Figure 1.
Production of Gng5 mutant mice.
A. Wild type Gng5 allele (top bar) illustrating the three Gng5 exons (black boxes), homology arms of the targeting vector (grey boxes) and 5′ and 3′ probes for Southern blotting (striped boxes). Floxed Gng5 allele (middle bar) illustrating insertion of loxP sites (triangles) and neomycin resistance cassette (NeoR). Deleted Gng5 allele (bottom bar) illustrating deletion of sequence between 1st and 3rd loxP site including the first exon of Gng5 and NeoR cassette. Also illustrated are expected sizes of fragments following digestion with BamHI (B) or XhoI (X), along with the primers used for PCR (a,b,c). Primer sequences can be found in Table S1. B. Southern blot of DNA prepared from five properly-targeted ES cell clones (C3-H8) and one wild type clone (WT). DNA was digested with BamHI and probed with the 5′-probes. Properly targeted clones displayed both the 7.8 kb floxed allele and the 11.4 kb wild type allele. C. DNA from these same clones was digested with XhoI and probed with the 3′-probe. Properly targeted clones displayed both the 13 kb floxed allele and the 18.9 kb wild type allele. D. PCR analysis of DNA from tail biopsy of a wild mouse Gng5+/+, a floxed mouse Gng5+/fl, and offspring of a cross between a floxed mouse and an Tg(EIIa-cre) mouse showing varying degrees of recombination Gng5+/fl:del or Gng5+/del. The first lane is the φX HaeIII molecular weight marker. Primers a and b produce a 241-bp band from the wild type (+) allele or a 333-bp band from the floxed (fl) allele. Primers a and c produce a 454-bp band from the deleted (−) allele resulting from recombination between the 1st and 3rd loxP site. E. RT-PCR of Gng5 (primers d and e) and eukaryotic elongation factor (EF) from two wild type (Gng5+/+), two heterozygous (Gng5+/−), and two homozygous knockout (Gng5−/−) embryos, confirming the absence of Gng5 mRNA transcript in knockout embryos.
Figure 2.
Successful targeting of the Gng5 locus.
A. Region of mouse chromosome 3∶146,110,000–147,170,000 containing the Gng5 locus from the UCSC genome browser. B. The top bar of this schematic illustrates the arrangement of exons of Gng5 (orange boxes), Ctbs (blue boxes), and Spata1 (yellow boxes). The bars underneath illustrate the Gng5 mRNA transcript, the two Ctbs splice variants, the two Ctbs-Gng5 splice variants, and the three Spata1 splice variants (not drawn to scale). Letters indicate RT-PCR primers which can be found in Table S1. C. RT-PCR of the Ctbs-Gng5 fusion transcript (primers f and g) from the same embryos shown in Fig. 1E, confirming that expression of this fusion transcript is preserved in knockout embryos. Identification of the amplified products marked Ctbs-Gng5 was confirmed by DNA sequence analysis (Figure S1). The first lane on both gels is a molecular weight marker, φX digested with HaeIII. D. RT-PCR of Spata1 from the same embryos (primers f and h), demonstrating that expression of Spata1 is preserved in knockout embryos.
Table 1.
Genotype distribution of embryos from Gng5+/− intercrosses.
Figure 3.
Developmental expression of Gng5.
A, Ventral view of e8.0 mouse embryo after whole mount RNA in situ hybridization (blue signal) to detect Gng5 mRNA. White arrowheads denote location of cardiac progenitors. HF, head fold. B, Section of e8.5 embryo through the region of the pharynx. Brown signal indicates Gng5 transcripts in HF, endoderm (En), ectoderm (Ec) and cardiac progenitors (black arrowheads). There is little signal in the heart proper (RV, right ventricle; OFT, outflow tract). C, Magnification of the region boxed in B containing cardiac progenitors in splanchnic mesoderm (SM). D, Left lateral view of e9.5 mouse embryo after whole mount RNA in situ hybridization (blue signal) to detect Gng5 mRNA. Widespread expression is present with less in the heart (white line denotes dorsal inflow region). E, Section of e9.5 embryo through the region of the pharynx. Brown signal indicates Gng5, endoderm (En), ectoderm (Ec) and cardiac progenitors (black arrowhead). There is also some signal in the heart (HT). F, Magnified view of pharyngeal and splanchnic mesoderm corresponding to boxed region in E.
Figure 4.
Gng5−/− mutants fail to form the cardiac outflow tract and right ventricle and have severely hypoplastic pharyngeal arches.
Panels A–D images of e8.5 whole mount wild type and mutant embryos. By comparing the left lateral (A, C) and ventral (B, D) views of intact wild type and Gng5−/− embryos, respectively, the unlooped heart tube is clearly evident (B, D). Panels E–H, images of e9.5 wild type and mutant embryos. The left parasagittal section from a control embryo shows the inflow tract and left side of common atrium, endocardial cushion in the atrioventricular canal, and left ventricle (E). The red arrowheads mark the first pharyngeal arch, also labeled 1. The midline section shows the outflow tract connecting to aortic sac in the second pharyngeal arch (bracket), right ventricle, the right portion of the common atrium, and the sinus venosus in the control embryo (F). The left parasagittal section of a Gng5−/− mutant shows severely hypoplastic but vascularized first pharyngeal arch (red arrowhead), dilated heart tube with atrial chamber caudal to ventricle, narrow inflow, and paucity of cells in the pharyngeal mesoderm (G). The midline section shows the unlooped, dilated heart tube and no outflow tract; cardiac chamber opens directly into dilated aortic sac (bracket) in a Gng5−/− embryo (H). TB, tail bud; HF, head fold; OFT, outflow tract; RV, right ventricle; LV, left ventricle; A, atrium V, ventricle; EC, endocardial cushion; SV, sinus venosus.
Figure 5.
Gng5−/− mutants show loss of second heart field and reduced expression of proliferative markers.
Panels A–C, control and mutant embryos stained for Fgf8 expression in the second heart field. As shown by in situ hybridization for Fgf8 transcripts, the e8.5 control embryo (A) displays numerous pharyngeal mesodermal cells exhibiting Fgf8 expression (brown staining; black arrowheads). These cells represent progenitors of the right ventricle (RV) and outflow tract (OFT) that reside dorsal to the heart. Fgf8 expression is also detected in the lateral pharyngeal endoderm (En) and pharyngeal ectoderm (Ec). In stark contrast, The Gng5−/− mutants (B,C) have fewer pharyngeal mesoderm cells and reduced Fgf8 expression in this region. The red arrow (B) indicates the most rostral portion of heart tube and the absence of OFT; the black arrowhead denotes absent Fgf8 expression in the thin layer of pharyngeal mesoderm dorsal to the heart. In a more caudal section (C), the heart tube (HT) is visible and faint Fgf8 expression is detected in the mesoderm adjacent to the heart tube (black arrowhead) and in the most proximal portion of the left side of the heart tube and adjacent mesoderm (red arrowhead). HF, head fold, P, pharynx; En, endoderm; Ec, ectoderm. Panel D, relative expression of proliferative markers in pharyngeal region of e9.0 control and Gng5−/− mutant embryos. As shown by qPCR analysis, Gng5−/− mutant embryos display reduced expression of three proliferative markers in the second heart field (* p<0.002; ** p<0.0001 by Student’s t-test), using elongation factor 1 (Eef1a1) as the housekeeping gene. All primer sequences can be found in Table S1.
Figure 6.
Gng5−/− mutants have decreased cell proliferation and survival.
Sagittal cryosections of e9.5 control and mutant embryos after immunohistochemical staining for DNA (DAPI, blue), cells in mitosis (anti-pHH3, green), and cells undergoing apoptosis (TUNEL, red). In all panels, rostral is at right, ventral at top. Panels A, D represent 10X views of control (A) and mutant (D) embryos, while Panels B,C,E,F show 20x views of control (B,C) and mutant (E,F) sections. The red arrowheads in C and F denote second heart field pharyngeal mesoderm and adjacent endoderm. The pharyngeal arches are numbered. H, heart; T, telencephalon; HB, hindbrain; O, otocyst; OFT, outflow tract; IFT; inflow tract; HT, heart tube (mutant only).
Figure 7.
Non-redundant function of Gng5 gene during development.
As shown by qPCR analysis on a normalized mouse cDNA panel containing different gestational stages (Mouse MTC Panel1, Clontech), multiple Gng family members are expressed at developmental stages relevant to neural and cardiac development. All primer sequences can be found in Table S1.