doi: 10.1128/MCB.25.16.7042-7053.2005.
Mice deficient in the fused homolog do not exhibit phenotypes indicative of perturbed hedgehog signaling during embryonic development
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- PMID:16055716
- PMCID: PMC1190231
- DOI: 10.1128/MCB.25.16.7042-7053.2005
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Mice deficient in the fused homolog do not exhibit phenotypes indicative of perturbed hedgehog signaling during embryonic development
Miao-Hsueh Chen et al. Mol Cell Biol.2005 Aug.
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Abstract
Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. To understand how a single Hh signal is capable of generating distinct readouts in Hh-responsive cells requires elucidation of the signal transduction cascade at the molecular level. Key components that mediate Hh signal transduction downstream of the receptor include Fused (Fu), Suppressor of fused (Sufu), and Costal-2 (Cos2) or the vertebrate homologs Kif27/Kif7. Studies with both invertebrates and vertebrates have led to a model in which a protein complex composed of Fu, Sufu, and Cos2 controls the processing, activity, and subcellular distribution of the Ci/Gli transcription factors responsible for Hh target gene activation. These converging results obtained with different species reaffirm the prevailing view of pathway conservation during evolution. Genetic studies of Fu, Sufu, and Kif27/Kif7 in mice are required to provide further verification of Hh pathway conservation. To this end, we generated a gene-targeted allele of Fu in mice. Surprisingly, our analysis indicates that Fu-deficient mice do not exhibit any embryonic phenotypes indicative of perturbed Hh signaling. This could be due to either functional redundancy or Hh pathway divergence and clearly indicates greater complexity of Hh signaling in vertebrates.
Figures
Expression ofFu during mouse embryonic development. (A to C, G to I, and M to O) Whole-mount in situ hybridization using digoxigenin-labeled riboprobes on wild-type embryos at 8.5 (A and G), 9.5 (B and H), and 10.5 (C, I, and M) dpc andFuΔE1-5 embryos at 10.5 dpc (N and O). All views are lateral, with the exception of M and N, which are dorsal views of the forelimb.Shh is expressed in several signaling centers, whileFu is broadly expressed in wild-type embryos, including in domains of Hh-responsive cells. Only backgroundFu signal could be detected inFuΔE1-5 embryos. Embryos in panels A and G; B and H; C, I, and O; and M and N were photographed at the same magnification, respectively. (D to F, J to L, and P to R) Isotopic in situ hybridization using [33P]UTP-labeled riboprobes (pink) on paraffin sections of 10.5-dpc wild-type (D and J) andFuΔE1-5 (P) embryos at the forelimb-heart level, 13.5-dpc wild-type (E and K) andFuΔE1-5 (Q) embryos, and 18.5-dpc wild-type (F and L) andFuΔE1-5 (R) lungs. Panels D, J, and P; E, K, and Q; and F and R were photographed at the same magnification, respectively. Panel L was photographed at a higher magnification than panels F and R.Fu signal is absent inFuΔE1-5 embryos. TheFu probe used for in situ hybridization is derived fromFu cDNA sequences that correspond approximately to exons 2 to 9. Probes derived from several other regions ofFu cDNA, including exons 2 to 5 and exons 27 and 28, gave identical expression patterns (data not shown). TheFu sense probes did not yield signals above the background (data not shown). nt, neural tube; fp, floor plate; nc, notochord; lg, lung; lv, liver.
Alternative splicing ofFu transcripts during mouse embryogenesis. (A) Northern blot analysis of poly(A)+ RNA isolated from 7-, 11-, 15-, and 17-dpc wild-type mouse embryos (Clontech). Two major species ofFu transcripts, approximately 4.7 and 4.1 kb, respectively (arrows), were detected. In adult mice,Fu is mainly expressed in testis (data not shown). TheFu probe used for hybridization is derived fromFu cDNA sequences that correspond approximately to exons 2 to 9. The same membrane was rehybridized with aGAPDH probe, which serves as the loading control. (B) RT-PCR using RNA derived from wild-type 10.5-dpc mouse embryos in combination with primers spanning the indicated exons. Alternative splicing of the region containing exons 11 to 16 was detected by PCR. This was subsequently verified to be alternative splicing of exons 13 to 15 by sequencing. The white arrow points to the PCR product without exons 13 to 15, which is much less abundant than the upper band containing exons 13 to 15. No alternative splicing of exon 8 or exon 23, as reported for humanFu transcripts, was detected in this assay. A PCR product was amplified with primers derived from exons 1 and 5, respectively. Sequence analysis of the PCR product revealed that it contains exons 1 and 3 to 5, and whether it represents the dominant form during mouse embryogenesis remains to be further investigated. RT-PCR using primers derived from exons 2 and 5 failed to produce a product (data not shown). (C) Schematic diagram of theFu genomic locus and possible alternative splicing. TheFu genomic locus consists of 28 exons (E1 to E28). The ATG translation start codon is predicted to reside in the third exon (E3), and the TGA stop codon is in the 28th exon (E28). Alternative splicing of exons 13 to 15 is labeled in red and blue, respectively, and potential alternative splicing of exons 1 and 2 is labeled with lime and olive, respectively. Splicing of common exons is not labeled. The longerFu transcript, containing exons 13 to 15, appears to constitute the major species during mouse embryonic development based on the relative abundance of the transcripts.
Targeted disruption of the mouseFu gene. The schematic diagram shows theFu genomic locus, the targeting vector, and the mutant allele. The top line shows a partial restriction map of the mouseFu genomic locus on chromosome 1. TheFu genomic locus consists of 28 exons (E1 to E28), and only the first 7 exons are shown for simplicity. The translation start ATG is predicted to reside in the third exon (E3). The regions between the dotted lines represent the 5′ and 3′ regions of homology used in gene targeting, respectively, and the symbol × indicates events of homologous recombination. Germ line-transmitting chimeric males carrying the targetedFu locus were mated with β-actin::Cre mice to remove sequences between the twoloxP sites (including the PGK-neo-pA selection cassette) and generate theFuΔE1-5 allele in which the first five exons ofFu are removed. As a result, β-galactosidase was brought under the control of putative upstreamFu regulatory elements. The locations of the fragments used as the 5′ or 3′ external probes in Southern blotting are shown, as well as the sizes of the restriction fragments detected for wild-type and targetedFuΔE1-5 alleles. TheRnf25 (ring finger protein 25) gene is divergently transcribed immediately upstream ofFu, suggesting that targeted disruption ofFu could potentially remove regulatory elements that controlRnf25 expression.
FuΔE1-5 animals exhibit growth retardation and postnatal lethality, andFu transcript levels are barely detectable inFuΔE1-5 embryos. (A) Wild-type andFuΔE1-5 animals photographed at postnatal day 14. The mutant is significantly smaller than its wild-type littermate. Some toes in both animals were clipped for numbering and genotyping. (B) Northern blot analysis of poly(A)+ RNA isolated from 10.5-dpc wild-type,FuΔE1-5/+, andFuΔE1-5 embryos. The twoFu probes used for hybridization gave identical results, and they correspond approximately to the last 775 bp ofFu transcripts and the 624-bp genomic sequences (exons 2 to 5) deleted in the gene-targetedFuΔE1-5 allele (data not shown). A ∼4.7-kb upper band with stronger intensity and a ∼4.1-kb lower band were detected in both wild-type andFuΔE1-5/+ embryos but were completely absent inFuΔE1-5 embryos. The expression level of the housekeeping geneGAPDH serves as the loading control with the same membrane reprobed. (C) RT-PCR using RNA derived from 10.5-dpc wild-type (wt) andFuΔE1-5 mouse embryos in combination with primers spanning the exons indicated. While PCR products of the predicted sizes were detected from RNA derived from wild-type embryos, no PCR products were generated using RNA obtained fromFuΔE1-5 mutants. The same number of PCR cycles (37 cycles) was employed for all the PCRs shown in this panel. The faint band, corresponding to exons 24 to 28, seen inFuΔE1-5 was not detected when a lower number of PCR cycles (less than 33) was applied (data not shown). PCR products corresponding to other genes such asShh,Fgf10, andGAPDH (not shown) were detected at similar levels using RNA produced from wild-type orFuΔE1-5 mutants, suggesting that the absence of aFu signal inFuΔE1-5 was not due to technical difficulties in RNA preparation or RT-PCR.
Development of major tissues and organs does not appear to be affected inFuΔE1-5 mutants. (A to T) Hematoxylin-and-eosin-stained sections of major tissues and organs of wild-type andFuΔE1-5 animals at embryonic dpc 18.5 and postnatal day P8. A to D, coronal sections through the forebrain; E to H, cross sections through the thoracic cavity; I to R, cross sections through the abdominal cavity; S and T, longitudinal sections through the proximal ulna. AlthoughFuΔE1-5 mutants were significantly smaller than their wild-type littermates at day P8, no obvious developmental defects or pathological changes in major tissues and organs were detected inFuΔE1-5 mutants. Multiple wild-type andFuΔE1-5 embryos were examined. In certain regions of the sections, the minor differences in morphology between wild-type andFuΔE1-5 animals were due to planes of sections. sp, spinal cord; lg, lung; h, heart; s, stomach; si, small intestine; li, large intestine; lv, liver; k, kidney; b, bladder. The magnification of each section is indicated.
Expression of Hh targets is not perturbed inFuΔE1-5 mutants. (A to P) Isotopic in situ hybridization using [33P]UTP-labeled riboprobes (pink) on paraffin sections of wild-type (A, C, E, G, I, K, M, and O) andFuΔE1-5 (B, D, F, H, J, L, N, and P) 10.5-dpc embryos at the forelimb-heart level (A, B, C, D, G, H, K, L, O, and P) or the hind limb level (E, F, I, J, M, and N). The axis for orientation of the specimens is dorsal upward and ventral downward.Shh is expressed in several signaling centers such as the notochord and floor plate, while Hh targets, includingPtch1,Hip1, andGli1, are expressed in the ventral neural tube and the somite in response to Hh signaling.Shh is also expressed in the epithelium of many developing organs, including the foregut endoderm, whilePtch1,Hip1, andGli1 are expressed in the surrounding mesenchyme. Multiple sections were examined in multiple rounds of in situ hybridization, and expression of Hh targets does not appear to be affected inFuΔE1-5 mutants. nt, neural tube; fp, floor plate; nc, notochord; s, somite; fg, foregut; h, heart.
References
- Alcedo, J., M. Ayzenzon, T. Von Ohlen, M. Noll, and J. E. Hooper. 1996. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell 86:221-232. - PubMed
- Alves, G., B. Limbourg-Bouchon, H. Tricoire, J. Brissard-Zahraoui, C. Lamour-Isnard, and D. Busson. 1998. Modulation of Hedgehog target gene expression by the Fused serine-threonine kinase in wing imaginal discs. Mech. Dev. 78:17-31. - PubMed
- Aza-Blanc, P., H. Y. Lin, A. Ruiz i Altaba, and T. B. Kornberg. 2000. Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities. Development 127:4293-4301. - PubMed
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