Defects in Limb, Craniofacial, and Thymic Development in Jagged2 Mutant Mice
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Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice Rulang Jiang,1,3 Yu Lan,1,3 Harry D. Chapman,1 Carrie Shawber,2 Christine R. Norton,1 David V. Serreze,1 Gerry Weinmaster,2 and Thomas Gridley1,4 1The Jackson Laboratory, Bar Harbor, Maine 04609 USA; 2Department of Biological Chemistry, University of California, Los Angeles (UCLA), The School of Medicine, Los Angeles, California 90024 USA The Notch signaling pathway is a conserved intercellular signaling mechanism that is essential for proper embryonic development in numerous metazoan organisms. We have examined the in vivo role of the Jagged2 (Jag2) gene, which encodes a ligand for the Notch family of transmembrane receptors, by making a targeted mutation that removes a domain of the Jagged2 protein required for receptor interaction. Mice homozygous for this deletion die perinatally because of defects in craniofacial morphogenesis. The mutant homozygotes exhibit cleft palate and fusion of the tongue with the palatal shelves. The mutant mice also exhibit syndactyly (digit fusions) of the fore- and hindlimbs. The apical ectodermal ridge (AER) of the limb buds of the mutant homozygotes is hyperplastic, and we observe an expanded domain of Fgf8 expression in the AER. In the foot plates of the mutant homozygotes, both Bmp2 and Bmp7 expression and apoptotic interdigital cell death are reduced. Mutant homozygotes also display defects in thymic development, exhibiting altered thymic morphology and impaired differentiation of gd lineage T cells. These results demonstrate that Notch signaling mediated by Jag2 plays an essential role during limb, craniofacial, and thymic development in mice. [Key Words: Notch signaling; Jagged2; Serrate2; limb development; cleft palate; T cell differentiation] Received December 15, 1997; revised version accepted February 2, 1998. The Notch signaling pathway is an evolutionarily con- Notch signaling pathway have been implicated in cancer served signaling mechanism, and mutations in its com- and in two inherited disease syndromes (Ellisen et al. ponents disrupt cell fate specification and embryonic 1991; Joutel et al. 1996; Li et al. 1997; Oda et al. 1997). development in organisms as diverse as insects, nema- Four genes encoding ligands for the Notch family of todes, and mammals (for recent reviews, see Artavanis- receptors have been cloned in mammals: Jagged1 (Jag1; Tsakonas et al. 1995; Gridley 1997; Robey 1997; Wein- Lindsell et al. 1995), Jagged2 (Jag2; Shawber et al. 1996; master 1997). This signaling pathway was first identified these genes are also referred to as Serrate1 and 2), Delta- and studied in Drosophila. The Notch gene of Dro- like1 (Dll1; Bettenhausen et al. 1995), and Delta-like3 sophila encodes a large transmembrane receptor that, at (Dll3; Dunwoodie et al. 1997). All of these Notch family the extracellular surface of a cell, interacts with mem- ligands are transmembrane proteins that, in their extra- brane-bound ligands encoded by the Delta and Serrate cellular domain, contain multiple EGF-like motifs as genes. The signal induced by ligand binding is then well as a second conserved motif termed the DSL do- transmitted at the intracellular surface in a process in- main (named after three invertebrate Notch ligands; volving proteolysis of the receptor and interactions with Delta, Serrate, Lag-2). The DSL domain is required for several novel cytoplasmic and nuclear proteins (Fortini interaction of ligands with Notch family receptors and Artavanis-Tsakonas 1994; Jarriault et al. 1995; Mat- (Henderson et al. 1994; Muskavitch 1994). Two of these suno et al. 1995; Kopan et al. 1996; Blaumueller et al. ligands, Jagged1 and Jagged2, have been shown to acti- 1997; Pan and Rubin 1997). Genes homologous to mem- vate Notch1 in mammalian cells (Lindsell et al. 1995; bers of the Notch signaling pathway have been cloned Luo et al. 1997). from numerous vertebrate organisms, and many have We have been studying the role of Notch signaling been shown to be essential for normal embryonic devel- during embryonic development in mice. To study the opment. In humans, the importance of Notch signaling biological role of the Jag2 gene, we made a targeted mu- for growth and development is underscored by the find- tation that removes exons encoding the DSL domain of ing that mutations in genes encoding components of the the Jagged2 protein. Mice homozygous for this deletion die at birth because of defects in craniofacial morpho- genesis. The mutant mice have cleft palate, and dorsal 3These authors contributed equally to this work. 4Corresponding author. portions of the tongue are fused to the unelevated palatal E-MAIL [email protected]; FAX (207) 288-6077. shelves. In addition, the mutant mice exhibit impaired 1046 GENES & DEVELOPMENT 12:1046–1057 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Analysis of Jag2 mutant mice differentiation of gd lineage T cells and altered thymic morphology. The Jag2 mutant homozygotes also exhibit syndactyly of the fore- and hindlimbs. Previously, we mapped the Jag2 gene to distal Chromosome 12 (Lan et al. 1997), near two mutations that exhibit limb and/or craniofacial de- fects, syndactylism (sm; Gru¨neberg 1956) and legless (lgl; Singh et al. 1991). Sidow et al. (1997) recently reported that the sm mutation is a missense mutation in the Jag2 gene. Our results suggest that sm is a hypomorphic allele of Jag2 because mice homozygous for our targeted DSL domain deletion exhibit completely penetrant perinatal lethality and syndactyly that is more severe than in sm homozygous mice, many of which survive to adulthood. These results demonstrate that Notch signaling medi- ated by Jag2 plays an essential role during limb, cranio- facial, and thymic development in mice. Results Figure 1. Targeted disruption of the mouse Jag2 gene. (A) Tar- Targeted disruption of the mouse Jag2 gene geting scheme. The top line shows the genomic organization of a portion of the Jag2 gene. Exons are indicated by black boxes. Recently, we described the isolation of a gene, Jag2, Additional uncharacterized exons are present 38 of the exons which encodes a protein whose amino acid sequence and indicated. The middle line represents the structure of the tar- expression pattern during rat embryogenesis suggest that geting vector. A 5.0-kb deletion was created that removes exons it functions as a ligand for the Notch family of receptors encoding the DSL domain and half of the first EGF repeat. Shown at the bottom is the predicted structure of the Jag2 locus (Shawber et al. 1996). Mouse Jag2 cDNA and genomic following homologous recombination of the targeting vector. clones were isolated by screening brain cDNA and geno- Probes used for Southern blot analysis are indicated. (E) EcoRI; mic libraries (see Materials and Methods). To investigate (N) NotI; (S) SacI; (Sp) SpeI. (B) DNA isolated from embryos of the biological role of the Jag2 gene, we created a deletion the intercross of Jag2+/DDSL heterozygous mice was digested allele by gene targeting. A Jag2 targeting vector was con- with EcoRI, blotted, and hybridized with the indicated probe. structed that deleted genomic sequence encoding the Genotypes of progeny are indicated at the top of the lane. Lack DSL domain of the Jagged2 protein, which is required for of hybridization of the DSL domain probe to DNA of the interaction of ligands with Notch family receptors in Jag2DDSL homozygotes confirms that the fragment containing both invertebrates (Henderson et al. 1994; Muskavitch the exon encoding the DSL domain is deleted in the transmitted DDSL 1994) and vertebrates (C. Lindsell and G. Weinmaster, Jag2 allele. unpubl.). Deletion of the DSL domain should create a null mutation in the Jag2 gene. We have named this DDSL mutant allele Jag2 (Fig. 1A). The linearized targeting birth. Caesarean delivery at E18 of pups from heterozy- vector was electroporated into ES cells, and germ-line gous intercrosses indicated that the majority of the DDSL transmission of the Jag2 mutant allele was obtained. Jag2DDSL homozygotes were unable to breathe, became Southern blot analyses confirmed the expected structure cyanotic, and died within a few minutes. A few of the of the transmitted allele, and also confirmed that the Jag2DDSL homozygotes were able to breathe, but all of fragment containing the exon encoding the DSL domain these died within a few hours. Autopsy of these Jag2DDSL DDSL was deleted in the transmitted Jag2 mutant allele homozygous neonates revealed that they contained large DDSL (Fig. 1B). Mice heterozygous for the Jag2 mutant al- amounts of air in the stomach and intestines. lele appeared normal. Further examination of the Jag2DDSL mutant neonates revealed a bilateral cleft of the secondary palate (Fig. 2). During normal palatal morphogenesis, the palatal Jag2DDSL mutant mice die at birth with cleft palate shelves (consisting of the palatine and maxillary shelves) To examine whether mice homozygous for the Jag2DDSL elevate and grow toward the midline of the embryo mutation were viable, heterozygous F1 animals were in- above the tongue. The shelves then undergo an epitheli- tercrossed, and the genotypes of F2 progeny were deter- al-mesenchymal transition to fuse and form the second- mined two to three weeks after birth. No mice homozy- ary palate, which ossifies and forms the roof of the oral gous for the mutation were found, indicating that the cavity. In Jag2DDSL homozygous embryos, the palatal Jag2DDSL mutation was lethal prior to this stage. To de- shelves did not elevate, and the tongue became wedged termine when the Jag2DDSL homozygotes were dying, between the shelves (Fig. 2A,B). In dorsal regions of the embryos from timed matings were isolated. This analy- mutant embryos, the unelevated palatal shelves had un- sis revealed that Jag2DDSL homozygous mutants could dergone an epithelial-mesenchymal transition and fused complete embryogenesis, but died at or shortly after on each side with the posterior portion of the tongue (Fig.