Genitocrural Island Perforator Flap (GIP-Flap): An ideal surgical technique for covering uni- or bilateral vulvar loss tissue.

Vulvar loss of soft tissue leads to urinary, sexual and morphological dysfunctions. Most patients affected are comorbid making it difficult to perform a flap, which is the most appropriate way to reconstruct. Our multidisciplinary plastic and gynecologic surgery team has developed a new technique using a pedicled internal pudendal island flap. Reconstruction is reliable, quick and applicable to all patients, with a highly satisfactory final appearance.

Pax6 modulates the dorsoventral patterning of the mammalian telencephalon.

The Pax6 gene encodes a transcription factor with a restricted expression in the ventricular zone of the pallium and subpallium. We tested whether the function of Pax6 is necessary for the correct patterning and morphogenesis of the vertebrate telencephalon. Homozygous embryos of the Pax6/Small eye mutant lack functional PAX6 protein because of a point mutation of the gene. In the mutant Small eye embryos we detected a ventralization of the molecular patterning of the telencephalon at two borders, the pallium/subpallium and the lateral/medial ganglionic eminence. The results indicate that Pax6 controls the lateral limit of the expression of Nkx2.1, Shh, and Lhx6 in the prechordal neural tube, the telencephalon. This finding is in agreement with previous studies and supports a model for a common genetic mechanism for modulation of the dorsoventral patterning of the prechordal and epichordal CNS. The pattern defects caused by the loss of Pax6 function result in multiple morphological abnormalities in the Small eye brain: dysgenesis of the piriform, insular, and lateral cortices, the claustrum-endopiriform nucleus, and a failure in the differentiation of a subpopulation of the cortical precursors. Together the results demonstrate that Pax6 has an essential role for the modulation of the dorsoventral patterning of the embryonic telencephalon, influencing thereby the forebrain morphogenesis.

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS.

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.

Vax1, a novel homeobox-containing gene, directs development of the basal forebrain and visual system.

The novel homeobox-containing gene Vax1, a member of the Emx/Not gene family, is specifically expressed in the developing basal forebrain and optic nerve. Here, we show that Vax1 is essential for normal development of these structures. Mice carrying a targeted mutation of Vax1 show dysgenesis of the optic nerve, coloboma, defects in the basal telencephalon, and lobar holoprosencephaly. With the help of molecular markers we determined that in the developing visual system, the absence of Vax1 results in a proximal expansion of the activity of Pax6 and Rx. This observation suggests that Vax1 may interfere negatively with the expression of Pax6 and Rx. In reciprocal gain-of-function experiments, injection of Xvax1 mRNA or Shh into Xenopus embryos primarily affects the brain at the level of the eye primordium. Consistent with the loss-of-function results, the injection of Xvax1 results in a down-regulation of Rx. Similarly, Shh injection expands the Vax1 and Pax2 territory at the expense of the Pax6 and Rx region. On the basis of these results, we propose a model for a molecular cascade involved in the establishment of structures of the visual system.

Vax1 is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain.

The vertebrate forebrain is formed at the rostral end of the neural plate under the regulation of local and specific signals emanating from both the endomesoderm and neuroectoderm. The development of the rostral and ventral forebrain in particular was difficult to study, mainly because no specific markers are available to date. Here, we report the identification of Vax1, a novel homeobox-containing gene identified in mouse, Xenopus and human. It is closely related to members of the Not and Emx gene families, all of which are required for the formation of structures where they are expressed. In mouse and Xenopus, Vax1 expression first occurs in the rostral neural plate, in the medial anterior neural ridge and adjacent ectoderm. Later, at midgestation in the mouse and tadpole stage in Xenopus, the expression remains confined in the derivatives of this territory which differentiate into rostromedial olfactory placode, optic nerve and disc, and anterior ventral forebrain. Together, these observations suggest that Vax1 could have an early evolutionary origin and could participate in the specification and formation of the rostral and ventral forebrain in vertebrates. Comparison of the limits of the expression territory of Vax1 with that of Dlx1, Pax6 and Emx1 indicates that the corticostriatal ridge is a complex structure with distinct identifiable genetic compartments. Besides, the study of Vax1 expression in Pax6-deficient homozygous brains indicates that its regulation is independent of Pax6, although the expression patterns of these two genes appear complementary in wild-type animals. Vax1 chromosomal location is mapped at the distal end of the mouse chromosome 19, linked with that of Emx2. These two genes may have arisen by tandem duplication. The Vax1 gene is thus an interesting new tool to study the rostral ventral forebrain patterning, morphogenesis and evolution as well as the terminal differentiation of the forebrain in mouse and Xenopus.

The chick/quail chimeric system: a model for early cerebellar development.

The chick/quail chimeric system is now extensively used to study the development of the central nervous system. Here we discuss data obtained by this powerful experimental approach by which to study several issues of the cerebellar ontogenesis. We first discuss experiments which have allowed redefinition of the localization of the cerebellar primordium in the early neural tube and which suggest that the cerebellum could originate from different morphogenetic units. Then, we discuss experiments testing the possible role of the homeobox containing gene En-2 in cerebellar specification and showing that the En-2 expressing cerebellar neuroepithelium can act as an organizer. Finally we discuss data obtained in chimeric embryos with partial cerebellar grafts used to reexamine the origin and settling of several types of cortical cerebellar cells, in particular granule cells, molecular layer interneurons and Purkinje cells.

Pax genes and their roles in cell differentiation and development.

Members of the Pax gene family are expressed in various tissues during ontogenesis. Evidence for their crucial role in morphogenesis, organogenesis, cell differentiation and oncogenesis is provided by rodent mutants and human diseases. Additionally, recent experimental in vivo and in vitro approaches have led to the identification of molecules that interact with Pax proteins.

Tracing neuroepithelial cells of the mesencephalic and metencephalic alar plates during cerebellar ontogeny in quail-chick chimaeras.

The quail-chick chimaera system was used to investigate the origin of the various neuronal cell types of the cerebellum from the mesencephalic and metencephalic brain vesicles at the 11- to 14-somite stage in the avian embryo. We have already demonstrated that the cerebellum is derived from both the mesencephalic and metencephalic brain vesicles. The mesencephalic contribution to the cerebellum is restricted to a mediodorsal territory inserted as a V-shaped area into the primitive metencephalic vesicle through complex morphogenetic movements taking place from day 2 to day 4 of embryonic development. Here we report that the cerebellar presumptive territory extends along the anteroposterior axis, over the caudal half of the mesencephalon and the rostral half of the metencephalon. Along the dorsoventral axis, the cerebellar anlage is located in the alar plates at the exclusion of the roof and basal plates, i.e. lies in the lateral walls of the neuroepithelium in the area included between approximately 25 and 120 degrees with respect to the sagittal plane. We also report that the neuroepithelium corresponding to the cerebellar presumptive territory also yields other brain structures (e.g. part of the optic tectum in the mesencephalon). The external granular layer (EGL) arises only from the rostral metencephalon, undergoing extensive tangential movements which we have analysed in detail: the more ventral the position of cells in the metencephalic alar plates the more rostral and lateral is their position in the EGL. Finally, we discuss the fact that the cerebellar cortex, an integrative structure of the brain, arises from the alar plates of the neural tube. This is consistent with the general spatial organization of the neural anlage of the vertebrate embryo, in which this part of the neuroepithelium is devoted to the production of interneurons, whereas the basal plate and the neural folds yield motor structures and primary sensory neurons respectively.

Studying brain development with quail-chick neural chimeras.

Avian embryonic neural chimeras are constructed by substituting defined areas of the neural epithelium of a chick embryo by their exact counterpart obtained from a quail embryo at the same developmental stage. The experiment can also be performed using the quail as a host and the chick as a graft donor. The stages elected are either the late neurula (0 to 3 somites) or the stage after cephalic vesicles formation (10 to 14 somites) but in all cases before the onset of vascularization of the neural primordium. Quail and chick territories can be recognized in the chimeras any time after the graft owing to the particular structure of the quail nucleus or by means of species-specific antibodies. Quail-chick chimeras have been instrumental in the study of the ontogeny of the medulla oblongata and of the cerebellum. The complex morphogenetic movements and cell migrations which occur during the development of this part of the brain have been worked out by this method. The main results obtained are described in the article.

Association of BEN glycoprotein expression with climbing fiber axonogenesis in the avian cerebellum.

In a previous study, we have identified an avian 100 kDa membrane glycoprotein that we called BEN and demonstrated that it is transiently present in the CNS and PNS on the cell somas and axons of neurons that establish the peripheral neuronal circuitry. We report here that in the developing chick cerebellar system BEN is selectively expressed on fibers whose ingrowth and synaptogenesis pattern corresponds to that described for climbing fibers. We have constructed quail-chick chimeras in which the chick mesencephalon and anterior metencephalon were replaced by their quail counterparts, thus generating a cerebellum and mesencephalon exclusively composed of quail cells whereas the main nuclei emitting afferent fibers to the cerebellar cortex were of chick origin. Then, using species-specific monoclonal antibodies we were able to show in double staining experiments that BEN protein is specifically expressed on fibers arising from the inferior olivary nucleus. The spatiotemporal pattern of BEN expression on the climbing fibers leads us to propose that this molecule is associated with the growth of these fibers and with the establishment of synapses between them and the Purkinje cell dendritic tree.

A new approach to the development of the cerebellum provided by the quail-chick marker system.

We have used the quail-chick chimera system to reveal the cell migrations and settling pattern involved in the construction of the cerebellum. Three types of orthotopic transplantations were carried out, between quail and chick embryos, at the 12-somite stage: exchanges of (i) the whole metencephalic vesicle, (ii) the lateral half of this vesicle and (iii) the diencephalic plus the mesencephalic vesicles. Histological study of chimeric embryos and young chicks provided the following results: longitudinal morphogenetic movements distort the embryonic neural tube as early as the fifth embryonic day, so that the dorsal limit of the mes-, met- and myelencephalic vesicles are displaced caudad and their ventral limits rostrad. This leads to a participation of mesencephalic vesicular material in the construction of the cerebellum. Cells originating in the mesencephalic vesicle are found in a rostromedial V-shaped region, in all the cerebellar cellular layers, except the external granular layer, the presumptive territory of which is entirely located in the metencephalic vesicle. The chimerism of the rostromedial part of the cerebellum allows the analysis of the origin of the various cerebellar cell types. We find (i) that the Purkinje cells always have the same cellular marker as the ventricular epithelium radially beneath them. This strongly suggests that these cells reach their final localization following strictly radial migrations. (ii) Most of the small cells surrounding the Purkinje neurons and most of the neurons and glial cells of the molecular layer are also of the same type as the ventricular epithelium they surmount, i.e. different from the type of the external granular layer cells. Therefore, they are not derived from the external granular layer and are not of the same origin as the granule cells as previously believed. Unilateral substitutions of the metencephalic vesicle revealed that transverse cell migrations occur across the sagittal plane. They have been observed mainly in the inner and external granular layers, but also, though to a lesser extent, in the molecular layer and in the cell layer located at the level of the Purkinje neurons. These observations show that the position of cerebellar cells is determined by both morphogenetic movements and cell type-specific active radial and tangential migrations. The quail-chick chimera system is thus able to provide new information both on the origin of cerebellar cells and how each cell type assumes its final position.