Gruss, P.

Treichel, D.

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Gene expression patternThe novel transcription factor gene Sp5 exhibits a dynamic and highlyrestricted expression pattern during mouse embryogenesisDieter Treichel, May-Britt Becker, Peter Gruss*Department of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 GoÈttingen, GermanyReceived 16 October 2000; received in revised form 6 November 2000; accepted 6 November 2000AbstractWe describe the sequence and expression pattern of Sp5, a novel member of the vertebrate Sp1 transcription factor gene family whichconsists of at least ®ve members. This gene family is characterized by a highly conserved domain which is formed by three Zn ®ngers, whichbind to the GC box or the GT/CACC box in the promoter of many genes. These boxes are important cis-acting elements required for theexpression of the respective genes. In vitro experiments indicate that the Sp1 transcription factors act by in¯uencing the methylation state ofthe DNA, or by direct interactions with other promoter speci®c transcription factors. Despite intensive research, the results from in vivoexperiments, including targeted gene inactivation, have been dif®cult to explain. This may be due to possible redundancies and interferenceswith other transcription factors of this gene family. Here, we report the isolation of the mouse Sp5 gene, a novel Sp1 homolog. Its sequenceindicates that Sp5 is a possible link between Sp1 and the closely related BTEB/KLF gene family. We provide detailed information of itshighly dynamic expression pattern during mouse embryogenesis in the developing brain, the spinal cord, the trigeminal ganglia, the somitesand additional sites outside the nervous system starting from embryonic day 7.25 (E7.25) up to E10.5. q 2001 Elsevier Science Ireland Ltd.All rights reserved.Keywords: Sp5; Sp1; BTEB; EKLF; LKLF; Mouse; Embryogenesis; Tail bud; Branchial arches; Pharyngeal pouch; Pharyngeal cleft; Central nervous system;Pretectum; Tuberal hypothalamus; Mammilary body; Mesencephalon; Red nucleus; Rubral nucleus; Midbrain±hindbrain boundary; Spinal cord; Trigeminalganglia; Somites; Otic vesicle; Limbs1. ResultsA PCR based approach was used to clone Sp1 genefamily homologs. Degenerate PCR primers were designedon the basis of the conserved Zn ®nger domain. The paircgccaggtgatcrctickcatraaic (5 0) and cacatctgccayatycciggit-gygarmg (3 0) ampli®ed on genomic mouse DNA a charac-teristic Sp1-type Zn ®nger box of 231 nucleotides(annealing at 478C; 40 cycles). This fragment was used toscreen an E8.5 mouse cDNA library (kindly provided byBrigid Hogan). We obtained a 1255 bp fragment of acDNA sequence including the poly(A). From the NCBIentry AF279479, 636 bp were added 5 0 to our sequence(length of the entry, 1825). Our sequence is identical toAF279479, except for an additional 53 bp at the 3 0end(TTCCAGGGTCTCCTCGGGAAGACCCCCACAGAA-AAAAAAAAAAAAAAAAAAAA).As shown in Fig. 1A,B, the Sp5 gene belongs to the Sp1transcription factor family, which is characterized by typicalZn ®nger and buttonhead regions (El-Baradi and Pieler,1991; Wimmer et al., 1993, 1996). Outside of thesedomains, the Sp5 protein shows little similarity to otherSp1 homologs, which contain S/T-rich and Q-rich stretches.Instead, in the latter region, Sp5 is similar to the evolution-ary homologous but distinct BTEB/KLF group, character-ized by a high content of proline residues (about 20%; Fig.1C; Philipsen and Suske, 1999). Members of the BTEB/KLF group have a distinct Zn ®nger domain and lack thebuttonhead domain. Therefore, the Sp5 gene has probablybeen generated during evolution by a domain swappingbetween a Sp1 and a BTEB/KLF homolog.The in situ hybridization (ISH) revealed a highly dynamicand speci®c expression pattern, which appears to be subdi-vided into two phases (Figs. 2 and 3): an early activity whichis connected to the process of gastrulation, and a secondwave of expression during organogenesis.1.1. Early expression of Sp5At E7.25 (Figs. 2A,B and 3A), Sp5 is expressed in theentire region of the primitive streak. In the cells of the embryoMechanisms of Development 101 (2001) 175±1790925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.PII: S0925-4773(00)00544-Xwww.elsevier.com/locate/modo* Corresponding author. Tel.: 149-551-2011361; fax: 149-551-2011504.E-mail address: pgruss@gwdg.de (P. Gruss).proper (primitive ectoderm and ingressing mesoderm), Sp5mRNA is transcribed, but not in the underlying visceral endo-derm (arrows in Fig. 3A). The lateral constriction of theexpression is unclear, since the lateral most mesoderm andectoderm are also negative (data not shown). Later on, thesignal remains con®ned to the primitive streak at the end ofthe embryo (Fig. 2E±J). At E8.25, the gene is expressed in theposterior end of the neural plate and the underlying meso-derm (Fig. 3C). The visceral endoderm is still negative (datanot shown). With the formation of the gut, the de®nitiveendoderm also becomes positive (arrow in Fig. 3C), as wellas the notochord (double arrow in Fig. 3C). At E10.25 andlater stages, the primary gastrulation has ®nished and thesecondary gastrulation starts in the posterior tip of theembryo, the tail bud. Its mesenchymal tissue stronglyexpresses Sp5 (Figs. 2J and 3D,E), while the surroundingectoderm is negative (arrow in Fig. 3E). Interestingly, afterthe switch in the gastrulation and notochord formation, thenotochord still expresses Sp5 (arrow in Fig. 3D), but exclu-sively in a single cell (double arrow in Fig. 3F) surrounded bySp5-negative notochordal cells (arrows in Fig. 3F).1.2. Sp5 expression in the developing brainAt E8.0, the expression during organogenesis starts in theCNS as a faint ISH signal in the mesencephalon and dien-cephalon (arrows in Figs. 2E,F and 4A). At E8.5, the mRNAlevel becomes very strong in the dorso-lateral regions of thesame domains (Fig. 4B,I). In the diencephalon, the Sp5-mRNA is restricted to the region of the prospective pretec-tum and epithalamus (Fig. 4B). The telencephalon shows nostaining up to E10.5.D. Treichel et al. / Mechanisms of Development 101 (2001) 175±179176Fig. 1. (A) Deduced amino acid sequence of the mouse Sp5 protein. The three Zn ®nger domains are highlighted in light green, the btd domain(RCRRCRCPNC) is in a darker green, and the proline residues are highlighted in light blue. (B) Comparison of the Zn ®nger and btd protein domains ofdifferent Sp1-like and BTEB/KLF-like proteins. All sequences are according to the protein translations given at NCBI for the mentioned genes (m, mouse; h,human). Note that the proteins Sp1±Sp5 all contain the conserved btd box about 18 amino acids in front of the Zn ®nger domains, while the more distantlyrelated genes belonging to the BTEB/KLF family lack this domain. In contrast, they contain conserved sequences (black boxes) that are not found in the Sp1-like proteins. (C) Schematic drawing representing the deduced proteins of the Sp1 and the BTEB/KLF family. The scheme is drawn to scale. The region of thebtd and Zn ®nger domains is symbolized by green boxes and represents the deduced sequences in (B). The purple boxes represent glutamine-rich domains, thered boxes serine/threonine-rich stretches, and the blue boxes represent proline-rich domains. Note that Sp5 is homologous to Sp1 regarding the btd and Zn®nger domain, while the rest of the protein is homologous to the BTEB/KLF family like BTEB2, BTEB3, TIEG1, TIEG2, EKLF or LKLF.Until stage E9.5, both the dorsal and lateral regions of themesencephalon are Sp5-positive (arrow in Fig. 2H), but thelateral expression gradually disappears between E10.0 andE10.5 (arrows in Figs. 2I,J and 4C). However, the midbrain±hindbrain boundary (MHB) shows a strong hybridizationsignal throughout its entire dorso-ventral axis, except forthe roofplate (arrow in Fig. 4D) and the ¯oorplate (arrowin Fig. 4F). In addition, in the midbrain, Sp5 starts to beexpressed from E10.0 onwards in the tegmentum (tm in Fig.4F±H), while the mesencephalic ¯oorplate is Sp5-negative(arrow in Fig. 4F).In contrast to the mesencephalon, the pretectum alsoexpresses Sp5 in lateral and ventral regions, at least untilE10.5. Here, the transcripts are detectable as a stripe at thebase of the pretectum and hypothalamus, within the anlageof the mammilary bodies (mb in Fig. 4F±H,J). Noteworthy,in the entire ¯oorplate of the brain, only the hypothalamus isSp5-positive (Fig. 4J).1.3. Sp5 expression in the developing spinal cordIn the spinal cord, Sp5 expression is ®rst detectablearound E8.0. The hybridization signal has a peak withinthe upper thoraxic region and fades towards the rhomben-D. Treichel et al. / Mechanisms of Development 101 (2001) 175±179 177Fig. 2. Sp5 shows two phases of expression during gastrulation and organogenesis. Whole-mount in situ hybridizations on mouse embryos from E7.25 to E10.5.The hybridizations were done with a PmlI fragment containing the 3 0 part of the cDNA up to nt 676 as previously described. In (A±D) the anterior is to the left,and the posterior is to the right. The arrows in (E,F) point to the starting CNS expression, the arrows in (H±J) point to the fading expression in the lateralmesencephalon.Fig. 3. Sp5 expression during: (A±C), primary; and (D±F), secondary gastrulation. (A) Cross-section through the embryo in (2A). The arrows are pointing toindividual visceral endoderm cells. (B) Cross-section through the posterior end of an E8.25 embryo. (C) Cross-section through the posterior end of the embryoin (2H). The arrow points to the de®nitive endoderm, the double arrow to the notochord. (D) Tail bud of a whole-mount in situ hybridized embryo; thenotochord expression is marked with an arrow. (E) Cross section through the tail in (D). The arrow points to the ectoderm. (F) Cross-section through thenotochord at the level of the arrow in (D). The double arrow points to the single Sp5-positive cell inside the notochord, the arrows to individual surroundingSp5-negative cells. (A±C,E,F) Fifteen micrometer paraf®n sections through whole-mount in situ hybridized embryos. d, dorsal; md, mesoderm; np, neuralplate; v, ventral.D. Treichel et al. / Mechanisms of Development 101 (2001) 175±179178Fig. 4. Sp5 expression during organogenesis. Sp5 expression in: (A±J) the brain; (K,L), the spinal cord; (M±O), the otic placode/vesicle; (P,Q), the trigeminalganglia; (R±T), the branchial pouches and clefts; (U±X), the somites; and (Y), the limbs (Y) at various stages of embryonic development. (A,B) Top view of thedeveloping brain. The arrow in (A) indicates the expression in the neural folds of the mesencephalon/diencephalon. (C) Lateral view of the head. The arrowpoints to the lateral mesencephalon which is at this stage Sp5-negative. (D) The arrow indicates the Sp5-negative roofplate at the MHB. (E) The arrow points tothe Sp5-negative roofplate of the anlage of the epithalamus and pretectum. (F) Top view of the developing brain after removal of the dorsal telencephalon. Thearrow points to the Sp5-negative ¯oorplate of the MHB. (G,H) Lateral and top view of the head after removal of the dorsal telencephalon, mesencephalon andanterior rhombencephalon, respectively. (I) Frontal section within the mesencephalon through the head of the embryo shown in (A). (J) Section showingspecimen in the plane of the tuberal hypothalamus and the anlage of the mammilary bodies. (K) Dorso-lateral view of the spinal cord of a stained embryo. (L)Cross section through the embryo shown in (K) at the level of the rostral spinal cord. The arrow points to the expression in the dorsal most alar plate of thespinal cord. (M) Lateral view of the embryo showing the otic vesicle as an additional site of expression. (N) Dorso-lateral view of the otic vesicle. (O) Frontalsection through the otic vesicle in (N). (P) Dorso-lateral view of the embryo head. The arrow points to the beginning of expression in the trigeminal ganglia. (Q)Cross section through the trigeminal ganglion of an E10.50 embryo. (R) Lateral view of the embryo head. The arrow points to the ®rst branchial cleft. (S)Frontal section through the branchial cleft and pouch in (R). (T) Lateral view showing Sp5 expression into the branchial clefts 1, 2 and 3. (U) Dorsal view ofE8.25; and (V), dorso-lateral view of E10.00. The arrows point to the Sp5 expressing somites. (W) Sagittal section through the embryo at the level of the heart.The arrow points to the Sp5 expression in the somites. (X) Cross-section through the embryo at the level of the arrow in (V). The arrow points to the epaxialdermomyotome lip. (Y) dorsal view of the forelimb. The arrow points to Sp5 expressing cells within the mesenchymal tissue of the limb bud. (A±H,K,M,N,P,R,T±W,Y) Whole-mount ISH; (I,S,X), paraf®n sections (18 mm); and (J,L,O,Q,W), vibratome sections (35 mm). ant, anterior; d, dorsal; e, anlageof the epithalamus; ec, ectoderm; en, endoderm; m, mesencephalon; mb, anlage of the mammilary body; nt, neural tube; ov, otic vesicle; p, pretectum; post,posterior; sc, spinal cord; t, telencephalon; tec, prospective tectum of the mesencephalon; tm, tegmentum of the mesencephalon; 1, 2, 3, 4, branchial arches; 4v,fourth ventricle. ISH was performed according to Wilkinson (1992). The anatomical analysis was done according to Puelles and Rubenstein (1993).cephalon and the tail bud. Along the dorso-ventral axis, theexpression is con®ned to the dorsal most part of the alarplate (Fig. 4K,L).1.4. Additional sites of Sp5 expressionAt around E10.0, the trigeminal ganglia are strongly posi-tive (arrow in Fig. 4P,Q). Furthermore, the neurogenic oticplacodes/vesicles express Sp5. The initially (E9.00) faintISH signal becomes very strong in the otic vesicles atE10.0, and is con®ned to its medio-dorsal part (Fig. 4M±O).In addition, the branchial (pharyngeal) arch system, thesomites and the limbs show a dynamic Sp5 expression.While the ®rst three branchial pouches (endoderm) andclefts (ectoderm) express the gene continuously betweenE8.5 and 10.5 (Fig. 4R±T), the fourth pouch and cleft areSp5-negative (Fig. 4T). In the somites, the mRNA is onlybrie¯y detectable. About 2 h after the generation of an indi-vidual somite, Sp5 is expressed in its dorsal tip which willdevelop into the epaxial dermomyotome lip. Sp5-mRNAlevels reach a peak after about 15 h (arrows in Fig. 4U±X)and gradually fade away during the maturation of the indi-vidual somite.In the limb buds, the Sp5 gene is ®rst expressed dorsallyin scattered cells within the mesenchyme at E10.50 (arrowin Fig. 4Y). Later on, Sp5-positive cells are also found in theventral part (data not shown).AcknowledgementsThe authors would like to thank A. Stoykova for her helpin identifying brain structures, L. Wittler for help in de®ningearly embryonic structures and G. Goudreau, A. Mansouriand M. Kessel for critical reading of the manuscript.ReferencesEl-Baradi, T., Pieler, T., 1991. Zinc ®nger proteins: what we know and whatwe would like to know. Mech. Dev. 35 (3), 155±169.Philipsen, S., Suske, G., 1999. A tale of three ®ngers: the family of mamma-lian Sp/XKLF transcription factors. Nucleic Acids Res. 27, 2991±3000.Puelles, L., Rubenstein, J.L., 1993. Expression patterns of homeobox genesin the embryonic mouse forebrain suggest a neuromeric organization.Trends Neurosci. 16 (11), 472±479.Wilkinson, D.G., 1992. Whole-mount in situ hybridization of vertebrateembryos. In: Wilkinson, D.G. (Ed.). In Situ Hybridization, A PracticalApproach, IRL Press, Oxford, pp. 75±84.Wimmer, E.A., Jackle, H., Pfei¯e, C., Cohen, S.M., 1993. A Drosophilahomologue of human Sp1 is a head-speci®c segmentation gene. Nature366 (6283), 690±694.Wimmer, E.A., Frommer, G., Purnell, B.A., Jackle, H., 1996. Buttonheadand D-Sp1: a novel Drosophila gene pair. Mech. Dev. 59 (1), 53±62.D. Treichel et al. / Mechanisms of Development 101 (2001) 175±179 179

The novel transcription factor gene Sp 5 exhibits a dynamic and highly restricted expression pattern during embryogenesis.

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The novel transcription factor gene Sp 5 exhibits a dynamic and highly restricted expression pattern during embryogenesis.

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