QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online December 12, 2006
doi: 10.1242/10.1242/dev.02701

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, C. Articles by Mansour, S. L. Search for Related Content PubMed PubMed Citation Articles by Li, C. Articles by Mansour, S. L. Social Bookmarking                         What's this?

Dusp6 (Mkp3) is a negative feedback regulator of FGF-stimulated ERK signaling during mouse development

Chaoying Li¹, Daryl A. Scott^1,*, Ekaterina Hatch¹, Xiaoyan Tian² and Suzanne L. Mansour^1,

¹ Department of Human Genetics, University of Utah, 15 N 2030 E RM 2100, Salt Lake City, UT 84112-5330, USA.
² Division of Radiobiology, University of Utah, 729 Arapeen Drive #2334, Salt Lake City, UT 84108-1218, USA.

View larger version (94K): [in this window] [in a new window]   Fig. 1. Dusp6 expression correlates with particular Fgfrs and with most areas of dpERK expression. (A,D,G) Whole-mount RNA in situ hybridization (WmISH) of mouse embryos with the Fgfr probes indicated at the bottom right of each panel. (B,E,H) WmISH of embryos with a Dusp6 probe. (C,F,I) Whole-mount immunostaining of embryos with antibody directed against dpERK. Embryo age is indicated at the bottom left of each panel. (J-S) Transverse sections of E10-10.5 WmISH embryos illustrating potential FGF signaling pathways in the limb bud (J-O) and branchial arches (P-S). Probes are noted at the bottom left of each panel. ba, branchial arches; ec, ectoderm; lb, limb bud; mes, mesenchyme; mhb, midhindbrain junction; op, olfactory pit; ov, otic vesicle; so, somites. Scale bars: 100 µm in O for J-O; 100 µm in S for P-S.

View larger version (99K): [in this window] [in a new window]   Fig. 2. Dusp6 expression depends on signaling through FGF receptors. WmISH of wild-type and homozygous Fgfr hypomorphic (h) mutant littermates with a Dusp6 probe (A,B) Fgfr1, E9.5; (C-H) Fgfr2IgIII, E8.5. Genotype of each embryo is indicated in the upper left of each panel.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Gene targeting at the Dusp6 locus. (A) Structure of the linearized Dusp6 targeting vector and depiction of the wild-type Dusp6 allele (Dusp6+), the correctly targeted mutant allele in ES cells (Dusp6LACN) and the targeted allele found in mice following expression of CRE in germlinetransmitting chimeras (Dusp6L). Mouse genomic Dusp6 DNA is depicted with solid thick lines; dotted lines indicate Dusp6 genomic DNA not present in the targeting vector; open boxes indicate untranslated regions; solid boxes indicate protein coding regions. The LacZ gene (nls-lacZpA) is shown as a dark gray box; the Cre/Neo `suicide cassette' (ACN) as a light gray box; the stop codon in the DUSP6 frame in exon 3 as an asterisk. Flanking thymidine kinase genes (TK1 and TK2, transcriptional orientation indicated by arrows) and the plasmid backbone are depicted as open boxes. Recognition sites for NdeI are indicated by `N'; probes used for Southern analysis by black bars. Numbered arrows indicate the identity, position and directionality of primers used in PCR assays. (B) Southern blot hybridization assay demonstrating correct targeting of Dusp6 in ES cells. NdeI-digested DNA from the R1 ES cell line (R1), a cell line with a random insertion of the targeting vector (Ran) and a targeted cell line (Tar) was probed sequentially with 3' and Cre probes. Correctly targeted cell lines had a novel 7.0 kb fragment that hybridized with both probes. (C) PCR assay used to detect the stop-codon-containing insertion in exon 3. DNAs isolated from correctly targeted ES cells (Tar), a control random integrant (Ran) and wild-type cells (R1) were PCR-amplified using primers 376 and 331. Targeted cell lines (TAR*) that produced both the wild-type (187 bp) and the insertion amplicon (198 bp) were selected for germline transmission. (D) PCR assay used to genotype offspring of Dusp6+/L intercrosses. Tail DNAs were PCR-amplified with primers 344, 309 and 315. The mutant allele yielded a 363 bp band and the wild-type allele yielded a 499 bp band. (E) Northern blot hybridization of mRNA isolated from E11.5 embryos of the indicated genotypes was probed with a fragment of Dusp6 3' UTR, revealing a wild-type transcript of 3 kb in +/+ and +/-samples that was absent from the -/-sample. A minor read-through transcript of 7.5 kb was evident in +/- and -/-samples, but due to the targeting strategy, it is incapable of encoding functional DUSP6. Rehybridization with a Gapdh probe is shown below.

View larger version (74K): [in this window] [in a new window]   Fig. 4. DUSP6 is a negative feedback regulator of the ERK pathway. (A-C) Immunostaining of wild-type and Dusp6-/- embryos with anti-dpERK. (A) An E9.5 Dusp6-/- embryo has increased levels of dpERK in the limb (black arrow) relative to that of a wild-type embryo (white arrow). (B) Limb bud of wild-type E10.5 embryo. (C) Limb bud of Dusp6-/- embryo with increased levels of dpERK. (D-F) RNA in situ hybridization of E10.5 embryos shows increasing levels of Erm transcripts as Dusp6 levels decrease. Genotype of each embryo is indicated in the lower left of each panel. Northern blot hybridization of mRNA isolated from pooled E11.5 embryos (G) or individual adult brains (H) of the indicated genotypes and probed sequentially with a Dusp6 5' UTR probe, a LacZ probe and Gapdh shows that embryos lacking Dusp6 have increased levels of transcripts initiated from the Dusp6 promoter. Note that the embryonic Gapdh panel is identical to the one shown in Fig. 3E, because it comes from the same blot. (I) Diagrams of the transcripts produced by wild-type (Dusp6+) and mutant (Dusp6L) alleles and the locations of the probes (bars above transcripts) used for hybridization. Open boxes, untranslated Dusp6 sequence; black boxes, DUSP6 coding sequence; gray box, LacZ cassette.

View larger version (98K): [in this window] [in a new window]   Fig. 5. Reduction or loss of Dusp6 can result in skeletal dwarfism, altered growth plates, coronal craniosynostosis and delayed ossification of the phalanges. (A) Small Dusp6+/L P10 pup (+/L) and wild-type (+/+) littermate. (B-E) Frontal sections (4 µm) of undecalcified P14 proximal tibia from wild-type and small Dusp6L/L pups stained with silver nitrate and basic fuchsin. (B,C) The ossification, hypertrophic and proliferation zones are indicated. Higher magnification views of Toluidine Blue-stained sections of wild type (D) and mutant (E) growth plates. (F-I) Dorsal views (anterior is to the left) of Alizarin Red (bone) and Alcian Blue (cartilage)-stained skulls of P5 and P10 pups. Black arrows in F and H indicate the open coronal sutures in wild-type skulls and white arrows in G and I indicate the fused coronal sutures in small homozygous and heterozygous mutant pups, respectively. (J-M) Dorsal views of P0 and P5 control and small heterozygous or homozygous mutant autopod skeletons. (J,K) Right hands, black and gray arrows indicate the presence and absence, respectively, of the primary ossification center of the middle phalanx of the fifth digit. (L,M) Right feet, black and gray arrows indicate the presence and absence, respectively, of the primary ossification centers of the middle phalangeal ossification centers for digits 2-4. Age and genotype are indicted to the lower left and lower right of each panel, respectively. Scale bars: 0.5 mm in B,C; 0.2 mm in D,E. hz, hypertrophic zone; oz, ossification zone; pz, proliferation zone.

View larger version (105K): [in this window] [in a new window]   Fig. 6. Staining for bone and cartilage reveals abnormalities of the otic capsule and ossicles in mature and developing small Dusp6-deficient animals. (A,B) Lateral views of left temporal bones of control (A) and small heterozygous hearing-impaired (B) P22 animals. The shape of the opening to the middle ear cavity is emphasized with a black line, and an abnormal notch in the heterozygote is indicated with an arrow. (C,D) Lateral views of left ears of P0 control (C) and small heterozygous (D) P0 animals are shown with some of the cochlea dissected away to enable better visualization of the ossicles. The malleus and stapes and incus are indicated. (E,F) Lateral views of right inner ears of P5 wild-type (E) and small Dusp6L/L (F) pups. Black arrow in E indicates a normal dorsal otic capsule, and gray arrow in F indicates failure of the corresponding region of the mutant otic capsule to form Alcian Blue-positive cartilage. (G,H) Right ossicles dissected from P5 wild-type (G) and small Dusp6L/L (H) pups. All three mutant ossicles have regions in which cartilage (blue) is missing or weakly staining. (I,J) Lateral views of left temporal bones from wild-type (I) and small, Dusp6L/L (J) P10 pups. Black arrow in I indicates normal development of the dorsal otic capsule, whereas gray arrow in J indicates that the corresponding region of the mutant capsule has failed to form Alizarin Red-positive bone. (K,L) Left middle ears from wild-type (K) and small Dusp6L/L (L) P10 pups. The mutant malleus is malformed. i, incus; m, malleus; s, stapes.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter