Alfonso Lavado

Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

The transcription factor Prox1 plays a crucial role in intermediate progenitor maintenance and hippocampal neuron differentiation during adult neurogenesis in the dentate gyrus region of the hippocampus.

Prox1 expression patterns in the developing and adult murine brain.

Prox1, a homeobox gene related to the Drosophila gene prospero, is necessary for retina, lens, liver, pancreas, and lymphatics development. However, not much is yet known about Prox1 expression during central nervous system development. Here we provide a detailed analysis of Prox1 mRNA and protein expression during prenatal and postnatal murine brain development. Prenatally, Prox1 is expressed in the subventricular zone or in early differentiating regions of the brain. At these stages, Prox1 mRNA, but not Prox1 protein, was also detected in several regions of the prethalamus and hypothalamus. At an early postnatal stage, Prox1 expression is mainly detected in several nuclei of the thalamus, the cerebellum, and the hippocampus. In adulthood, Prox1 expression remains only in the hippocampus and cerebellum. These complex patterns of expression suggest that Prox1 activity is differentially required during brain development and adulthood. Developmental Dynamics 236:518–524, 2007.

Six3 inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon

The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3-null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.

Tumor suppressor Nf2 limits expansion of the neural progenitor pool by inhibiting Yap/Taz transcriptional coactivators

Brain development requires a precise balance between expansion of the neural progenitor pool and the production of postmitotic neurons and glia. Disruption of this equilibrium results in a myriad of structural abnormalities and disorders of the nervous system. The molecular mechanism that restricts neural progenitor expansion is poorly understood. Here we show that the tumor suppressor neurofibromatosis 2 (Nf2; merlin) limits the expansion of neural progenitor cells (NPCs) in the mammalian dorsal telencephalon. Nf2 is localized at the apical region of NPCs. In the absence of Nf2, NPCs of the cortical hem, hippocampal primordium and neocortical primordium overexpand, while production of Cajal-Retzius cells and hippocampal neurons decreases, resulting in severe malformation of the hippocampus in adult mice. We further show that Nf2 functions by inhibiting the Yap/Taz transcriptional coactivators, probably through a mechanism that is distinct from the canonical Hippo pathway. Overexpressing human YAP in NPCs causes a hippocampal malformation phenotype that closely resembles that of Nf2 mutants and, importantly, deleting Yap in the Nf2 mutant background largely restores hippocampal development. Our studies uncover Nf2 as an important inhibitor of neural progenitor expansion and establish Yap/Taz as key downstream effectors of Nf2 during brain development.

Canal cristae growth and fiber extension to the outer hair cells of the mouse ear require Prox1 activity.

Background The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons. Methodology/Principal Findings We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons. Conclusions/Significance These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.

Prox1 Regulates the Subtype-Specific Development of Caudal Ganglionic Eminence-Derived GABAergic Cortical Interneurons

Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties. SIGNIFICANCE STATEMENT Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking. Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to direct the migration, differentiation, circuit integration, and maintenance programs within distinct subtypes of CGE-derived interneurons.

Jagged1 is necessary for postnatal and adult neurogenesis in the dentate gyrus.

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.

Six3 is required for ependymal cell maturation

Ependymal cells are part of the neurogenic niche in the adult subventricular zone of the lateral ventricles, where they regulate neurogenesis and neuroblast migration. Ependymal cells are generated from radial glia cells during embryonic brain development and acquire their final characteristics postnatally. The homeobox gene Six3 is expressed in ependymal cells during the formation of the lateral wall of the lateral ventricles in the brain. Here, we show that Six3 is necessary for ependymal cell maturation during postnatal stages of brain development. In its absence, ependymal cells fail to suppress radial glia characteristics, resulting in a defective lateral wall, abnormal neuroblast migration and differentiation, and hydrocephaly.

New animal models to study the role of tyrosinase in normal retinal development

Albino animals display a hypopigmented phenotype associated with several visual abnormalities, including rod photoreceptor cell deficits, abnormal patterns of connections between the eye and the brain and a general underdevelopment of central retina. Oculocutaneous albinism type I, a common form of albinism, is caused by mutations in the tyrosinase gene. In mice, the albino phenotype can be corrected by functional tyrosinase transgenes. Tyrosinase transgenic animals not only show normal pigmentation but the correction of all visual abnormalities associated with albinism, confirming a role of tyrosinase, a key enzyme in melanin biosynthesis, in normal retinal development. Here, we will discuss recent work carried out with new tyrosinase transgenic mouse models, to further analyse the role of tyrosinase in retinal development. We will first report a transgenic model with inducible tyrosinase expression that has been used to address the regulated activation of this gene and its associated effects on the development of the visual system. Second, we will comment on an interesting yeast artificial chromosome (YAC)-tyrosinase transgene, lacking important regulatory elements, that has highlighted the significance of local interactions between the retinal pigment epithelium (RPE) and developing neural retina.

A strategy to study tyrosinase transgenes in mouse melanocytes

BackgroundA number of transgenic mice carrying different deletions in the Locus Control Region (LCR) of the mouse tyrosinase (Tyr) gene have been developed and analysed in our laboratory. We require melanocytes from these mice, to further study, at the cellular level, the effect of these deletions on the expression of the Tyr transgene, without potential interference with or from the endogenous Tyr alleles. It has been previously reported that it is possible to obtain and immortalise melanocyte cell cultures from postnatal mouse skin.ResultsHere, we describe the efforts towards obtaining melanocyte cultures from our Tyr transgenic mice. We have bred our Tyr transgenic mice into Tyrc-32DSDmutant background, lacking the endogenous Tyr locus. In these conditions, we failed to obtain immortalised melanocytes. We decided to include the inactivation of the Ink4a-Arf locus to promote melanocyte immortalisation. For this purpose, we report the segregation of the Ink4a-Arf null allele from the brown (Tyrp1b) mutation in mice. Finally, we found that Ink4a-Arf+/- and Ink4a-Arf-/- melanocytes had undistinguishable tyrosine hydroxylase activities, although the latter showed reduced cellular pigmentation content.ConclusionThe simultaneous presence of precise genomic deletions that include the tyrosinase locus, such as the Tyrc-32DSDallele, the Tyr transgene itself and the inactivated Ink4a-Arf locus in Tyrp1Bgenetic background appear as the crucial combination to perform forthcoming experiments. We cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, subsequent experiments with melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus.