Gonzalo Alvarez-Bolado

Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development.

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.

Comprehensive expression atlas of fibroblast growth factors and their receptors generated by a novel robotic in situ hybridization platform

A recently developed robotic platform termed “Genepaint” can carry out large‐scale nonradioactive in situ hybridization (ISH) on tissue sections. We report a series of experiments that validate this novel platform. Signal‐to‐noise ratio and mRNA detection limits were comparable to traditional ISH procedures, and hybridization was transcript‐specific, even in cases in which probes could have hybridized to several transcripts of a multigene family. We established an atlas of expression patterns of fibroblast growth factors (Fgfs) and their receptors (Fgfrs) for the embryonic day 14.5 mouse embryo. This atlas provides a comprehensive overview of previously known as well as novel sites of expression for this important family of signaling molecules. The Fgf/Fgfr atlas was integrated into the transcriptome database (www.genepaint.org), where individual Fgf and Fgfr expression patterns can be interactively viewed at cellular resolution and where sites of expressions can be retrieved using an anatomy‐based search. Developmental Dynamics 234:371–386, 2005.

The Repulsive Guidance Molecule RGMa Is Involved in the Formation of Afferent Connections in the Dentate Gyrus

In the developing dentate gyrus, afferent fiber projections terminate in distinct laminas. This relies on an accurately regulated spatiotemporal network of guidance molecules. Here, we have analyzed the functional role of the glycosylphosphatidylinositol (GPI)-anchored repulsive guidance molecule RGMa. In situ hybridization in embryonic and postnatal brain showed expression of RGMa in the cornu ammonis and hilus of the hippocampus. In the dentate gyrus, RGM immunostaining was confined to the inner molecular layer, whereas the outer molecular layers targeted by entorhinal fibers remained free. To test the repulsive capacity of RGMa, different setups were used: the stripe and explant outgrowth assays with recombinant RGMa, and entorhino–hippocampal cocultures incubated either with a neutralizing RGMa antibody (Ab) or with the GPI anchor-digesting drug phosphatidylinositol-specific phospholipase C. Entorhinal axons were clearly repelled by RGMa in the stripe and outgrowth assays. After disrupting the RGMa function, the specific laminar termination pattern in entorhino–hippocampal cocultures was lost, and entorhinal axons entered inappropriate hippocampal areas. Our data indicate an important role of RGMa for the layer-specific termination of the perforant pathway as a repulsive signal that compels entorhinal fibers to stay in their correct target zone.

Pax2/5 and Pax6 subdivide the early neural tube into three domains.

The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. We demonstrate that, in Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), towards the caudal diencephalon. Our results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle.

Distribution of calbindin and parvalbumin in the developing somatosensory cortex and its primordium in the rat: an immunocytochemical study

SummaryImmunocytochemical techniques were used to analyze the distribution of the calcium-binding proteins calbindin and parvalbumin during the pre- and postnatal development of the rat somatosensory cortex. Calbindin occurs in most early differentiated neurons that form the primordial plexiform layer at embryonic day 14. This expression in transient; during the perinatal period, calbindin becomes immunologically undetectable within the structures derived from the primordial plexiform layer, i.e., the prospective layers I and VIb. Immunoreactive neurons are also absent from adult layers I and VIb. Calbindin is also detected in a second population of neurons which, from embryonic day 18 onwards, distributes diffusely within the cortical plate. Some neurons of this population show morphological traits of immaturity, while others show complete dendritic arborization. The definitive pattern of distribution of calbindin-immunoreactive neurons is achieved by postnatal day 22. Infragranular layers contain intensely-immunoreactive cells whose numerical density decreases during postnatal development, whereas in supragranular layers similar neurons are interspersed among numerous faintly-stained neurons.Parvalbumin is detected for the first time at postnatal day 6, within a small group of neurons located in cortical layer V, and extends afterwards through the whole thickness of the cerebral cortex. At this same postnatal stage, groups of immunoreactivepuncta are also found in layer IV of the somatosensory cortex; these puncta increase in density progressively and, at embryonic day 13, immunoreactive cells appear also grouped at this level. At this postnatal age, parvalbumin immunostaining delineates the somatosensory map in cortical layer IV. From this stage to adulthood, the number of immunoreactive neurons increases in the whole thickness of the somatosensory cortex. Barrels in layer IV become less distinct as immunoreactive cells and processes invade the septa. Layer IV in the adult somatosensory cortex appears more densely populated by parvalbumin immunoreactive neurons and puncta than in the surrounding areas.

Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration

TRIM RING finger proteins have been shown to play an important role in cancerogenesis, in the pathogenesis of some human hereditary disorders, and in the defense against viral infection, but the function of the majority of TRIM proteins remains unknown. Here, we show that TRIM RING finger protein TRIM2, highly expressed in the nervous system, is an UbcH5a-dependent ubiquitin ligase. We further demonstrate that TRIM2 binds to neurofilament light subunit (NF-L) and regulates NF-L ubiquitination. Additionally, we show that mice deficient in TRIM2 have increased NF-L level in axons and NF-L-filled axonal swellings in cerebellum, retina, spinal cord, and cerebral cortex. The axonopathy is followed by progressive neurodegeneration accompanied by juvenile-onset tremor and ataxia. Our results demonstrate that TRIM2 is an ubiquitin ligase and point to a mechanism regulating NF-L metabolism through an ubiquitination pathway that, if deregulated, triggers neurodegeneration.

Role of Neuroepithelial Sonic hedgehog in Hypothalamic Patterning

The hypothalamus is a region of the diencephalon with particularly complex patterning. Sonic hedgehog (Shh), encoding a protein with key developmental roles, shows a peculiar and dynamic diencephalic expression pattern. Here, we use transgenic strategies and in vitro experiments to test the hypothesis that Shh expressed in the diencephalic neuroepithelium (neural Shh) coordinates tissue growth and patterning in the hypothalamus. Our results show that neural Shh coordinates anteroposterior and dorsoventral patterning in the hypothalamus and in the diencephalon–telencephalon junction. Neural Shh also coordinates mediolateral hypothalamic patterning, since it is necessary for the lateral hypothalamus to attain proper size and is required for the specification of hypocretin/orexin cells. Finally, neural Shh is necessary to maintain expression of differentiation markers including survival factor Foxb1.

Expression of Meis2, a Knotted-related murine homeobox gene, indicates a role in the differentiation of the forebrain and the somitic mesoderm

Knotted (Kn) genes are expressed within restricted domains of the plant meristems and play a key role in the control of plant morphogenesis. We have isolated the Kn‐related gene Meis2 in mouse, which labels the lateral somitic compartment and its derivatives during early mouse embryogenesis and later becomes a marker for the dorso‐ectodermal region overlying cells of the paraxial mesoderm. Meis2 is also highly expressed in specific areas of the developing central nervous system from embryonic day 9 (e9) onward. In later developmental stages, a strong expression is detectable in differentiating nuclei and regions of the forebrain, midbrain, hindbrain, and spinal cord. This temporal and spatial expression pattern suggests that Meis2 may play an important role in the cascade of induction leading to somitic differentiation as well as in brain regionalization and histogenesis. Dev. Dyn. 1997;210:184–190.

The Role of Sonic Hedgehog of Neural Origin in Thalamic Differentiation in the Mouse

The specification of the intricate neuronal assemblies that characterize the forebrain is not well understood. The ventral spinal cord is specified through a concentration gradient of Sonic hedgehog (Shh) protein secreted by the notochord. Shh is expressed also in the forebrain neuroepithelium (neural Shh) and the underlying notochord and prechordal plate. Neural Shh is essential for the development of the prethalamus (ventral thalamus), but its effects on the thalamus (dorsal thalamus) are still unclear. We hypothesized that neural Shh would act on a previously regionalized dorsal diencephalic region to promote the emergence of specific thalamic nuclear and histological traits. To find out, we generated a conditional mouse mutant line specifically lacking Shh expression in the diencephalic neuroepithelium. We show that the transcription factor Gbx2, required for thalamic development downstream Shh, is expressed in our mutant in a restricted thalamic region and is necessary and sufficient for the differentiation of the medial and intralaminar thalamic nuclei. In the rest of the thalamus, neural Shh is required to promote neuronal aggregation into nuclei as well as axonal extension. In this way, the individual thalamic nuclei show differential dependence on Shh, Gbx2, or both for their differentiation. Additionally, Gbx2 is required for the survival of thalamic neurons.

Regulatory Pathway Analysis by High-Throughput In Situ Hybridization

Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing ∼1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades.