Generation of Rgs4 deficient mice. (A) Targeting strategy. (Top) Wild-type Rgs4 allele with exons 1 to 5 (boxes, coding sequences in dark shade, non coding sequences in light shade). The RGS domain is encoded by exons 3, 4, and 5. Selected sites for EcoRI (E), BamHI (B), KpnI (K), and SwaI (S) are indicated. (Middle) Targeting vector. A loxP/BamHI/FRT cassette has been inserted in the SwaI site. A cassette (loxP site/splice acceptor [Sa]/promoterless lacZ open reading frame/FRT site/neo open reading frame under the control of the pGK promoter/FRT site/three out-of-phase stop codons) replaces the KpnI/BamHI fragment downstream of the fifth exon. A thymidine kinase cassette under the control of the hsv promoter was added at the 3′ end of the construct. (Bottom) Recombined locus. (B) PCR genotyping strategy of the wild-type, targeted, and Cre recombined loci. Primers are represented by arrowheads. (C) Southern blot of the DNA of a recombinant (IIID3) and a wild-type (IIB4) ES clone, digested by EcoRI and probed with the external probe depicted in panel A. floxed, recombined allele. (D) PCR analysis of tail DNA of wt, Rgs4^loxFRTlacZ/+ (wt/flox), and Rgs4 ^{loxFRTlacZ/loxFRTlacZ} (flox/flox) pups with the primers depicted in panel B. (E) PCR analysis of tail DNA of wild-type, Rgs4^+/lacZ (wt/lacZ), and Rgs4^lacZ/lacZ (lacZ/lacZ) pups with the primers depicted in panel B (see Materials and Methods for details). (F) Transverse sections through wild-type and homozygous mutant E11.75 embryos, showing that the expression of Rgs4 mRNA is lost in the mutants, even though the ganglionic cells are present, as assessed by β-tubulin expression. nt, neural tube.

Expression pattern of Rsg4 monitored by X-Gal staining in Rgs4^lacZ/+ embryos and mice. (A) Whole mounts of Rgs4^loxFRTlacZ/+ E11 embryos without (left) or with (middle) the action of the Cre recombinase (provided by a PGK-Cre female). (Right) In situ hybridization (ISH) with an Rgs4 probe on a wild-type embryo. A slight localized leakage of lacZ expression from the Rgs4^loxFRTlacZ locus is visible in the mandibles of Rgs4^loxFRTlacZ/+ embryos. The action of the Cre recombinase triggers lacZ expression in a pattern indistinguishable from that of Rgs4. (B) Flat mount of a hindbrain from an Rgs4^lacZ/+ E11 embryo showing expression of the Rgs4 locus in the forming trigeminal (nV) and facial (nVII) motor nuclei, as well as in the locus coeruleus (lc) and unidentified interneurons in the metencephalon (star). (C) Section of the thorax of an E11 embryo showing expression of Rgs4^lacZ in the dorsal root ganglia and in the anlagen of sympathetic ganglia surrounding the dorsal aorta (da). (D) Coronal section of the forebrain of an adult Rgs4^lacZ/+ mouse. Cx, cortex; Hi, hippocampus; Th, thalamus; 3V, third ventricle. The expression in hippocampus is comparable to that in cortex and stronger than reported by Gold et al. (). (E) Section of the spinal cord of an adult Rgs4^lacZ/+ mouse showing expression of lacZ in the dorsal horns. (F) In the retina, a subset of ganglion cells (arrowhead) express Rgs4. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (G) Sagittal section of the heart of an adult Rgs4^lacZ/+ mouse treated with X-Gal, showing intense expression in the aorta (ao) and pulmonary trunk (pt) but none in the heart muscle. LA, left atrium; RA, right atrium; RV, right ventricle. (H) Close-up of Rgs4^lacZ expression in a whole mount of aorta, suggestive of smooth muscular expression. (I) Rgs4 expression in blood vessels on the surface of the cerebellum.

Absence of a neuronal developmental defect in Rgs4^lacZ/lacZ embryos. (A and A') Combined in situ hybridization for lacZ and immunohistochemistry for the motoneuronal marker Islet1/2 on transverse sections at the level of the fourth rhombomere, showing an intact differentiation of facial motoneurons. (B and B') Transverse sections through the pons of adult mice stained with X-Gal and showing morphologically intact facial nuclei in the homozygote mutants. (C to D') Sagittal sections of E13.5 embryos hybridized with a lacZ or Dbh probe, showing intact morphology and Dbh expression of the paravertebral (black arrowhead) and prevertebral (red arrowhead) sympathetic chains and organ of Zuckerkandl (z) in homozygous (C' to D') compared to heterozygous (C to D) mutants. (E and E') Nissl stain of sections of the cortex, showing normal cytoarchitecture in Rgs4 null mutants.

Naloxone-precipitated morphine withdrawal syndrome in wild-type and Rgs4 null mice. Data are expressed as means ± standard errors of the means. Black stars, morphine- versus saline-treated animals of the same genotype; white stars, wild-type versus mutant groups receiving the same treatment (two-tailed Student t test). One star, P < 0.05; two stars, P < 0.01.

Startle reactivity and prepulse inhibition in wild-type and Rgs4 null mice. (A) The startle amplitude is measured at the background noise (BN) level or at indicated intensities. (B) The PPI is measured as percent startle response to the 120-dB pulse for prepulses of indicated intensities. Values are expressed as means ± standard errors of the means.