A role for TENM1 mutations in congenital general anosmia.

Congenital general anosmia (CGA) is a neurological disorder entailing a complete innate inability to sense odors. While the mechanisms underlying vertebrate olfaction have been studied in detail, there are still gaps in our understanding of the molecular genetic basis of innate olfactory disorders. Applying whole-exome sequencing to a family multiply affected with CGA, we identified three members with a rare X-linked missense mutation in the TENM1 (teneurin 1) gene (ENST00000422452:c.C4829T). In Drosophila melanogaster, TENM1 functions in synaptic-partner-matching between axons of olfactory sensory neurons and target projection neurons and is involved in synapse organization in the olfactory system. We used CRISPR-Cas9 system to generate a Tenm1 disrupted mouse model. Tenm1(-/-) and point-mutated Tenm1(A) (/A) adult mice were shown to have an altered ability to locate a buried food pellet. Tenm1(A) (/A) mice also displayed an altered ability to sense aversive odors. Results of our study, that describes a new Tenm1 mouse, agree with the hypothesis that TENM1 has a role in olfaction. However, additional studies should be done in larger CGA cohorts, to provide statistical evidence that loss-of-function mutations in TENM1 can solely cause the disease in our and other CGA cases.

Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body.

Fibroblast growth factors and receptors are intimately connected to the extracellular matrix by their affinity to heparan sulfate proteoglycans. They mediate multiple processes during embryonic development and adult life. In this study, embryonic stem cell-derived embryoid bodies were used to model fibroblast growth factor signaling during early epithelial morphogenesis. To avoid redundancy caused by multiple receptors, we employed a dominant negative mutation of Fgfr2. Mutant-derived embryoid bodies failed to form endoderm, ectoderm, and basement membrane and did not cavitate. However, in mixed cultures they displayed complete differentiation induced by extracellular products of the normal cell. Evidence will be presented here that at least one of these products is the basement membrane or factors connected to it. It will be shown that in the mutant, collagen IV and laminin-1 synthesis is coordinately suppressed. We will demonstrate that the basement membrane is required for embryoid body differentiation by rescuing columnar ectoderm differentiation and cavitation in the mutant by externally added basement membrane proteins. This treatment induced transcription of Eomesodermin, an early developmental gene, suggesting that purified basement membrane proteins can activate inherent developmental programs. Our results provide a new paradigm for the role of fibroblast growth factor signaling in basement membrane formation and epithelial differentiation.

Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.

The aim of this study was to clarify the role of Fgfr2 during later stages of embryonic development. Of two previously reported gene-targeting experiments, the more extensive Fgfr2 deletion was lethal shortly after implantation, because of trophoblast defects, whereas the less extensive one survived until midgestation with placental insufficiency and defective limb outgrowth [Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. & Deng, C. (1998) Development (Cambridge, U.K.) 125, 753-765]. Fgfr2 in the early embryo is expressed in the trophectoderm, and this extra-embryonic localization persists into mid- and late gestation, when Fgfr2 also is expressed in multiple developing organs. To gain insight into the later functions of Fgfr2, fusion chimeras were constructed from homozygous mutant embryonic stem cells and wild-type tetraploid embryos. This allowed survival until term and revealed that Fgfr2 is required for both limb outgrowth and branching lung morphogenesis. The use of fusion chimeras demonstrated that early lethality was indeed because of trophectoderm defects and indicated that in the embryonic cell lineages Fgfr2 activity manifests in limb and lung development. Highly similar lung and limb phenotypes were detected recently in the loss of function mutation of Fgf10, a ligand of Fgfr2. It is likely, therefore, that whereas during early development Fgfr2 interacts with Fgf4, in limb and lung development interactions between Fgf10 and Fgfr2 may be required. Possible epithelial-mesenchymal interactions between the splicing alternatives of Fgfr2 and their specific ligands will be discussed.

Expression of Fgfr2 in the early mouse embryo indicates its involvement in preimplantation development.

We report that the IIIc transcriptional alternative of Fgfr2 is transcribed in the unfertilized egg and that during early zygotic transcription, messages encoded by both Fgfr2 alternatives (IIIc and IIIb) are present. The Fgfr2 protein was first detected in peripheral blastomeres of compacted morulae. Trophectoderm specificity of Fgfr2 became obvious in the early blastocyst and with maturation its localization underwent further specification, Fgfr2 concentration increased at the abembryonic pole and decreased at the embryonic pole. Moreover Fgfr2 expression became markedly asymmetrical along the animal-vegetal axis of the mature blastocyst. Our observations indicate a role for Fgfr2 in trophectoderm growth and specification and in the orientation and polarity of the preimplantation conceptus.

Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.