Pathophysiological analyses of cortical malformation using gyrencephalic mammals.

One of the most prominent features of the cerebral cortex of higher mammals is the presence of gyri. Because malformations of the cortical gyri are associated with severe disability in brain function, the mechanisms underlying malformations of the cortical gyri have been of great interest. Combining gyrencephalic carnivore ferrets and genetic manipulations using in utero electroporation, here we successfully recapitulated the cortical phenotypes of thanatophoric dysplasia (TD) by expressing fibroblast growth factor 8 in the ferret cerebral cortex. Strikingly, in contrast to TD mice, our TD ferret model showed not only megalencephaly but also polymicrogyria. We further uncovered that outer radial glial cells (oRGs) and intermediate progenitor cells (IPs) were markedly increased. Because it has been proposed that increased oRGs and/or IPs resulted in the appearance of cortical gyri during evolution, it seemed possible that increased oRGs and IPs underlie the pathogenesis of polymicrogyria. Our findings should help shed light on the molecular mechanisms underlying the formation and malformation of cortical gyri in higher mammals.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules and RTK molecules in general. Since RTK dimerization regulates RTK signaling, our findings advance our knowledge of RTK activity in health and disease. The utility of the QI-FRET method is not restricted to RTKs, and we thus hope that in the future the QI-FRET method will be applied to other classes of membrane proteins, such as channels and G protein-coupled receptors.

Mutant activated FGFR3 impairs endochondral bone growth by preventing SOX9 downregulation in differentiating chondrocytes.

Fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the control of endochondral ossification, and bone growth and mutations that cause hyperactivation of FGFR3 are responsible for a collection of developmental disorders that feature poor endochondral bone growth. FGFR3 is expressed in proliferating chondrocytes of the cartilaginous growth plate but also in chondrocytes that have exited the cell cycle and entered the prehypertrophic phase of chondrocyte differentiation. Achondroplasia disorders feature defects in chondrocyte proliferation and differentiation, and the defects in differentiation have generally been considered to be a secondary manifestation of altered proliferation. By initiating a mutant activated knockin allele of FGFR3 (FGFR3K650E) that causes Thanatophoric Dysplasia Type II (TDII) specifically in prehypertrophic chondrocytes, we show that mutant FGFR3 induces a differentiation block at this stage independent of any changes in proliferation. The differentiation block coincided with persistent expression of SOX9, the master regulator of chondrogenesis, and reducing SOX9 dosage allowed chondrocyte differentiation to proceed and significantly improved endochondral bone growth in TDII. These findings suggest that a proliferation-independent and SOX9-dependent differentiation block is a key driving mechanism responsible for poor endochondral bone growth in achondroplasia disorders caused by mutations in FGFR3.

The thanatophoric dysplasia type II mutation hampers complete maturation of fibroblast growth factor receptor 3 (FGFR3), which activates signal transducer and activator of transcription 1 (STAT1) from the endoplasmic reticulum.

The K650E substitution in the fibroblast growth factor receptor 3 (FGFR3) causes constitutive tyrosine kinase activity of the receptor and is associated to the lethal skeletal disorder, thanatophoric dysplasia type II (TDII). The underlying mechanisms of how the activated FGFR3 causes TDII remains to be elucidated. FGFR3 is a transmembrane glycoprotein, which is synthesized through three isoforms, with various degrees of N-glycosylation. We have studied whether immature FGFR3 isoforms mediate the abnormal signaling in TDII. We show that synthesis of TDII-FGFR3 presents two phosphorylated forms: the immature non-glycosylated 98-kDa peptides and the intermediate 120-kDa glycomers. The mature, fully glycosylated 130-kDa forms, detected in wild type FGFR3, are not present in TDII. Endoglycosidase H cleaves the sugars on TDII intermediates thus indicating their intracellular localization in the endoplasmic reticulum. Accordingly, TDII-FGFR3-GFP co-localizes with calreticulin in the endoplasmic reticulum. Furthermore, following TDII transfection, signal transducer and activator of transcription 1 (STAT1) is phosphorylated in the absence of FGFR3 ligand and brefeldin A does not inhibit its activation. On the contrary, the cell membrane-anchored FRS2alpha protein is not activated in TDII cells. The opposite situation is observed in stable TDII cell clones where, despite the presence of phosphorylated mature receptor, STAT1 is not activated whereas FRS2alpha is phosphorylated. We speculate that the selection process favors cells defective in STAT1 activation through the 120-kDa TDII-FGFR3, thus allowing growth of the TDII cell clones. Accordingly, apoptosis is observed following TDII-FGFR3 transfection. These observations highlight the importance of the immature TDII-FGFR3 proteins as mediators of an abnormal signaling in TDII.

Displasia tanatofórica: presentación de un caso

Se presenta el caso de una mujer de 26 años, que asiste a consulta prenatal cursando embarazo de 23 semanas y a quien se le hizo el diagnóstico prenatal por ultrasonografía de una feto con displasia tanatofórica. Se mencionan los diferentes hallazgos así como los diagnósticos diferenciales que deben hacerse en estos casos teniendo en mente que en la totalidad de los casos el pronóstico es letal

Atelosteogenesis I and boomerang dysplasia: a question of nosology.

We report a patient whose clinical, radiologic and histopathologic findings are compatible with severe atelosteogenesis (AT-I). The patient is compared with previously reported cases of AT-I, as well as with patients reported as having "boomerang" dysplasia. We conclude that it is reasonable to consider AT-I and boomerang dysplasia as part of a spectrum, probably reflecting a common etiology. More and detailed clinical, radiologic and histopathologic reports are needed to further clarify the relationship of AT-II and AT-III in this family of skeletal dysplasias.

A chromosome 17q de novo paracentric inversion in a patient with campomelic dysplasia; case report and etiologic hypothesis.

The campomelic syndrome is a skeletal dysplasia with a characteristic pattern of deformity involving the proximal and distal extremities, pelvic and shoulder girdles, thoracic cage and palate. Respiratory compromise often leads to death in early infancy. Etiology has not been determined although evidence suggests genetic heterogeneity in patients with campomelia. Cytogenetic analysis in the past have revealed an unexpectedly high incidence of a 46, XY karyotype in phenotypic females. We report here on a patient with a typical case of campomelic dysplasia in whom a de novo paracentric inversion of chromosome 17q was identified. Review of the genetic map of the inverted region identified potential "structural" genes including the Hox-2-homeobox gene and the collagen gene, COLIA1, which may be involved in the pathogenesis of campomelic syndrome.

Displasia tanatofórica: observación de dos casos / Thanatophoric dysplasia, presentation of 2 cases

Se describen las principales características de dos recién nacidos afectados de displasia tanatofórica, quienes fallecen en las primeras horas de vida debido a insuficiencia respiratoria por distrofia torácica. Se realiza el diagnóstico diferencial con acondroplasia y acondrogénesis, entidades que tienen distinto patrón hereditario genético.