For adult brain studies, femalerats (Harlan Sprague-Dawley) weremaintained on a laboratory chow dietand euthanized at approximately 100days of age. All animals were main-tained according to conditions undera research protocol approved by theInstitutional Animal Care and UseCommittee at the University of Wis-consin-Madison.

Riboprobe Generation and In

Situ Hybridization

The polymerase chain reaction (PCR)was used to amplify the rat ClmncDNA using the following primers:upstream 50-TTTTCGGTTGAACAACATAGCG-30 and downstream 50-ACAGACGAAGACCTTGTCAGGC-30

to generate a 788-bp product. Theproduct was subcloned into the ZeroBlunt TOPO PCR vector (Invitrogen),and was sequenced to verify the iden-tity of the insert cDNA. For whole-mount in situ hybridization, embryoswere fixed in 4% PFA overnight at4�C and studies were performed aspreviously described (Merrill et al.,2004; See et al., 2008). A subset ofembryos after staining were embed-ded in 3% agarose in phosphate buf-fered saline (PBS) and sectioned at athickness of 40 mm using a LeicaVT1000S vibrating microtome.

Antibody Production and

Immunoblotting

A polyclonal antibody was generatedin rabbit to a 15-mer CLMN peptide(VQLRNAADLDDRRNR; amino acids947-961, NCBI accession no.AB047978) conjugated to keyhole lim-pet hemocyanin. To test antibodyspecificity, the full-length murineClmn cDNA (3.0 kb) was amplifiedfrom reverse transcribed cDNA fromthe murine neuroblastoma Neuro2A(N2A) cell line with upstream 50-TCCATGGCTGCTCAGGAGT-30 anddownstream 50-GAGCCGGCTGACATCAAGCT-30 primers and cloned intothe EcoRI-ScaI site of pIRES-hrGFP-1a (Stratagene) and sequenced, andencodes for a protein with a predicted

molecular weight of 112 kDa contain-ing a 3x C-terminal flag tag (CLMN-3xFl).N2A cells were transfected with

CLMN-3xFl or pIRES, lysed in buffercontaining 0.05 M Hepes (pH 7.4),0.15 M NaCl, and 1% NP40 with pro-tease inhibitors (5 mg/ml leupeptin, 25mM benzamidine, and 1 mM phenhyl-methyl sulfonyl fluoride), and proteinwas analyzed by BCA assay (Pierce).The sample was mixed 1:1 in 2�Laemmli sample buffer (60 mM TrisHCl, pH 6.8, 5% sodium dodecyl sul-fate (SDS), 5% b-mercaproethanol,10% glycerol, and 0.05% bromophenolblue). Protein (5 mg) was resolved on a10% SDS-polyacrylamide gel, andproteins were transferred to a nitro-cellulose membrane (Bio-Rad). Mem-branes were blocked with 8% nonfatdry milk powder in PBS with 0.2%Tween-20 for CLMN antibody or non-immune serum and 5% nonfat drymilk powder in PBS with 0.1%Tween-20 for the M2 anti-flag anti-body (Sigma, 1:2,000, Catalog F1804)and the anti–a-tubulin antibody(Clone B-5-12, Sigma, 1:10,000) for 1hr at room temperature, followed byprimary antibody incubation in block-ing buffer for 1 hr. Membranes werewashed three times in PBS beforeincubating with secondary antibodiesconjugated to horseradish peroxidasein blocking buffer for 1 hr. Antigen-antibody conjugates were visualizedby SuperSignal West Pico Chemilumi-nescent kit (Pierce) and exposed to X-ray film. Rat and mouse brain tissueswere lysed in homogenization buffer(1 mg of tissue: 4 ml of buffer; 20 mMTris-HCl, pH 8.0, 2 mM ethylenedia-minetetraacetic acid, 2 mM ethylene-glycoltetraacetic acid, 10 mMNa2HPO4, 25 mg/ml soybean trypsininhibitor, 5 mg/ml leupeptin, 2 mM di-thiothreitol, 25 mM benzamidine, and1 mM phenylmethylsulfonyl fluoride)using the Tekmar Tissuemizer (Tek-mar Co.) and lysed in 10% SDS Buffer(62.5 mM Tris-HCl, pH 6.8, 10% glyc-erol, 10% SDS, 0.03% bromophenolblue, and 10% b-mercaptoethanol).

Immunohistochemical and

Immunofluorescence Studies

Embryos (E18.5) and adult brain tis-sues were incubated in Dent’s fixativefor 1 week. Embryos were taken

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through ethanol and xylene washes,and transferred to xylene: paraffin(1:1) overnight at 60�C. Samples werethen incubated in 100% paraffin at60�C for 7 hr with three exchanges,and, during the final incubation, wereplaced under 15 mmHg pressure for 1hr at 60�C. Paraffin blocks were sec-tioned at 10 mm in series on a MicromHM325 microtome.

Tissue sections were deparaffinizedthrough a series of xylene washes toPBS and incubated in 0.3% H2O2 /1�PBS for 30 min at room temperatureto quench endogenous peroxidase ac-tivity. Sections were blocked with 2%goat serum, 1% bovine serum albu-min (BSA), 0.1% cold fish skin gelatin,0.05% Tween-20, and 0.05% NaN3 inPBS for 30 min in a humidified cham-ber. Samples were then incubatedwith primary antibodies in 1% BSA,0.1% fish skin gelatin, and 0.05% thio-merosal in PBS overnight at 4�C in ahumidified chamber. Primary anti-bodies included anti-calbindin D28K(Clone CB-955, Sigma, mouse IgG,1:1,000), anti-Ki67 (Clone B56, BDPharmingen, 1:400), anti-islet 1 (clone40.2D6 supernatant, DevelopmentalStudies Hybridoma Bank), rabbit IgG(Sigma, 1:200), and anti-CLMN (rab-bit polyclonal immune serum). TheVectastain ABC kit (mouse IgG orrabbit IgG) and 3,30-diaminobenza-dine tetrahydrochloride with metalenhancer was used to evaluate anti-body binding.

For immunofluorescence studies,tissue sections were prepared asabove except the hydrogen peroxideincubation was eliminated. Primaryantibodies included anti-GFAP (CloneG-A-5, Sigma, 1:1,000), anti-NeuN(Clone A60, Millipore, 1:1,000), anti-MAP2 (Clone AP-20, Sigma, 1:100)and anti-CLMN (rabbit polyclonalimmune serum). After overnight pri-mary antibody incubation, sectionswere incubated in Alexa-conjugatedsecondary antibodies (Alexa Fluor594 goat-anti-mouse IgG [Invitrogen,1: 200]; Alexa Fluor 488 goat anti-rab-bit IgG [Invitrogen, 1:200]).

ACKNOWLEDGMENTSWe thank Jamie Ahrens and VivianPoon for their contribution to the cellbiological experiments. We also thank

Mary Kaiser for assistance in the gen-eration of embryo samples and in situhybridization studies, and JeanChungfor embryonic retinoid analysis.

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