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. 2000 Feb 15;19(4):655-61.
doi: 10.1093/emboj/19.4.655.

Transcription factor Sp3 is essential for post-natal survival and late tooth development

P Bouwman  1 , H Göllner, H P Elsässer, G Eckhoff, A Karis, F Grosveld, S Philipsen, G Suske
Affiliations

Transcription factor Sp3 is essential for post-natal survival and late tooth development

P Bouwman et al. EMBO J. .

Abstract

Sp3 is a ubiquitously expressed transcription factor closely related to Sp1 (specificity protein 1). We have disrupted the mouse Sp3 gene by homologous recombination. Sp3-deficient embryos are growth retarded and invariably die at birth of respiratory failure. The cause for the observed breathing defect remains obscure since only minor morphological alterations were observed in the lung, and surfactant protein expression is indistinguishable from that in wild-type mice. Histological examinations of individual organs in Sp3(-/-) mice show a pronounced defect in late tooth formation. In Sp3 null mice, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblast-specific gene products. Comparison of the Sp1 and Sp3 knockout phenotype shows that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy.

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Figures

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Fig. 1. Targeted disruption of the mouse Sp3 gene. (A) Schematic representation of the Sp3 protein structure. The glutamine-rich activation domains A and B, the inhibitory domain (ID) and the zinc fingers (black bars) of the DNA-binding domain (DBD) are indicated. Connecting lines with the corresponding murine Sp3 gene regions indicate the derivation of the N–terminal part of the Sp3 protein. Both glutamine-rich activation domains (A and B) are encoded by a single large exon. In the targeting vector, this exon was replaced by a cassette containing a splice acceptor site (SA), an internal ribosomal entry site (IRES) and a lacZ–neo fusion gene (LacZ Neo) (Mountford et al., 1994). The positions of relevant restriction sites (B, BamHI; E, EcoRI; H, HindIII; P, PstI; N, NotI) and the probe used for Southern blotting are indicated. Restriction of genomic DNA with EcoRI and hybridization with the indicated probe detects a >11 kbp fragment of the wild-type allele and a 5.4 kbp fragment of the mutated allele. (B) Southern blot analysis of targeted ES cells. (C) PCR analysis of mouse embryos. The primers produce a 273 bp DNA fragment from the wild-type and a 593 bp fragment from the targeted allele.
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Fig. 2. Sp3 protein expression in wild-type and Sp3 mutant mice. (A) Western blot analysis of wild-type, Sp3+/– and Sp3–/– animals. Nuclear extracts (6 μg of protein) from brains of wild-type (WT), heterozygous (Sp3+/–) and Sp3-deficient (Sp3–/–) mice were fractionated through 7.5% SDS–polyacrylamide gels, stained with Coomassie Blue (lanes 1–4; M, marker lane) or blotted on nitrocellulose filter and incubated with Sp3-specific antibodies (lanes 5–7). (B) Electrophoretic mobility shift assay of GC box binding activity in wild-type (WT) and Sp3-deficient (Sp3–/–) mouse embryonic fibroblasts. Crude nuclear extracts (1.25 μg of protein) were incubated with 32P-labeled GC box oligonucleotide in the absence (lanes 8 and 11) or presence of antisera against Sp1 (lanes 9 and 12, αSp1) or a mixture of antisera against Sp1 and Sp3 (lanes 10 and 13, αSp1/3).
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Fig. 3. Sp3 mutant E18.5 embryos. Genotyped E18.5 embryos of one litter that are wild-type (+/+), heterozygous (+/–) or homozygous (–/–) for the targeted Sp3 gene locus. Homozygous mutants are ∼25% smaller than wild-type and heterozygous embryos.
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Fig. 4. Histological and ultrastructural analyses of lung tissue in wild-type and Sp3–/– mice. Lung tissue at E18.5 of wild-type (WT) (A, C and E) and Sp3 knockout mice (–/–) (B, D and F) is shown in histological sections stained with hematoxylin and eosin (A–D), or in ultrastructural sections (E and F). At low magnification (A and B), lung tissue from Sp3–/– mice is more compact, with a smaller mean diameter of alveolar spaces and a thicker septum between individual alveoli. At higher magnification (C and D), capillaries are regularly seen at the inner surface of the alveoli in wild-type mice (C, arrow). Lung tissue from Sp3-deficient mice exhibits small alveoli with the inner surface lined with cuboid pale cells (D, arrowheads). Ultrastructural analysis revealed that the apical membranes of these cells together with a small rim of cytoplasm (F, large arrows) are disrupted from their original location near the nucleus (N, small arrows in F). These lead to artificial intracellular spaces (asterisks) filled with slightly electron-dense material. In wild-type lung tissue, a regular air–blood boundary (between arrowheads in E) consisting of an endothelial cell, a basement membrane and a pneumocyte type I is observed. An erythrocyte (‘Er’ in E) filling the space of a capillary and surfactant (‘S’ in E and F) are indicated. Bar in (A) and (B), 100 μm; in (C) and (D), 10 μm; in (E) and (F), 0.5 μm.
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Fig. 5. Expression of putative Sp3 target genes in the lung. RNA was extracted from lungs of wild-type (+/+), Sp3+/– (+/–) and Sp3-deficient (–/–) E18.5 embryos, subjected to electrophoresis through 1.2 or 0.8% formaldehyde–agarose gels and transferred to nylon membranes. The filters were hybridized with cDNA fragments of surfactant proteins A (SP-A), B (SP-B), C (SP-C) and D (SP-D), uteroglobin/Clara cell secretory protein (UG/CCSP; two different membranes), lung Krüppel-like factor (LKLF) and thyroid transcription factor (TTF–1). As a control, the filters were probed with an 18S rRNA-specific oligonucleotide.
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Fig. 6. Histological analysis of developing teeth in wild-type and Sp3–/– mice. Teeth at E18.5 of wild-type (A and C) and Sp3-deficient mice (B and D) are shown in histological sections stained with PAS. At low power magnification, the developing tooth of wild-type mice shows a continuous row of ameloblasts (‘Am’, arrow) and odontoblasts (‘Od’, arrow) interrupted by small vessels interspersed between the odontoblasts (black arrowheads in C). Between the ameloblasts and odontoblasts, a sheet of pink and purple stained material that contains dentin and enamel is visible (marked in A and C by two white arrowheads). This layer of secretory products has been disrupted from the ameloblasts, leaving an artificial space (asterisks in A and C) that is regularly observed in wild-type mice of this age, but not in Sp3 null mice. In contrast, Sp3 knockout mice show only a pink layer (white arrowheads in B and D). In addition, only a few vessels are observed in the dental papilla, leaving the odontoblasts in a continuous row (‘Od’ with arrow in B and bottom cell layer in D). (E–H) PAS-stained histological sections of E18.5 tissues with no obvious morphological alterations in knockout mice. (E and F) Pancreas. (G and H) Liver; the arrow in (H) points to a megakaryocyte. Liver cells have a pale cytoplasm and are interspersed by small clusters of hematopoetic cells. Bar in (A) and (B), 100 μm; in (C) and (D), 10 μm; in (E–H), 30 μm.
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Fig. 7. Expression of putative Sp3 target genes in teeth. RNA was extracted from head or jaws of wild-type (+/+), Sp3+/– (+/–) and Sp3-deficient (–/–) newborn or E18.5 embryos, subjected to electrophoresis through 1.2% formaldehyde–agarose gels and transferred to nylon membranes. The filters were hybridized with cDNA fragments encoding ameloblastin (A and E), amelogenin (B and F), DMP1 (C) and tuftelin (D). As a control, the filters were probed with an 18S rRNA-specific oligonucleotide.
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