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.

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.

During limb outgrowth, signaling by bone morphogenetic proteins (BMPs) must be moderated to maintain the signaling loop between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Gremlin, an extracellular Bmp antagonist, has been proposed to fulfill this function and therefore be important in limb patterning. We tested this model directly by mutating the mouse gene encoding gremlin (Cktsf1b1, herein called gremlin). In the mutant limb, the feedback loop between the ZPA and the AER is interrupted, resulting in abnormal skeletal pattern. We also show that the gremlin mutation is allelic to the limb deformity mutation (ld). Although Bmps and their antagonists have multiple roles in limb development, these experiments show that gremlin is the principal BMP antagonist required for early limb outgrowth and patterning.

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.

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.Leukemia inhibitory factor (LIF) or DIA (differentiation inhibitory activity) is a 45-to 56-kDa secreted glycoprotein (1, 2) that has multiple activities on various in vitro culture systems. These include the induction of the acute-phase response in hepatocyte cultures (3), the regulation of the differentiation and proliferation of certain hematopoietic cell lines (4-6), the establishment of the cholinergic phenotype in rat sympathetic neurons (7), the remodeling of bone (8,9), and the inhibition of embryonic stem cell differentiation in vitro (10, 11). Little is known about the in vivo function(s) of LIF, although mice that have high levels of circulating LIF undergo excessive bone formation, become cachexic, and eventually die (12). The observation that LIF inhibits embryonic stem cell differentiation in vitro also suggests that it may play a role in regulating the growth and development of early mouse embryos, since embryonic stem cells are derived from the inner cell mass of blastocysts. Derivation of embryonic stem cells from the inner cell mass is dependent either on the presence of a fibroblast "feeder" cell layer that secretes LIF or on exogenous LIF added to the culture medium (13-17).To [a-32P]dUTP by transcription using the T7 promoter. The mouse 3-actin probe, supplied by Ambion, was used as a control. RNA levels were quantified by using a Cliniscan densitometer (Helena Laboratories).In situ RNA hybridization and histological analysis were performed, essentially under conditions described by Wilkinson et al. (20). For histological analysis, sections were stained with hemotoxylin and eosin.Mice Undergoing Delayed Implantation. Derivation of mice was performed surgically or induced naturally as described by Bergstrom (21). Implantation was initiated in these mice on day 6 or 7 after plugging (days 3 or 4 after ovariectomy) by a single intraperitoneal injection of 0.2 1g of 3-estradiol (Sigma) dissolved in ethanol and diluted to the final concentration in corn oil (Mazola). In naturally induced mice, implantation was initiated by removing the suckling pups. RESULTS LIF mRNA Expression. Total RNA was isolated from adult and neonatal tissues and from postimplantation embryos starting on day 8 of gestation (day of plug = day 1 of pregnancy). RNAs were analyzed by using either the blot hybridization or RNase protection procedures. The majority of tissues and embryonic stages analy...

During skull development, the cranial connective tissue framework undergoes intramembranous ossification to form skull bones (calvaria). As the calvarial bones advance to envelop the brain, fibrous sutures form between the calvarial plates. Expansion of the brain is coupled with calvarial growth through a series of tissue interactions within the cranial suture complex. Craniosynostosis, or premature cranial suture fusion, results in an abnormal skull shape, blindness and mental retardation. Recent studies have demonstrated that gain-of-function mutations in fibroblast growth factor receptors (fgfr) are associated with syndromic forms of craniosynostosis. Noggin, an antagonist of bone morphogenetic proteins (BMPs), is required for embryonic neural tube, somites and skeleton patterning. Here we show that noggin is expressed postnatally in the suture mesenchyme of patent, but not fusing, cranial sutures, and that noggin expression is suppressed by FGF2 and syndromic fgfr signalling. Since noggin misexpression prevents cranial suture fusion in vitro and in vivo, we suggest that syndromic fgfr-mediated craniosynostoses may be the result of inappropriate downregulation of noggin expression.

Olfactory neurons expressing the same odorant receptor converge to a small number of glomeruli in the olfactory bulb. In turn, mitral and tufted cells receive and relay this information to higher cortical regions. In other sensory systems, correlated neuronal activity is thought to refine synaptic connections during development. We asked whether the pattern of connections between olfactory sensory axons and mitral cell dendrites is affected when odor-evoked signaling is eliminated in mice lacking functional olfactory cyclic nucleotide-gated (CNG) channels. We demonstrate that olfactory sensory axons converge normally in the CNG channel mutant background. We further show that the pruning of mitral cell dendrites, although slowed during development, is ultimately unperturbed in mutant animals. Thus, the olfactory CNG channel-and by inference correlated neural activity--is not required for generating synaptic specificity in the olfactory bulb.

Summary Background Soon after birth all mammals must initiate milk suckling to survive. In rodents, this innate behavior is critically dependent on uncharacterized maternally-derived chemosensory ligands. Recently the first pheromone sufficient to initiate suckling was isolated from the rabbit. Identification of the olfactory cues that trigger first suckling in the mouse would provide the means to determine the neural mechanisms that generate innate behavior. Results Here we use behavioral analysis, metabolomics, and calcium imaging of primary sensory neurons and find no evidence of ligands with intrinsic bioactivity, such as pheromones, acting to promote first suckling in the mouse. Instead, we find that the initiation of suckling is dependent on variable blends of maternal ‘signature odors’ that are learned and recognized prior to first suckling. Conclusions As observed with pheromone-mediated behavior, the response to signature odors releases innate behavior. However, this mechanism tolerates variability in both the signaling ligands and sensory neurons which may maximize the probability that this first essential behavior is successfully initiated. These results suggest that mammalian species have evolved multiple strategies to ensure the onset of this critical behavior.