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Expression of BEN, a member of TFII-I family of transcription factors, during mouse pre- and postimplantation development
Gene Expression Patterns 3 (2003) 579–589 www.elsevier.com/locate/modgep
Expression of BEN, a member of TFII-I family of transcription factors, during mouse pre- and postimplantation development Dashzeveg Bayarsaihana,b,*, Natalia Bitchevaiaa, Badam Enkhmandakha, Maria Isabel Tussie-Lunac, James F. Leckmanb, Ananda Royc, Frank Ruddlea a
Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney avenue, New Haven, CT 06520, USA b Child Study Center, School of Medicine, Yale University, 230 South Frontage Road, New Haven, CT, USA c Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, USA Received 15 April 2003; received in revised form 29 May 2003; accepted 2 June 2003
Abstract BEN is a member of the TFII-I family of transcription factors, characterized by the presence of multiple helix – loop– helix repeat domains. Our immunohistochemical analysis demonstrated broad and extensive expression of BEN during mouse pre- and postimplantation development, with highest levels occuring during early to midgestation. Maternally expressed BEN is present in both the cytoplasm and nuclei of the zygote; however, it retains a predominantly nuclear localization between the two-cell stage of development and early blastocyst stages. This nuclear expression is observed in most tissues throughout development. Although, it is interesting to note that at E4.5 – 6.5, during early gastrulation stage, BEN is localized in the cytoplasm. At later stages, BEN retains an extensive expression pattern in a variety of developing systems implicating its involvement in tissue development and organogenesis. q 2003 Elsevier B.V. All rights reserved. Keywords: TFII-I; Preimplantation; Immunohistochemistry; Blastocyst; Transcription factor; Helix–loop–helix; Two-cell embryo; Williams syndrome
1. Results and discussion BEN and TFII-I proteins represent a novel class of transcription factors that are characterized by the presence of distinct helix – loop – helix repeat domains and a leucine zipper motif (Roy, 2001). These helix – loop –helix repeats are encoded separately on adjacent exons and were generated by independent genomic rearrangements (Bayarsaihan et al., 2002). The repeat domains and the leucine zipper are important in specific DNA recognition and dimerization activities (Vullhorst and Buonanno, 2003). TFII-I promotes the formation of stable higher-order complexes with different nuclear factors in response to various signals, and induces transcription of downstream target genes (Roy, 2001). TFII-I and BEN interact with HDAC3, a member of the class I histone deacetylases which are implicated in transcriptional repression through * Corresponding author. Address: Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney avenue, New Haven, CT 06520, USA. Tel.: þ 203-432-3525; fax: þ 203-432-5690. E-mail address: [email protected] (D. Bayarsaihan). 1567-133X/03/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1567-133X(03)00118-2
deacetylation of acetyllysines in amino-terminal tails of core histones. They also interact with PIASxb, a member of the E3 ligase family involved in the SUMO pathway (TussieLuna et al., 2002). BEN works as a specific repressor of TFII-I by preventing its entry into the nucleus (Tussie-Luna et al., 2001). Both proteins are encoded by GTF2IRD1 and GTF2I paralogs, which are closely linked at 7q11.23. This chromosomal region is hemizygously deleted in individuals with Williams syndrome, a genetic disorder characterized by distinctive physical and neurodevelopmental deficits (Morris and Mervis, 2000). In the mouse genome both paralogs extend over 200 kb and are separated by a 40 kb intergenic region (Bayarsaihan et al., 2002). The recently described deletion of the 50 region of mouse Gtf2ird1, including the first exon, results in the reduced level of RNA expression, although the homozygous mice have no obvious phenotype (Durkin et al., 2001). The expression domain of GTF2IRD1 was shown to be ubiquitous in vertebrate tissues -(Ring et al., 2002; Bayarsaihan and Ruddle, 2000; Osborne et al., 1999; Tassabehji et al., 1999; Franke et al., 1999). The Xenopus ortholog of GTF2IRD1 has a broad maternal expression and displays a more restricted zygotic expression pattern later in
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Table 1 Early expression of BEN Zygote þ pronuclear cytoplasmic
Two-cell þ nuclear
Four-cell þ nuclear
Eight-cell þ nuclear
Morula þ nuclear
Early blastocyst Trophoblast þ nuclear
E5.5–6.5 cytoplasmic embryonic and extraembryonic endoderm
Implanted blastocyst ICM þ nuclear
Trophoblast þ cytoplasmic
ICM –
E7.0–8.5 nuclear neuroepithelium, neural crest, somite, heart, foregut diverticulum, amnion, allantois, lateral mesoderm
development (Ring et al., 2002). However, little is known about its expression pattern during mammalian embryonic development. In this immunolocalization study, we found a very extensive expression pattern of BEN, a product of Gtf2ird1, during mouse pre- and postimplantation development (summarized in Tables 1 – 5).
Early expression. A low level of maternally expressed BEN was detected both in the cytoplasm and pronuclei of the zygote (Fig. 1A). Then, during the two-cell stage of development when zygotic gene activation begins, BEN preferentially localizes in the nucleus (Table 1). The nuclear immunostaining remained constant throughout the following
Table 2 BEN expression in the brain and spinal cord Region CNS
E8.5
Prosencephalon (forebrain) Telencephalon Neocortex Diencephalon Thalamus Hypothalamus
þþ
E9.5
E10.5
E11.5
E12.5
E13.5
E14.5
E15.5
E17.5
E18.5
P1
P10
þ þþ
þþ þ
þþ þþ
þ þ
þ þ
þ þ
þ þ
2 2
2 2
þ
2
þþ þþ
þ þ
þ þ
þ þ
þ þ
2 2
2 2
þ þ
2 2
þ þþ
þþ
þ
þ
þ
2
2
2
þþ þ
þ þ
þ þ
þ þ
þ þ
2 2
2 2
þ þ
þ þ
þ
þ
þ
þ
2
2
2
2
2
þ þþ þ þ þþ þþ þþ 2
þþ þ þ þþ þ þþ þþ 2
þþ 2 þ þþ þ þ 2
þ 2 þ þ þ 2
þ 2 þ þ 2 2
2
þþ þ
Mesencephalon (midbrain)
þ þþ
þþ þ
Rhombencephalon (hindbrain) Metencephalon Cerebellum Pons Myelencephalon Medulla
þþ
þþ þ þþ
þþ þþ
Spinal cord (neural tube) Ventricular zone Motor neurons Inter neurons Root plat Floor plate
þ þ
þ þþ þ þþ þþ þ 2
þþ þ þ þþ þ þþ þþ 2
2 2 2 2
(þ) intensity of labeling; (2) no labeling; ( ) not studied.
Table 3 BEN expression in the developing eye
Lens Neural retina Inner nuclear layer Outer nuclear layer Pigment retina Cornea epithelium Iris Eyelid
E10.5
E11.5
E12.5
E13.5
E14.5
E15.5
E17.5
E18.5
P1
þþþ þþþ 2 2 þþþ 2
þþþ þþþ 2 2 þþ 2
þþþ þþ 2 2 þ þ
þþþ þþ 2 2 þ þ
þ þþ þþ 2 2 2 þ
þ þþ þþ 2 2 2 2
þ þþ
þ þþ
þ þþ
2 þ þþ 2 2
2 þ þþ 2 2
2
2
2
2
2
2
2
2 þ þþ 2 2 þ 2
(þ) intensity of labeling; (2) no labeling; ( ) not studied.
2
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Table 4 BEN expression in the developing teeth Early bud stage
Oral epithelium/dental opithelium Inner dental epithelium Outer dental epithelium Primary enamel knot Secondary enamel knot Dental mesenchyme/dental papilla Ameloblast Odontoblast
Late bud st.
Cap stage
Bell stage
E10.5
E11.5
E12.5
E13.5
E14.5
E15.5
E17.5
E18.5
P1
þþ 2 2 2 2 þ
þþ 2 2 2 2 þ
þþ 2 2 2 2 þ
þþ 2 2 2 2 þ
þ 2 2 þþ 2 þ
þ 2 2 þþ 2 þ
2 2 þþ þ 2 þþ þ þ
2
2 2 2 2 2 þ þ þþ þþ
þþ þ 2 þþ þ þ
(þ) intensity of labeling; (2) no labeling; ( ) not studied.
stages of cleavage and compaction (8- to 16-cell and morula stages) (Fig. 1B – E). The protein was found in the nucleus of the ICM and trophectoderm at E3.5, the early blastocyst stage (Fig. 1F). However, one day later at the stage of implantation (E4.5), BEN was detected only in the cytoplasm of trophoblast cells. No significant level of staining was
detected in the ICM at this stage (Fig. 1G and H). These results indicate that BEN is expressed in the nuclei of twocell stage embryos and in the nuclei of the morulas until early blastocyst stages. However, BEN displays a restricted localization in the cytoplasm of trophoblast cells of implantation stage embryos.
Table 5 BEN expression in the different developing systems Systems Peripheral NS Trigeminal ganglia Dorsal ganglion Respiratory system Lung Bronchial epithelium Digestive system Gut Liver Esophagus Stomach Intestine Tongue Teeth Cardiovascular system Heart Vascular walls Urogenital system Urogenital ridge Kidney Testis Ovary Uninary bladder Penis Skeletal system Cephalic mesenchyme/skull Somite mesoderm Cartilage Ribs Limb Surface ectoderm/epidermis Hair follicles
E8.5
E9.5
E11.5
E10.5
E12.5
E13.5
E14.5
E15.5
E17.5
þþ 2
þ þþ 2
þ þþ þ þþ
þþ þ þþ þ
þþ þþ þ
þþ þ þþ
þþ þ þþ
þþ þþ
þ
2 2
þþ þþ
þþ þþ
þþ 2
þþ 2
þ 2
þ 2
þþ
þ þþ 2 þ
þ þþ þ þ
þþ þþ þþ 2
þ þþ þ þþ 2 þ þþ
þþ
P1
P10
þ 2
þ
þ
þ þþ þ þþ 2 þ þþ
þþ þ þ þþ 2 þ þþ
þ þþ
þ þþ
þþ
þþ
2 2
þ
þ
þ
þ 2
þ þ
þ þ
þþ
þþ
þþ þ þþ þþ 2 þ þþ
þ þþ þ
þ þþ þ
þþ þ þ
þþ þ
þ 2
2 2
2 2
þ þþ
þ þþ
þþ þ þ þ þ 2 2
þ þ þ 2 2
þ þ þ 2 2
2 2
2 2
2 2
2 2
þþ þþ þþ 2 2
þþþ
þ þþ
þ þþ
þþ þ
þþ
þþ
þ
þ
þ þ þ þ 2
þþ þþ þ þ 2
þþ þþ þ þ 2
þþ þ þ þ 2
þþ þ þ þ 2
þþ þ þ þ þ
þþ þ 2 þ þ
þþ þ 2 þ þ
(þþ þ) strong signal; (þ þ) clear signal; (þ) low but positive signal; (2) not signal; no symbol/not studied.
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Fig. 1. BEN expression during early stages of embryogenesis. BEN is detected by indirect immunofluorescence in A–F and H or stained with 3,30 diaminobenzidine (DAB) and counterstained in hematoxylin (G, J–L). (A) The maternally expressed BEN was detected in the cytoplasm and pronuclei of the zygote. (B –E) BEN displayed nuclear staining at the two-cell stage and stages of cleavage and compaction (8- to 16-cell and morula). (F) The protein is present in the nucleus of the ICM and trophectoderm at the early blastocyst stage at E3.5. (G,H) At the stage of implantation (E4.5) BEN is detected only in the cytoplasm of trophoblast cells. DNA was stained with DAPI (H). BEN is expressed in the cytoplasm of the embryonic and extraembryonic endoderm (sagittal section) at E5.5 (I) and E6.5 (J). (K) At E7.5, BEN is expressed in the nuclei of cells in the anterior and posterior neural ectoderm, embryonic mesoderm and amnion (sagittal section). (L) At E8.5, BEN is found in the nuclei of the neuroepithelium of the procencephalon, mesencephalon, myelencephalon, headfold mesenchyme, somites, heart (endocardium and miocardium), primitive streak, lateral and paraxial mesoderm (sagittal section). Abbreviations: a, amnion; een, embryonic endoderm; eec, embryonic ectoderm; ep, ectoplacental cone; ex, extraembryonic endoderm; exc, extraembryonic ectoderm; em, embryonic mesoderm; h, heart; lm; lateral mesoderm; met, metencephalon; mes, mesencephalon; my, myelencephalon; ne, neural ectoderm; ng, neural groove; pro, prosencephalon; ps, primitive streak; pm, presomitic mesoderm; pxm, paraxial mesoderm; s, somites; ys, yolk sac.
At E5.5 and E6.5, BEN is localized in the cytoplasm of the embryonic and extraembryonic endoderm, a component of the visceral yolk sac (Fig. 1I and J). BEN appears again in the nucleus of the neural ectoderm and embryonic mesoderm during the differentiation of the germ layers (Fig. 1K). By E8.5 (5 – 6 somites), a high level of BEN expression is observed in the cephalic region (neuroepithelium and headfold mesenchyme) and the dorsal areas of the neural folds including the neural crest, somites, heart and foregut diverticulum (Fig. 1L). BEN is also expressed in the amnion, allantois, primitive streak and lateral plate mesoderm. BEN expression was not observed in the presomitic mesoderm. Expression in the developing brain. At E8.5, BEN is first expressed in the rostral neuroepithelium of the prosencephalon, mesencephalon, and rhombencephalon (Table 2). A particularly high level of expression was present in
the mesencephalon (Figs. 1L and 2A). At E9.5 –11.5, BEN is observed in the dorsal – lateral region of the forebrain. Specifically, high levels of BEN expression are present in the diencephalon, including the optic stalk, ventral thalamus and hypothalamus (Fig. 2B and not shown). Expression is also observed in the neuroepithelium of the developing midbrain. From E8.5 to E11.5, BEN is detected in the rhombencephalon, particularly in the ventrolateral region and in Rathke’s pouch (not shown). Furthermore, BEN is expressed in the neurons of the cortical and basal layers of the developing brain (not shown). During later stages of embryogenesis (E12.5 – 15.5), BEN expression decreases in all brain regions. By E17.5, BEN expression is no longer observed in the developing brain. Postnatally, BEN is detected in the P1 mouse brain, where it is preferentially localized in the postmitotic neurons of the ventricular zone of the telencephalon. It is also expressed in zones of young postmitotic
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Fig. 2. Expression of BEN in the developing brain. BEN is detected by indirect immunofluorescence (A,B and I–IV) or immunostained with DAB (C and E). (A) At E8.5 BEN is localized to the neuroepithelium of the head fold (transverse section). (B) At E9.5 BEN is present in the neuroepithelium of the telencephalon, cephalic mesenchyme and surface ectoderm (transverse section). (C) BEN expression in the telencephalon of a P1 mouse (frontal section). The protein is detected in the ventricular zone, intermedial zone and cortical plate, but not in the subventricular zone. (D) BEN expression in the P1 brain (saggital section). The nuclear localization of BEN was detected in the young postmitotic neurons of the cortical plate and intermedial zone of the telencephalon (I) and olfactory bulb (II). Strong expression was observed in the ventricular and subventricular zone, and a weak signal was detected in the intermedial zone and cortical plate of the dorsal midbrain (III) and in the cerebellum (IV). (E) BEN expression in the cerebellum (white matter) and pons of a P10 mouse brain (transverse section). Abbreviations: a, amnion; cm, cephalic mesenchyme; cp, cortical plate; cr, cerebellum; imz, intermedial zone; lv, left ventricle; nf, neuroepithelium of head fold; p, pons; te, telencephalon; se, surface ectoderm; svz, subventricular zone; vz, ventricular zone; wm, white matter; 1(egl), external germinal layer; 2(ml), molecular layer; 3(PI), Purkinje cell layer; 4(igl), internal granule layer; 5(wm), white matter.
neurons located in the intermedial zone and cortical plate (Fig. 2C). Expression of BEN is observed in different areas of the developing brain, including the olfactory bulb, telencephalon and midbrain (Fig. 2D –I, II and III). BEN is not detected in the mitotically active subventricular zone of the telencephalon, a source of forebrain astrocytes and oligodendrocytes. One exception is a small level of protein expression in the subventricular zone of the midbrain (Fig. 2D – III). Moreover, BEN is present in the cerebellum and pons of P1 mice (Fig. 2D – IV). In the cerebellum, BEN is specifically expressed in the external germinal layer, Purkinje cell layer, internal granule layer, molecular layer, and white matter (Fig. 2D –IV). By P10, BEN is detected only in the cerebellum and pons (Fig. 2E). Expression in the neural tube. At E8.5 BEN is found in most cells along the dorsoventral and rostrocaudal regions of the neural tube (Fig. 3A, Table 2). From E9.0
to E14.5 BEN localization is more restricted; predominantly, to neurons of the ventrolateral region and floor plate (Fig. 3B – D and data not shown). The dorsoventral extent of BEN expression in the ventrolateral region of the neural tube is restricted from the cervical to lumbar regions. It is well known that the ventral neuroblasts give rise to motor neurons and interneurons. BEN displays an overlapping domain of expression with the Hoxc8 protein, a marker of motor neurons in the thoracic region of neural tube (Fig. 3E – G). A weak BEN signal is also detected in the ventral, but not the dorsal region, of the sacral portion of the neural tube (not shown). The protein was not detected in the caudal part of the neural tube. After E15.5, BEN showed reduced nuclear staining in the cells of the spinal cord with no significant levels of expression at later stages of development, including E17.5 and P1.
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Fig. 3. BEN expression in the neural tube (transverse section), detected by indirect immunofluorescence (A,E–G) or stained with DAB (B,C,D) At E8.5–9, BEN is found in the dorsoventral region of the developing neural tube. (C) At E9.5, BEN expression is restricted to the ventrolateral region and floor plate of the neural tube. (D) At E12.5, BEN is expressed in the ventral horn of the spinal cord. The signal was not detected in the dorsal region. (E,F) BEN and Hoxc8 expression at E10.5 in the neural tube. (G) Overlap of the nuclear expression of BEN and Hoxc8 in the developing motor neurons. Abbreviations: dm, dermomyotome; dg, dorsal root ganglia; fg, foregut; mn, motor neurons; nt, neural tube; n, notochord; pm, presomitic mesoderm; sc, sclerotome; so, somatopleure; sp, splanchnopleure; vh, ventral horn.
Expression in the eye. BEN is expressed in forebrain neuroepithelium, including those areas that evaginate to form the optic vesicle (Table 3). In the early stages of eye development (E9.0), the optic vesicles invaginate from the ventral forebrain and make contact with the overlying surface ectoderm. At E9.5, BEN protein was found in the optic vesicles and the ventral diencephalon (data not shown). Subsequently, the optic vesicles invaginate to form the optic cups, while the overlying surface ectoderm gives rise to the lens placode. At E10.5, BEN is localized in all areas of the developing eye, including the lens placode ectoderm, the prospective neuroretina, and the prospective pigment epithelium (Fig. 4A). Analysis of the different
stages of eye development showed that BEN is expressed throughout the development of the lens (E10.5 – 18.5 and P1) (Table 3). Furthermore, BEN is detected in the lens fibers, consisting of postmitotic cells of the equatorial region which subsequently produce lens-specific crystallins. Throughout lens growth, BEN expression coincides with the movement of fibers from the equator to the outer cortex of the lens (Fig. 4B and C). BEN is detected in the entire neural retina beginning at E10.5 (Fig. 4A). By E13.5, strong expression is seen in the ganglion cell layer (Fig. 4B), a zone with intensive mitotic activity. However, after cellular differentiation at E17.5, BEN expression is restricted to the outer nuclear layer of the retina (Fig. 4C). This expression
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Fig. 4. BEN expression in the eyes and teeth. The indirect immunofluorescence (A,D,E) or DAB staining was used (B,C,F). (A) At E10.5, BEN is present in the optic cup area (lens ectoderm, neural retina, pigmental epithelium) and neuroepithelium of the diencephalon (transverse section). (B) At E13.5, BEN is detected in the lens fibers and ganglion cells of the neural retina (transverse section). (C) At E17.5, BEN localization is restricted to the outer nuclear layer and lens (frontal section). (D) At E12.5, BEN is expressed in the dental epithelium and mesenchymal cells (transverse section). (E) At E14.5, BEN is detected in the dental follicle, primary enamel knot, and dental papilla (transverse section). (F) At E18.5, BEN is expressed in the dental papilla, odontoblasts, and ameloblasts (frontal section). Abbreviations: a, ameloblasts; de, dental epithelium; dc, diencephalon; dp, dental papilla; ek, enamel knot; f, dental follicle; inl, inner nuclear layer of retina; lf, lens fibers; o, odontoblasts; nt, neural tube; mn, motor neurons; l, lens; le, lens ectoderm; nr, neural retina; onl, outer nuclear layer of retina; pe, pigmental epithelium; r, retina.
data suggests that during early retina development BEN might be involved in proliferation; thus controlling eye size as well as the relationship of all layers. Later in development, the localization of protein to the outer nuclear layer suggests that BEN might function in cellular differentiation. Expression in the developing teeth. BEN expression during dentition is summarized in Table 4. The protein was first present at E10.5 in the oral epithelium of the maxillary and mandibular components of the first branchial arch (Fig. 4D). At E12.5, during the early bud stage when invagination of the epithelium occurs, BEN expression was observed in the thickening dental epithelium and in the mesenchyme beneath the oral epithelium (Fig. 4E). The dental mesenchyme has been shown to control tooth identity. Thus the expression of BEN in these particular mesenchymal cells may be important in tooth patterning. At the cap stage (E14.5 – 15.5), BEN was localized in the primary enamel knot, the wall of the dental cap, and the dental papilla (Fig. 4F). The enamel knot is a known signaling center that regulates tooth shape. At the subsequent bell stage (E16.5 –18.5), BEN was detected in the outer dental epithelium and the secondary enamel knot. Also at this stage, the intensity of BEN expression was decreased in the inner dental epithelium and in the dental papillum mesenchyme, including the preodontoblasts and differentiating odontoblasts. In postnatal (P1) mice, BEN was detected in the dental papilla and in differentiating odontoblasts (data not shown). These results suggest that
BEN is involved in the regulation of tooth morphogenesis, specifically mouse molar development, at later stages. Expression in the gut, lung, liver and tongue. BEN is expressed in the visceral mesoderm, the endodermal epithelium and the mesenchymal components of associated organ primordia during gut formation (Table 5). At the onset of organogenesis (E9.5) BEN expression is observed in the splanchnic mesoderm contacting the embryonic gut and in the presumptive lung bud (Fig. 5A). As the bronchial tree continues to form (E13.5), the lung mesenchyme exhibits clear BEN immunoreactivity. This specific expression persists postnatally at P1 (data not shown) and P10 (Fig. 5B). At E11.5, transverse sections at the level of the posterior foregut reveal intense BEN staining in both the undifferentiated epithelium and the associated mesenchyme of the stomach, as well as in the mesenchyme of the outgrowing pancreatic bud (data not shown). In subsequent stages, the distribution of BEN in the developing stomach is progressively restricted to the mucosal epithelium and the smooth-muscle layer (E14.5), but is not observed in the intestine (Fig. 5C). By E10.5, BEN is clearly expressed in the hepatic primordia. This expression remains intense until at least P10 (Fig. 5D). The fetal liver is a hematopoietic organ with maximal hematopoietic activity occuring at E15.5. It is possible that BEN participates in the proliferation of hepatocytes. However, we have not ascertained which cells are BEN-positive or the involvement of BEN in hematopoiesis. At E12.5, the expression
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Fig. 5. BEN expression in the gut diverticulum during development (transverse sections) detected by DAB staining. (A) At E9.5, BEN is detected in the lung bud. (B,C) BEN expression at P10 in lung the mesenchyme. (D) At E14.5, restricted expression is observed in the stomach (mucosal epithelium and smoothmuscle layer). In P10 mice, a high level of BEN expression was observed in the liver (E,F) and in the secretory cells (G,H) of the foundation tongue (frontal section). Abbreviations: en, endoderm; fg, foregut; lb, lung bud; mu, smooth-muscle layer; m, mucosa; pc, peritonial cavity; s, serosa.
domain of BEN is localized to the foundation of the developing tongue prior to the differentiation of tongue cells (data not shown). After differentiation, BEN is clearly detected in the secretory cells of the tongue in P10 mice (Fig. 5E). Expression in the heart. As early as E8.5, BEN is expressed in the endocardium and the epicardium of the folding heart tube (Fig. 6A, Table 5). Expression is also evident after cardiac looping (E9.5) in cardiomyocytes located in the atria and ventricular walls (Fig. 6B), and in the ventricular septum at E10.5 – 13.5 (Fig. 6C). BEN expression is dramatically reduced at later stages of cardiac development, as no protein was detected after E14.5. Weak expression was detected in newborn (P1) and P10 mice in the wall of ventricle, but not in the atrium (Fig. 6D). Pertaining to the heart vessels, BEN is expressed in the epithelium of the sinus venus and the vascular structure at early stages (E9.5 – 10.5) of cardiac morphogenesis. However, expression is not observed in the wall of the aorta. After E13.5, BEN expression was extinguished in the vessels.
Expression in the urogenital system and kidney. BEN is expressed in the urogenital system throughout various stages of development (E9.5 until P10) (Table 5). At E9.5, BEN is expressed in the developing mesonephric mesenchyme and gonadal primordia (Fig. 6E). During later stages (E10.5 – 15.5), a decreasing signal was detected in the mesonephric mesenchyme and tubules, and in the mesenchyme of the gonads of both male and female embryos (data not shown). At P10, BEN expression was not detected in the testis (ovaries were not studied) (Table 5). However, a small amount of BEN protein was detected in the glomerular capillaries of P1 and P10 mice (Fig. 6F). Expression in the somites, limb buds, branchial arches, bone, dorsal root ganglia, trigeminal ganglia and skin. BEN is expressed in the newly forming somites at E8.5 (Fig. 7A, Table 5). Strong signal was detected in the sclerotome at later stages (E9.5 –15.5) (Fig. 3D). BEN was present in the dermatome from E10.5 until E15.5. At E17.5, strong BEN expression was detected specifically in the axial muscles but not in the sclerotome (Fig. 7B). Later stages (from E18.5 onward) were not studied. A high level of BEN was also
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Fig. 6. Expression of BEN in the developing heart and urogenital system (transverse section). (A) At E8.5, BEN is expressed in the endocardium and epicardium. (B) At E9.5, a strong signal is detected in the atrium and ventricle. (C) At E13.5, BEN is present in the wall of the ventricle and in the ventricular septum. (D) In P10 mice, a weak signal was detected in the wall of ventricle. (E) At E9.5, BEN is detected in the urogenital bud. (F) In P10 mice, a signal was detected only in the glomerular capillaries. Abbreviations: a, atrium; da, dorsal aorta; f, foregut; gl, glomerular capillaries; gm, gut of mesentery; h, heart; nt, neural tube; ugb, urogenital bud; v, ventricle.
detected in the mesenchymal cells of the developing limb buds and in the branchial arches at E9.5 –11.5 (data not shown). BEN is expressed in the developing skull, ribs and cartilage. The signal is strong in the hypertrophic and prehypertrophic regions, but not in the proliferative chondrogenic region (Fig. 7C – E). After E15.5, we failed to detect BEN in the developing bones. BEN expression is observed in the dorsal root ganglia at E10.5 (Fig. 3D) and in the trigeminal ganglia at E15.5 (Fig. 7E). By E15.5, the intensity of signal in the dorsal root ganglia was significantly decreased. No staining was detected after E17.5. BEN was detected in the notochord between E9.5 –15.5 (Fig. 3D and data not shown). Moreover, BEN positive cells were detected in the surface ectoderm of the head, limbs, and trunk from E8.5 until late stages of embryogenesis (Fig. 7F). At P1 and P10, BEN positive immunostaining included epidermal cells and hair follicles (Fig. 7G and H). We noticed staining specifically in the outer keratinized layer of the skin.
This dynamic spatial and temporal expression pattern suggests that BEN is involved in the determination and/or differentiation of a number of cell types during embryogenesis.
2. Materials and methods CD-1 mouse embryos were obtained from timedpregnant mice and fixed overnight at 4 8C in freshly prepared 4% PFA/PBS. The fixed embryos were equilibrated overnight with 30% sucrose/PBS, then with 30% sucrose/OCT (1:1) at 4 8C. Embryos were transferred to embedding chambers, covered with OCT (Tissue-Tek Ost4583 compound), and stored at 2 70 8C. Frozen and paraffin sections were treated according to the standard protocols. Rabbit-anti-BEN polyclonal affinity-purified primary antibodies were described elsewhere (Bayarsaihan and Ruddle, 2000). Tissue sections were incubated
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Fig. 7. Other origins of BEN expression during mouse embryogenesis (transverse section). (A) At E8.5, a strong signal was detected in the somites (saggital section); (B) At E17.5, BEN expression is restricted to the axial muscle. (C) At E14.5, BEN is expressed in the bone of the limb. At E15.5, BEN is expressed in the developing rib (D), cartilage, and ganglia (E). (F) At E12.5, BEN expression is restricted to the surface ectoderm of limb. (G) In P10 mice, it is expressed in the skin. Abbreviations: c, cartilage; dg, dorsal ganglion; ep, epidermal cells; g, ganglia; hf, hair follicle; mu, musculus masses; r, rib; sc, spinal cord; sm, skeletal muscle.
with these antibodies (dilution 1:500 with 1% ngs/0.1% Tween-20/PBS) overnight at 4 8C. We used the Hoxc8 antibodies as a marker for motor neurons. The anti-rabbit Alexa fluor 488 IgG (Molecular Probes; dilution 1:200), or anti-rabbit Texas Red IgG (Molecular Probes; dilution 1:100) were used as secondary antibodies. We also used biotin-SP-conjugated goat-anti-rabbit secondary antibody (Jackson ImmunoPeseach Laboratories; dilution 1:500), peroxidase-conjugated Streptavidin (Jackson ImmunoResearch Laboratories; dilution 1:500). Staining with 3,30 Diaminobenzidine (DAB) (Sigma) and counter-staining with Harris’ hematoxylin was performed essentially as recommended by the manufacturer. We used the same protocols without addition of primary antibodies for negative controls. Acknowledgements The authors would like to thank Flora Vaccarino and Sterhanie Brewer for critical reading of the manuscript. This work was supported by the NIH grant NS43525.
References Bayarsaihan, D., Ruddle, F.H., 2000. Isolation and characterization of BEN, a member of the TFII-I family of DNA-binding proteins containing distinct helix– loop–helix domains. Proc. Natl. Acad. Sci. USA 97, 7342–7347. Bayarsaihan, D., Dunai, J., Greally, J.M., Kawasaki, K., Sumiyama, K., Enkhmandakh, B., Shimizu, N., Ruddle, F.H., 2002. Genomic organization of the genes Gtf2ird1, Gtf2i, and Ncf1 at the mouse chromosome 5 region syntenic to the human chromosome 7q11.23 Williams syndrome critical region. Genomics 79, 137–143. Durkin, M.E., Keck-Waggoner, C.L., Popescu, N.C., Thorgeirsson, S.S., 2001. Integration of a c-myc transgene results in disruption of the mouse Gtf2ird1 gene, the homologue of the human GTF2IRD1 gene hemizygously deleted in Williams–Beuren syndrome. Genomics 73, 20 –27. Franke, Y., Peoples, R.J., Francke, U., 1999. Identification of GTF2IRD1, a putative transcription factor within the Williams–Beuren syndrome deletion at 7q11.23. Cytogenet. Cell Genet. 86, 296– 304. Morris, C.A., Mervis, C.B., 2000. Williams syndrome and related disorders. Annu. Rev. Genom. Hum. Genet. 1, 461–484. Osborne, L.R., Campbell, T., Daradich, A., Scherer, S.W., Tsui, L.C., 1999. Identification of a putative transcription factor gene (WBSCR11) that is commonly deleted in Williams–Beuren syndrome. Genomics 57, 279 –284.
D. Bayarsaihan et al. / Gene Expression Patterns 3 (2003) 579–589 Ring, C., Ogata, S., Meek, L., Song, J., Ohta, T., Miyazono, K., Cho, K.W., 2002. The role of a Williams–Beuren syndrome-associated helix– loop–helix domain-containing transcription factor in activin/nodal signaling. Genes Dev. 16, 820 –835. Roy, A.L., 2001. Biochemistry and biology of the inducible multifunctional transcription factor TFII-I. Gene 274, 1–13. Tassabehji, M., Carette, M., Wilmot, C., Donnai, D., Read, A.P., Metcalfe, K., 1999. A transcription factor involved in skeletal muscle gene expression is deleted in patients with Williams syndrome. Eur. J. Hum. Genet. 7, 737 –747.
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Tussie-Luna, M.I., Bayarsaihan, D., Ruddle, F.H., Roy, A.L., 2001. Repression of TFII-I-dependent transcription by nuclear exclusion. Proc. Natl. Acad. Sci. USA 98, 7789–7794. Tussie-Luna, M.I., Bayarsaihan, D., Seto, E., Ruddle, F.H., Roy, A.L., 2002. Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxbeta. Proc. Natl. Acad. Sci. USA 99, 12807–12812. Vullhorst, D., Buonanno, A., 2003. Characterization of general transcription factor 3, a transcription factor involved in slow muscle-specific gene expression. J. Biol. Chem. 278, 8370–8379.
Expression of BEN, a member of TFII-I family of transcription factors, during mouse pre- and postimplantation development Dashzeveg Bayarsaihana,b,*, Natalia Bitchevaiaa, Badam Enkhmandakha, Maria Isabel Tussie-Lunac, James F. Leckmanb, Ananda Royc, Frank Ruddlea a
Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney avenue, New Haven, CT 06520, USA b Child Study Center, School of Medicine, Yale University, 230 South Frontage Road, New Haven, CT, USA c Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, USA Received 15 April 2003; received in revised form 29 May 2003; accepted 2 June 2003
Abstract BEN is a member of the TFII-I family of transcription factors, characterized by the presence of multiple helix – loop– helix repeat domains. Our immunohistochemical analysis demonstrated broad and extensive expression of BEN during mouse pre- and postimplantation development, with highest levels occuring during early to midgestation. Maternally expressed BEN is present in both the cytoplasm and nuclei of the zygote; however, it retains a predominantly nuclear localization between the two-cell stage of development and early blastocyst stages. This nuclear expression is observed in most tissues throughout development. Although, it is interesting to note that at E4.5 – 6.5, during early gastrulation stage, BEN is localized in the cytoplasm. At later stages, BEN retains an extensive expression pattern in a variety of developing systems implicating its involvement in tissue development and organogenesis. q 2003 Elsevier B.V. All rights reserved. Keywords: TFII-I; Preimplantation; Immunohistochemistry; Blastocyst; Transcription factor; Helix–loop–helix; Two-cell embryo; Williams syndrome
1. Results and discussion BEN and TFII-I proteins represent a novel class of transcription factors that are characterized by the presence of distinct helix – loop – helix repeat domains and a leucine zipper motif (Roy, 2001). These helix – loop –helix repeats are encoded separately on adjacent exons and were generated by independent genomic rearrangements (Bayarsaihan et al., 2002). The repeat domains and the leucine zipper are important in specific DNA recognition and dimerization activities (Vullhorst and Buonanno, 2003). TFII-I promotes the formation of stable higher-order complexes with different nuclear factors in response to various signals, and induces transcription of downstream target genes (Roy, 2001). TFII-I and BEN interact with HDAC3, a member of the class I histone deacetylases which are implicated in transcriptional repression through * Corresponding author. Address: Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney avenue, New Haven, CT 06520, USA. Tel.: þ 203-432-3525; fax: þ 203-432-5690. E-mail address: [email protected] (D. Bayarsaihan). 1567-133X/03/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1567-133X(03)00118-2
deacetylation of acetyllysines in amino-terminal tails of core histones. They also interact with PIASxb, a member of the E3 ligase family involved in the SUMO pathway (TussieLuna et al., 2002). BEN works as a specific repressor of TFII-I by preventing its entry into the nucleus (Tussie-Luna et al., 2001). Both proteins are encoded by GTF2IRD1 and GTF2I paralogs, which are closely linked at 7q11.23. This chromosomal region is hemizygously deleted in individuals with Williams syndrome, a genetic disorder characterized by distinctive physical and neurodevelopmental deficits (Morris and Mervis, 2000). In the mouse genome both paralogs extend over 200 kb and are separated by a 40 kb intergenic region (Bayarsaihan et al., 2002). The recently described deletion of the 50 region of mouse Gtf2ird1, including the first exon, results in the reduced level of RNA expression, although the homozygous mice have no obvious phenotype (Durkin et al., 2001). The expression domain of GTF2IRD1 was shown to be ubiquitous in vertebrate tissues -(Ring et al., 2002; Bayarsaihan and Ruddle, 2000; Osborne et al., 1999; Tassabehji et al., 1999; Franke et al., 1999). The Xenopus ortholog of GTF2IRD1 has a broad maternal expression and displays a more restricted zygotic expression pattern later in
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Table 1 Early expression of BEN Zygote þ pronuclear cytoplasmic
Two-cell þ nuclear
Four-cell þ nuclear
Eight-cell þ nuclear
Morula þ nuclear
Early blastocyst Trophoblast þ nuclear
E5.5–6.5 cytoplasmic embryonic and extraembryonic endoderm
Implanted blastocyst ICM þ nuclear
Trophoblast þ cytoplasmic
ICM –
E7.0–8.5 nuclear neuroepithelium, neural crest, somite, heart, foregut diverticulum, amnion, allantois, lateral mesoderm
development (Ring et al., 2002). However, little is known about its expression pattern during mammalian embryonic development. In this immunolocalization study, we found a very extensive expression pattern of BEN, a product of Gtf2ird1, during mouse pre- and postimplantation development (summarized in Tables 1 – 5).
Early expression. A low level of maternally expressed BEN was detected both in the cytoplasm and pronuclei of the zygote (Fig. 1A). Then, during the two-cell stage of development when zygotic gene activation begins, BEN preferentially localizes in the nucleus (Table 1). The nuclear immunostaining remained constant throughout the following
Table 2 BEN expression in the brain and spinal cord Region CNS
E8.5
Prosencephalon (forebrain) Telencephalon Neocortex Diencephalon Thalamus Hypothalamus
þþ
E9.5
E10.5
E11.5
E12.5
E13.5
E14.5
E15.5
E17.5
E18.5
P1
P10
þ þþ
þþ þ
þþ þþ
þ þ
þ þ
þ þ
þ þ
2 2
2 2
þ
2
þþ þþ
þ þ
þ þ
þ þ
þ þ
2 2
2 2
þ þ
2 2
þ þþ
þþ
þ
þ
þ
2
2
2
þþ þ
þ þ
þ þ
þ þ
þ þ
2 2
2 2
þ þ
þ þ
þ
þ
þ
þ
2
2
2
2
2
þ þþ þ þ þþ þþ þþ 2
þþ þ þ þþ þ þþ þþ 2
þþ 2 þ þþ þ þ 2
þ 2 þ þ þ 2
þ 2 þ þ 2 2
2
þþ þ
Mesencephalon (midbrain)
þ þþ
þþ þ
Rhombencephalon (hindbrain) Metencephalon Cerebellum Pons Myelencephalon Medulla
þþ
þþ þ þþ
þþ þþ
Spinal cord (neural tube) Ventricular zone Motor neurons Inter neurons Root plat Floor plate
þ þ
þ þþ þ þþ þþ þ 2
þþ þ þ þþ þ þþ þþ 2
2 2 2 2
(þ) intensity of labeling; (2) no labeling; ( ) not studied.
Table 3 BEN expression in the developing eye
Lens Neural retina Inner nuclear layer Outer nuclear layer Pigment retina Cornea epithelium Iris Eyelid
E10.5
E11.5
E12.5
E13.5
E14.5
E15.5
E17.5
E18.5
P1
þþþ þþþ 2 2 þþþ 2
þþþ þþþ 2 2 þþ 2
þþþ þþ 2 2 þ þ
þþþ þþ 2 2 þ þ
þ þþ þþ 2 2 2 þ
þ þþ þþ 2 2 2 2
þ þþ
þ þþ
þ þþ
2 þ þþ 2 2
2 þ þþ 2 2
2
2
2
2
2
2
2
2 þ þþ 2 2 þ 2
(þ) intensity of labeling; (2) no labeling; ( ) not studied.
2
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Table 4 BEN expression in the developing teeth Early bud stage
Oral epithelium/dental opithelium Inner dental epithelium Outer dental epithelium Primary enamel knot Secondary enamel knot Dental mesenchyme/dental papilla Ameloblast Odontoblast
Late bud st.
Cap stage
Bell stage
E10.5
E11.5
E12.5
E13.5
E14.5
E15.5
E17.5
E18.5
P1
þþ 2 2 2 2 þ
þþ 2 2 2 2 þ
þþ 2 2 2 2 þ
þþ 2 2 2 2 þ
þ 2 2 þþ 2 þ
þ 2 2 þþ 2 þ
2 2 þþ þ 2 þþ þ þ
2
2 2 2 2 2 þ þ þþ þþ
þþ þ 2 þþ þ þ
(þ) intensity of labeling; (2) no labeling; ( ) not studied.
stages of cleavage and compaction (8- to 16-cell and morula stages) (Fig. 1B – E). The protein was found in the nucleus of the ICM and trophectoderm at E3.5, the early blastocyst stage (Fig. 1F). However, one day later at the stage of implantation (E4.5), BEN was detected only in the cytoplasm of trophoblast cells. No significant level of staining was
detected in the ICM at this stage (Fig. 1G and H). These results indicate that BEN is expressed in the nuclei of twocell stage embryos and in the nuclei of the morulas until early blastocyst stages. However, BEN displays a restricted localization in the cytoplasm of trophoblast cells of implantation stage embryos.
Table 5 BEN expression in the different developing systems Systems Peripheral NS Trigeminal ganglia Dorsal ganglion Respiratory system Lung Bronchial epithelium Digestive system Gut Liver Esophagus Stomach Intestine Tongue Teeth Cardiovascular system Heart Vascular walls Urogenital system Urogenital ridge Kidney Testis Ovary Uninary bladder Penis Skeletal system Cephalic mesenchyme/skull Somite mesoderm Cartilage Ribs Limb Surface ectoderm/epidermis Hair follicles
E8.5
E9.5
E11.5
E10.5
E12.5
E13.5
E14.5
E15.5
E17.5
þþ 2
þ þþ 2
þ þþ þ þþ
þþ þ þþ þ
þþ þþ þ
þþ þ þþ
þþ þ þþ
þþ þþ
þ
2 2
þþ þþ
þþ þþ
þþ 2
þþ 2
þ 2
þ 2
þþ
þ þþ 2 þ
þ þþ þ þ
þþ þþ þþ 2
þ þþ þ þþ 2 þ þþ
þþ
P1
P10
þ 2
þ
þ
þ þþ þ þþ 2 þ þþ
þþ þ þ þþ 2 þ þþ
þ þþ
þ þþ
þþ
þþ
2 2
þ
þ
þ
þ 2
þ þ
þ þ
þþ
þþ
þþ þ þþ þþ 2 þ þþ
þ þþ þ
þ þþ þ
þþ þ þ
þþ þ
þ 2
2 2
2 2
þ þþ
þ þþ
þþ þ þ þ þ 2 2
þ þ þ 2 2
þ þ þ 2 2
2 2
2 2
2 2
2 2
þþ þþ þþ 2 2
þþþ
þ þþ
þ þþ
þþ þ
þþ
þþ
þ
þ
þ þ þ þ 2
þþ þþ þ þ 2
þþ þþ þ þ 2
þþ þ þ þ 2
þþ þ þ þ 2
þþ þ þ þ þ
þþ þ 2 þ þ
þþ þ 2 þ þ
(þþ þ) strong signal; (þ þ) clear signal; (þ) low but positive signal; (2) not signal; no symbol/not studied.
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Fig. 1. BEN expression during early stages of embryogenesis. BEN is detected by indirect immunofluorescence in A–F and H or stained with 3,30 diaminobenzidine (DAB) and counterstained in hematoxylin (G, J–L). (A) The maternally expressed BEN was detected in the cytoplasm and pronuclei of the zygote. (B –E) BEN displayed nuclear staining at the two-cell stage and stages of cleavage and compaction (8- to 16-cell and morula). (F) The protein is present in the nucleus of the ICM and trophectoderm at the early blastocyst stage at E3.5. (G,H) At the stage of implantation (E4.5) BEN is detected only in the cytoplasm of trophoblast cells. DNA was stained with DAPI (H). BEN is expressed in the cytoplasm of the embryonic and extraembryonic endoderm (sagittal section) at E5.5 (I) and E6.5 (J). (K) At E7.5, BEN is expressed in the nuclei of cells in the anterior and posterior neural ectoderm, embryonic mesoderm and amnion (sagittal section). (L) At E8.5, BEN is found in the nuclei of the neuroepithelium of the procencephalon, mesencephalon, myelencephalon, headfold mesenchyme, somites, heart (endocardium and miocardium), primitive streak, lateral and paraxial mesoderm (sagittal section). Abbreviations: a, amnion; een, embryonic endoderm; eec, embryonic ectoderm; ep, ectoplacental cone; ex, extraembryonic endoderm; exc, extraembryonic ectoderm; em, embryonic mesoderm; h, heart; lm; lateral mesoderm; met, metencephalon; mes, mesencephalon; my, myelencephalon; ne, neural ectoderm; ng, neural groove; pro, prosencephalon; ps, primitive streak; pm, presomitic mesoderm; pxm, paraxial mesoderm; s, somites; ys, yolk sac.
At E5.5 and E6.5, BEN is localized in the cytoplasm of the embryonic and extraembryonic endoderm, a component of the visceral yolk sac (Fig. 1I and J). BEN appears again in the nucleus of the neural ectoderm and embryonic mesoderm during the differentiation of the germ layers (Fig. 1K). By E8.5 (5 – 6 somites), a high level of BEN expression is observed in the cephalic region (neuroepithelium and headfold mesenchyme) and the dorsal areas of the neural folds including the neural crest, somites, heart and foregut diverticulum (Fig. 1L). BEN is also expressed in the amnion, allantois, primitive streak and lateral plate mesoderm. BEN expression was not observed in the presomitic mesoderm. Expression in the developing brain. At E8.5, BEN is first expressed in the rostral neuroepithelium of the prosencephalon, mesencephalon, and rhombencephalon (Table 2). A particularly high level of expression was present in
the mesencephalon (Figs. 1L and 2A). At E9.5 –11.5, BEN is observed in the dorsal – lateral region of the forebrain. Specifically, high levels of BEN expression are present in the diencephalon, including the optic stalk, ventral thalamus and hypothalamus (Fig. 2B and not shown). Expression is also observed in the neuroepithelium of the developing midbrain. From E8.5 to E11.5, BEN is detected in the rhombencephalon, particularly in the ventrolateral region and in Rathke’s pouch (not shown). Furthermore, BEN is expressed in the neurons of the cortical and basal layers of the developing brain (not shown). During later stages of embryogenesis (E12.5 – 15.5), BEN expression decreases in all brain regions. By E17.5, BEN expression is no longer observed in the developing brain. Postnatally, BEN is detected in the P1 mouse brain, where it is preferentially localized in the postmitotic neurons of the ventricular zone of the telencephalon. It is also expressed in zones of young postmitotic
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Fig. 2. Expression of BEN in the developing brain. BEN is detected by indirect immunofluorescence (A,B and I–IV) or immunostained with DAB (C and E). (A) At E8.5 BEN is localized to the neuroepithelium of the head fold (transverse section). (B) At E9.5 BEN is present in the neuroepithelium of the telencephalon, cephalic mesenchyme and surface ectoderm (transverse section). (C) BEN expression in the telencephalon of a P1 mouse (frontal section). The protein is detected in the ventricular zone, intermedial zone and cortical plate, but not in the subventricular zone. (D) BEN expression in the P1 brain (saggital section). The nuclear localization of BEN was detected in the young postmitotic neurons of the cortical plate and intermedial zone of the telencephalon (I) and olfactory bulb (II). Strong expression was observed in the ventricular and subventricular zone, and a weak signal was detected in the intermedial zone and cortical plate of the dorsal midbrain (III) and in the cerebellum (IV). (E) BEN expression in the cerebellum (white matter) and pons of a P10 mouse brain (transverse section). Abbreviations: a, amnion; cm, cephalic mesenchyme; cp, cortical plate; cr, cerebellum; imz, intermedial zone; lv, left ventricle; nf, neuroepithelium of head fold; p, pons; te, telencephalon; se, surface ectoderm; svz, subventricular zone; vz, ventricular zone; wm, white matter; 1(egl), external germinal layer; 2(ml), molecular layer; 3(PI), Purkinje cell layer; 4(igl), internal granule layer; 5(wm), white matter.
neurons located in the intermedial zone and cortical plate (Fig. 2C). Expression of BEN is observed in different areas of the developing brain, including the olfactory bulb, telencephalon and midbrain (Fig. 2D –I, II and III). BEN is not detected in the mitotically active subventricular zone of the telencephalon, a source of forebrain astrocytes and oligodendrocytes. One exception is a small level of protein expression in the subventricular zone of the midbrain (Fig. 2D – III). Moreover, BEN is present in the cerebellum and pons of P1 mice (Fig. 2D – IV). In the cerebellum, BEN is specifically expressed in the external germinal layer, Purkinje cell layer, internal granule layer, molecular layer, and white matter (Fig. 2D –IV). By P10, BEN is detected only in the cerebellum and pons (Fig. 2E). Expression in the neural tube. At E8.5 BEN is found in most cells along the dorsoventral and rostrocaudal regions of the neural tube (Fig. 3A, Table 2). From E9.0
to E14.5 BEN localization is more restricted; predominantly, to neurons of the ventrolateral region and floor plate (Fig. 3B – D and data not shown). The dorsoventral extent of BEN expression in the ventrolateral region of the neural tube is restricted from the cervical to lumbar regions. It is well known that the ventral neuroblasts give rise to motor neurons and interneurons. BEN displays an overlapping domain of expression with the Hoxc8 protein, a marker of motor neurons in the thoracic region of neural tube (Fig. 3E – G). A weak BEN signal is also detected in the ventral, but not the dorsal region, of the sacral portion of the neural tube (not shown). The protein was not detected in the caudal part of the neural tube. After E15.5, BEN showed reduced nuclear staining in the cells of the spinal cord with no significant levels of expression at later stages of development, including E17.5 and P1.
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Fig. 3. BEN expression in the neural tube (transverse section), detected by indirect immunofluorescence (A,E–G) or stained with DAB (B,C,D) At E8.5–9, BEN is found in the dorsoventral region of the developing neural tube. (C) At E9.5, BEN expression is restricted to the ventrolateral region and floor plate of the neural tube. (D) At E12.5, BEN is expressed in the ventral horn of the spinal cord. The signal was not detected in the dorsal region. (E,F) BEN and Hoxc8 expression at E10.5 in the neural tube. (G) Overlap of the nuclear expression of BEN and Hoxc8 in the developing motor neurons. Abbreviations: dm, dermomyotome; dg, dorsal root ganglia; fg, foregut; mn, motor neurons; nt, neural tube; n, notochord; pm, presomitic mesoderm; sc, sclerotome; so, somatopleure; sp, splanchnopleure; vh, ventral horn.
Expression in the eye. BEN is expressed in forebrain neuroepithelium, including those areas that evaginate to form the optic vesicle (Table 3). In the early stages of eye development (E9.0), the optic vesicles invaginate from the ventral forebrain and make contact with the overlying surface ectoderm. At E9.5, BEN protein was found in the optic vesicles and the ventral diencephalon (data not shown). Subsequently, the optic vesicles invaginate to form the optic cups, while the overlying surface ectoderm gives rise to the lens placode. At E10.5, BEN is localized in all areas of the developing eye, including the lens placode ectoderm, the prospective neuroretina, and the prospective pigment epithelium (Fig. 4A). Analysis of the different
stages of eye development showed that BEN is expressed throughout the development of the lens (E10.5 – 18.5 and P1) (Table 3). Furthermore, BEN is detected in the lens fibers, consisting of postmitotic cells of the equatorial region which subsequently produce lens-specific crystallins. Throughout lens growth, BEN expression coincides with the movement of fibers from the equator to the outer cortex of the lens (Fig. 4B and C). BEN is detected in the entire neural retina beginning at E10.5 (Fig. 4A). By E13.5, strong expression is seen in the ganglion cell layer (Fig. 4B), a zone with intensive mitotic activity. However, after cellular differentiation at E17.5, BEN expression is restricted to the outer nuclear layer of the retina (Fig. 4C). This expression
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Fig. 4. BEN expression in the eyes and teeth. The indirect immunofluorescence (A,D,E) or DAB staining was used (B,C,F). (A) At E10.5, BEN is present in the optic cup area (lens ectoderm, neural retina, pigmental epithelium) and neuroepithelium of the diencephalon (transverse section). (B) At E13.5, BEN is detected in the lens fibers and ganglion cells of the neural retina (transverse section). (C) At E17.5, BEN localization is restricted to the outer nuclear layer and lens (frontal section). (D) At E12.5, BEN is expressed in the dental epithelium and mesenchymal cells (transverse section). (E) At E14.5, BEN is detected in the dental follicle, primary enamel knot, and dental papilla (transverse section). (F) At E18.5, BEN is expressed in the dental papilla, odontoblasts, and ameloblasts (frontal section). Abbreviations: a, ameloblasts; de, dental epithelium; dc, diencephalon; dp, dental papilla; ek, enamel knot; f, dental follicle; inl, inner nuclear layer of retina; lf, lens fibers; o, odontoblasts; nt, neural tube; mn, motor neurons; l, lens; le, lens ectoderm; nr, neural retina; onl, outer nuclear layer of retina; pe, pigmental epithelium; r, retina.
data suggests that during early retina development BEN might be involved in proliferation; thus controlling eye size as well as the relationship of all layers. Later in development, the localization of protein to the outer nuclear layer suggests that BEN might function in cellular differentiation. Expression in the developing teeth. BEN expression during dentition is summarized in Table 4. The protein was first present at E10.5 in the oral epithelium of the maxillary and mandibular components of the first branchial arch (Fig. 4D). At E12.5, during the early bud stage when invagination of the epithelium occurs, BEN expression was observed in the thickening dental epithelium and in the mesenchyme beneath the oral epithelium (Fig. 4E). The dental mesenchyme has been shown to control tooth identity. Thus the expression of BEN in these particular mesenchymal cells may be important in tooth patterning. At the cap stage (E14.5 – 15.5), BEN was localized in the primary enamel knot, the wall of the dental cap, and the dental papilla (Fig. 4F). The enamel knot is a known signaling center that regulates tooth shape. At the subsequent bell stage (E16.5 –18.5), BEN was detected in the outer dental epithelium and the secondary enamel knot. Also at this stage, the intensity of BEN expression was decreased in the inner dental epithelium and in the dental papillum mesenchyme, including the preodontoblasts and differentiating odontoblasts. In postnatal (P1) mice, BEN was detected in the dental papilla and in differentiating odontoblasts (data not shown). These results suggest that
BEN is involved in the regulation of tooth morphogenesis, specifically mouse molar development, at later stages. Expression in the gut, lung, liver and tongue. BEN is expressed in the visceral mesoderm, the endodermal epithelium and the mesenchymal components of associated organ primordia during gut formation (Table 5). At the onset of organogenesis (E9.5) BEN expression is observed in the splanchnic mesoderm contacting the embryonic gut and in the presumptive lung bud (Fig. 5A). As the bronchial tree continues to form (E13.5), the lung mesenchyme exhibits clear BEN immunoreactivity. This specific expression persists postnatally at P1 (data not shown) and P10 (Fig. 5B). At E11.5, transverse sections at the level of the posterior foregut reveal intense BEN staining in both the undifferentiated epithelium and the associated mesenchyme of the stomach, as well as in the mesenchyme of the outgrowing pancreatic bud (data not shown). In subsequent stages, the distribution of BEN in the developing stomach is progressively restricted to the mucosal epithelium and the smooth-muscle layer (E14.5), but is not observed in the intestine (Fig. 5C). By E10.5, BEN is clearly expressed in the hepatic primordia. This expression remains intense until at least P10 (Fig. 5D). The fetal liver is a hematopoietic organ with maximal hematopoietic activity occuring at E15.5. It is possible that BEN participates in the proliferation of hepatocytes. However, we have not ascertained which cells are BEN-positive or the involvement of BEN in hematopoiesis. At E12.5, the expression
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Fig. 5. BEN expression in the gut diverticulum during development (transverse sections) detected by DAB staining. (A) At E9.5, BEN is detected in the lung bud. (B,C) BEN expression at P10 in lung the mesenchyme. (D) At E14.5, restricted expression is observed in the stomach (mucosal epithelium and smoothmuscle layer). In P10 mice, a high level of BEN expression was observed in the liver (E,F) and in the secretory cells (G,H) of the foundation tongue (frontal section). Abbreviations: en, endoderm; fg, foregut; lb, lung bud; mu, smooth-muscle layer; m, mucosa; pc, peritonial cavity; s, serosa.
domain of BEN is localized to the foundation of the developing tongue prior to the differentiation of tongue cells (data not shown). After differentiation, BEN is clearly detected in the secretory cells of the tongue in P10 mice (Fig. 5E). Expression in the heart. As early as E8.5, BEN is expressed in the endocardium and the epicardium of the folding heart tube (Fig. 6A, Table 5). Expression is also evident after cardiac looping (E9.5) in cardiomyocytes located in the atria and ventricular walls (Fig. 6B), and in the ventricular septum at E10.5 – 13.5 (Fig. 6C). BEN expression is dramatically reduced at later stages of cardiac development, as no protein was detected after E14.5. Weak expression was detected in newborn (P1) and P10 mice in the wall of ventricle, but not in the atrium (Fig. 6D). Pertaining to the heart vessels, BEN is expressed in the epithelium of the sinus venus and the vascular structure at early stages (E9.5 – 10.5) of cardiac morphogenesis. However, expression is not observed in the wall of the aorta. After E13.5, BEN expression was extinguished in the vessels.
Expression in the urogenital system and kidney. BEN is expressed in the urogenital system throughout various stages of development (E9.5 until P10) (Table 5). At E9.5, BEN is expressed in the developing mesonephric mesenchyme and gonadal primordia (Fig. 6E). During later stages (E10.5 – 15.5), a decreasing signal was detected in the mesonephric mesenchyme and tubules, and in the mesenchyme of the gonads of both male and female embryos (data not shown). At P10, BEN expression was not detected in the testis (ovaries were not studied) (Table 5). However, a small amount of BEN protein was detected in the glomerular capillaries of P1 and P10 mice (Fig. 6F). Expression in the somites, limb buds, branchial arches, bone, dorsal root ganglia, trigeminal ganglia and skin. BEN is expressed in the newly forming somites at E8.5 (Fig. 7A, Table 5). Strong signal was detected in the sclerotome at later stages (E9.5 –15.5) (Fig. 3D). BEN was present in the dermatome from E10.5 until E15.5. At E17.5, strong BEN expression was detected specifically in the axial muscles but not in the sclerotome (Fig. 7B). Later stages (from E18.5 onward) were not studied. A high level of BEN was also
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Fig. 6. Expression of BEN in the developing heart and urogenital system (transverse section). (A) At E8.5, BEN is expressed in the endocardium and epicardium. (B) At E9.5, a strong signal is detected in the atrium and ventricle. (C) At E13.5, BEN is present in the wall of the ventricle and in the ventricular septum. (D) In P10 mice, a weak signal was detected in the wall of ventricle. (E) At E9.5, BEN is detected in the urogenital bud. (F) In P10 mice, a signal was detected only in the glomerular capillaries. Abbreviations: a, atrium; da, dorsal aorta; f, foregut; gl, glomerular capillaries; gm, gut of mesentery; h, heart; nt, neural tube; ugb, urogenital bud; v, ventricle.
detected in the mesenchymal cells of the developing limb buds and in the branchial arches at E9.5 –11.5 (data not shown). BEN is expressed in the developing skull, ribs and cartilage. The signal is strong in the hypertrophic and prehypertrophic regions, but not in the proliferative chondrogenic region (Fig. 7C – E). After E15.5, we failed to detect BEN in the developing bones. BEN expression is observed in the dorsal root ganglia at E10.5 (Fig. 3D) and in the trigeminal ganglia at E15.5 (Fig. 7E). By E15.5, the intensity of signal in the dorsal root ganglia was significantly decreased. No staining was detected after E17.5. BEN was detected in the notochord between E9.5 –15.5 (Fig. 3D and data not shown). Moreover, BEN positive cells were detected in the surface ectoderm of the head, limbs, and trunk from E8.5 until late stages of embryogenesis (Fig. 7F). At P1 and P10, BEN positive immunostaining included epidermal cells and hair follicles (Fig. 7G and H). We noticed staining specifically in the outer keratinized layer of the skin.
This dynamic spatial and temporal expression pattern suggests that BEN is involved in the determination and/or differentiation of a number of cell types during embryogenesis.
2. Materials and methods CD-1 mouse embryos were obtained from timedpregnant mice and fixed overnight at 4 8C in freshly prepared 4% PFA/PBS. The fixed embryos were equilibrated overnight with 30% sucrose/PBS, then with 30% sucrose/OCT (1:1) at 4 8C. Embryos were transferred to embedding chambers, covered with OCT (Tissue-Tek Ost4583 compound), and stored at 2 70 8C. Frozen and paraffin sections were treated according to the standard protocols. Rabbit-anti-BEN polyclonal affinity-purified primary antibodies were described elsewhere (Bayarsaihan and Ruddle, 2000). Tissue sections were incubated
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Fig. 7. Other origins of BEN expression during mouse embryogenesis (transverse section). (A) At E8.5, a strong signal was detected in the somites (saggital section); (B) At E17.5, BEN expression is restricted to the axial muscle. (C) At E14.5, BEN is expressed in the bone of the limb. At E15.5, BEN is expressed in the developing rib (D), cartilage, and ganglia (E). (F) At E12.5, BEN expression is restricted to the surface ectoderm of limb. (G) In P10 mice, it is expressed in the skin. Abbreviations: c, cartilage; dg, dorsal ganglion; ep, epidermal cells; g, ganglia; hf, hair follicle; mu, musculus masses; r, rib; sc, spinal cord; sm, skeletal muscle.
with these antibodies (dilution 1:500 with 1% ngs/0.1% Tween-20/PBS) overnight at 4 8C. We used the Hoxc8 antibodies as a marker for motor neurons. The anti-rabbit Alexa fluor 488 IgG (Molecular Probes; dilution 1:200), or anti-rabbit Texas Red IgG (Molecular Probes; dilution 1:100) were used as secondary antibodies. We also used biotin-SP-conjugated goat-anti-rabbit secondary antibody (Jackson ImmunoPeseach Laboratories; dilution 1:500), peroxidase-conjugated Streptavidin (Jackson ImmunoResearch Laboratories; dilution 1:500). Staining with 3,30 Diaminobenzidine (DAB) (Sigma) and counter-staining with Harris’ hematoxylin was performed essentially as recommended by the manufacturer. We used the same protocols without addition of primary antibodies for negative controls. Acknowledgements The authors would like to thank Flora Vaccarino and Sterhanie Brewer for critical reading of the manuscript. This work was supported by the NIH grant NS43525.
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