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First published online 17 December 2003
doi: 10.1242/dev.00946

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Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4 activity during forebrain development

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA

View larger version (52K): [in a new window]   Fig. 1. Genomic organization and expression of the Tsg gene. (A) Schematic representation of the wild-type and targeted alleles of Tsg. The 5' probe used for the Southern blot is indicated (probe). The oligonucleotides used for PCR genotyping are shown as arrows: Tsg22, Tsg23 and lacZ3. The position of the exons is indicated by black boxes and the recombination arms shown in red. The following restriction sites are indicated: B, BamHI; K, KpnI; N, NsiI; S, SacI. (B) Southern blot analysis of the 5' genomic region of the Tsg gene. DNAs extracted from wild-type, heterozygous and homozygous Tsg mutant animals were digested with SacI. The 170 bp SacI-NsiI probe fragment detected a wild-type band of 9.2 kb and a knock out (KO) band of 3.7 kb. (C) PCR genotyping of mice carrying the Tsg mutation. The wild-type band of 329 bp was obtained using the Tsg22 and Tsg23 primers. The KO band of 480 bp was obtained using the Tsg22 and lacZ3 primers. (D) RT-PCR analysis of RNAs extracted from E13.5 wild-type and Tsg-/- embryos showing the absence of Tsg mRNA in Tsg-/- embryos. HPRT expression was used as a control. (E-H,E'-H') Comparison of the expression pattern of Tsg by whole-mount in situ hybridization and ß-galactosidase staining. (E) Tsg is expressed in the anterior visceral endoderm and the primitive streak. (F,G) As gastrulation proceeds, Tsg is found in the mesodermal layer. (H) At E8.5 Tsg is present in the endoderm of the gut, the ventral neural tube and the dorsal region of the eye vesicle. (E'-H') The expression of the lacZ transgene recapitulates the expression of endogenous Tsg. (I) Transverse section through the anterior region of an E8.0 embryo showing Tsg mRNA in the endoderm of the foregut, ventral neuroectoderm and the head mesenchyme. (J) Section of an E8.5 embryo showing the expression of Tsg mRNA in the basal diencephalon. ave, anterior visceral endoderm; bd, basal diencephalon; bm, basal mesencephalon; ev, eye vesicle; en, endoderm; fg, foregut; hg, hindgut; hm, head mesenchyme; mw, mesodermal wings; ne, neuroectoderm; ps, primitive streak; A, anterior; L, left; P, posterior; R, right.

View larger version (61K): [in a new window]   Fig. 2. Skeletal abnormalities in Tsg-/- mice. (A) Comparison of 5-week-old wild-type and Tsg-/- mice. The Tsg-/- mouse is small and displays a short tail with multiple kinks. (B) X-ray of the caudal region of wild-type and Tsg-/- adult mice. The tail vertebrae in the mutant are shorter (compare tail vertebra 7, TV7) and have less X-ray opacity (inset). (C-C') Alcian Blue preparations of E14.5 wild-type and Tsg-/- embryos in lateral view. Note the defects in cervical and thoracic neural arch cartilages in Tsg-/- embryos. (D-D') Alcian Blue/Alizarin Red preparations of cervical vertebrae of 2-week old wild-type and Tsg-/- mice. The cervical neural arches are not fused dorsally in the mutant. (E-E') Alcian Blue/Alizarin Red preparations of the caudal regions of wild-type and Tsg-/- newborn mice. Note the presence of two centers of ossification in the mutant rather than a single one in the wild-type. The seventh tail vertebra is indicated. (F,F',G,G') Skeletal elements of the tail of 2-week old wild-type and Tsg-/- mice stained with Alcian Blue/Alizarin Red. (F') Abnormal vertebrae present a bow-tie shape and are shorter than the wild-type (compare TV7). (G') One ossification center has failed to form, producing hemi-vertebrae with misaligned articular surfaces, which cause the kinks. at, atlas; ax, axis; C3-7, cervical vertebrae 3-7; na, neural arch; T4, thoracic vertebra 4.

View larger version (72K): [in a new window]   Fig. 3. Development of vertebral defects in Tsg mutant embryos. (A-A') Hematoxylin and Eosin staining of sagittal sections of the tail of E12.5 wild-type and Tsg-/- embryos. (B-B') Mallory's tetrachrome staining of sagittal sections of E15.5 wild-type and Tsg-/- tails. Vertebral bodies (vb); intervertebral regions (iv) consisting of an outer annulus (oa) and inner annulus (ia); nucleus pulposus (np) containing the remnants of the notochord. In the wild type, the np is surrounded by the prechondrogenic mesenchyme of the ia. (B') The Tsg mutant vertebral bodies are more rectangular and narrower than wild type; the intervertebral region is expanded, notochordal cells are not resorbed in the vertebral bodies. (C-C') Alcian Blue staining of E14.5 wild-type and Tsg-/- embryos. Note the smaller vertebral bodies and persistence of the notochord (n) in the mutant. (D-D') ß-gal staining of the tail region of E14.5 wild-type and Tsg-/- embryos showing expanded Tsg expression in intervertebral prechondrogenic mesenchyme and in the notochord of the mutant. (E-E') Expression of Bmp4 at E14.5 in wild-type and Tsg-/- embryos. (E) Bmp4 is expressed in the intervertebral annulus fibrosus, which is expanded in the mutant (E'). cm, condensed mesenchyme; scl, sclerotome; s, somite; um; uncondensed mesenchyme; sp, spinal chord.

View larger version (129K): [in a new window]   Fig. 4. Tsg-/-;Bmp4+/- neonates display holoprosencephaly. (A-A') Lateral view of the heads of wild-type and Tsg-/-;Bmp4+/- newborn mice. Tsg-/- or Bmp4+/- are indistinguishable from wild type. The Tsg-/-;Bmp4+/- mutant lacks a mouth opening, has only one nostril, and the external ear has an abnormal setting. (B-B') Frontal sections through the snout region of the same neonates stained with Mallory's tetrachrome. The lack of nasal septum (ns) indicates holoprosencephaly. The lower jaw is reduced. gm, genioglossus muscle; lt, lower tooth; md, mandible; mu, muscle; nc, nasal cavity; sp, secondary palate; to, tongue; ut; upper tooth.

View larger version (62K): [in a new window]   Fig. 5. Holoprosencephaly in Tsg-/-;Bmp4+/- embryos at E15.5. (A-A') E15.5 wild-type and Tsg-/-;Bmp4+/- embryos. The plane of sections shown below are designated b,b',c and c'. (B,B',C,C') Histological sections were stained with Hematoxylin and Eosin. The lateral ganglionic eminence (lge) is missing and the floor of the diencephalon (fd) is greatly thickened in the mutant. Note the fusion of the lateral ventricles, owing to the lack of a medial wall (mw) and the lack of nasal septum (ns). In addition, note the empty orbits (ob), lack of lens (ls) and retina (re) of the eyes. di, diencephalon; hy, hypothalamus; ll, lower lid; lv, lateral ventricle; ol, olfactory epithelium; to, tongue; ul, upper lid; vi, follicles of vibrissae; 3V, third ventricle.

View larger version (92K): [in a new window]   Fig. 6. Comparative expression of Tsg, Bmp4 and Chd. Whole-mount in situ hybridization was performed between E8.0 and E10.0 using the gene markers indicated. (A) Tsg expression at E8.0, frontal view. (B,C) Frontal and lateral views of Bmp4 expression at E8.0. Inset shows expression in the surface ectoderm (se) at E7.75. (D) Chd expression at E8.0 in the node (no), notochord, prechordal plate and endoderm. (E) ß-Gal staining showing diffuse expression of Tsg at E9.0. (F) Expression of Bmp4 at E9.0. Inset shows expression in the branchial arch as early as E8.5. (G) Expression of Tsg at E10.0. (H) Expression of Bmp4 at E10.0. a, allantois; a1, first branchial arch; aip, anterior intestinal portal; bd, basal diencephalon; bm, basal mesencephalon; bt, basal telencephalon; dt, dorsal telencephalon; fn, frontonasal process; fp, floor plate; h, hyoid arch; lb, limb bud; m, mesoderm; md, mandibular; mx, maxillary; nf, neural fold; vm, ventral mesoderm.

View larger version (123K): [in a new window]   Fig. 7. Defective development of telencephalic vesicles and first pharyngeal arch in Tsg-/-;Bmp4+/- embryos. The phenotypes of Tsg-/- and Bmp4+/- were indistinguishable from wild-type. (A-A') Frontal view of E10.5 embryos stained with Dlx5. Insets show lateral views. The right mandibular component of the first arch is missing in the compound mutant (arrow). hy, hyoid arch; md, mandibular component of the first pharyngeal arch. (B,B',C,C') Lateral view of E8.5 and E8.25 embryos hybridized with Fgf8. (B) At E8.5 Fgf8 is expressed in the proximal ectoderm of the first pharyngeal arch (a1), isthmus (is), commissural plate (cp) and tailbud (tb). (B') Expression in the pharyngeal arch and the commissural plate (indicated by an asterisk) is missing in the mutant. (C-C') At E8.25, Fgf8 is present in the anterior neural ridge (anr), isthmus, pharyngeal endoderm (en) and tailbud; no differences in Fgf8 expression were observed at this stage. (D-D') At E8.5 Shh is expressed in the basal mesencephalon (bm), basal diencephalon (bd) and basal telencephalon (bt) and the notochord. Expression in the basal diencephalon and basal telencephalon is missing in the mutant (asterisk). (E-E'). Expression of mouse Cer1 in the anterior visceral endoderm (ave) of wild-type and Tsg-/-;Bmp4+/- embryos at E6.5. (F-F') Expression of Hex in the anterior endoderm (ae) and node (no) of wild-type and Tsg-/-;Bmp4+/- embryos at E7.5.