Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton.

Noggin is a bone morphogenetic protein (BMP) antagonist expressed in Spemann's organizer. Murine Noggin is expressed in condensing cartilage and immature chondrocytes, as are many BMPs. In mice lacking Noggin, cartilage condensations initiated normally but developed hyperplasia, and initiation of joint development failed as measured by the expression of growth and differentiation factor-5. The maturation of cartilage and Hoxd expression were unaffected. Excess BMP activity in the absence of Noggin antagonism may enhance the recruitment of cells into cartilage, resulting in oversized growth plates; chondrocytes are also refractory to joint-inducing positional cues.

General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.

Olfactory neurons transduce the binding of odorants into membrane depolarization. Two intracellular messengers, cyclic AMP (cAMP) and inositol trisphosphate (IP3), are thought to mediate this process, with cAMP generating responses to some odorants and IP3 mediating responses to others. cAMP causes membrane depolarization by activating a cation-selective cyclic nucleotide-gated (CNG) channel. We created a mutant "knockout" mouse lacking functional olfactory CNG channels to assess the roles of different second messenger pathways in olfactory transduction. Using an electrophysiological assay, we find that excitatory responses to both cAMP- and IP3-producing odorants are undetectable in knockout mice. Our results provide direct evidence that the CNG channel subserves excitatory olfactory signal transduction, and further suggest that cAMP is the sole second messenger mediating this process.

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation.

We have analyzed the expression of the cytokine leukemia inhibitory factor (LIF) during embryogenesis and in tissues of neonatal and adult mice. The site of the most abundant LIF expression is the uterine endometrial glands, specifically on day 4 of pregnancy. Analysis of LIF expression in pseudopregnant mice and in females undergoing delayed implantation showed that it is under maternal control and that its expression coincides with blastocyst formation and always precedes implantation. These results suggest that a principal function of LIF in vivo may be to regulate the growth and to initiate implantation of blastocysts.

Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor.

A critical point during mammalian pregnancy is the implantation of the blastocyst when the embryo attaches to the wall of the uterus. The autonomously developing preimplantation embryo then becomes dependent on the maternal environment for its continued development. Little is known about the regulation of implantation, except that a complex interaction between peptide and steroid hormones synchronizes the preparation of the uterus for implantation with the development of the embryo. Whether the implantation event is under maternal or embryonic control is also unclear (reviewed in refs 1, 2). We have previously shown that a cytokine, leukaemia inhibitory factor (LIF), is expressed in the uterine endometrial glands specifically on the fourth day of pregnancy. This burst of expression is under maternal control and always precedes implantation of the blastocyst. Here we report that transient expression of LIF in mice is essential for implantation. Females lacking a functional LIF gene are fertile, but their blastocysts fail to implant and do not develop. The blastocysts, however, are viable and, when transferred to wild-type pseudopregnant recipients, they can implant and develop to term.