Céline Plachez

The Transcription Factor Gene Nfib Is Essential for both Lung Maturation and Brain Development

ABSTRACT The phylogenetically conserved nuclear factor I (NFI) gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects, whereas Nfic-deficient mice have agenesis of molar tooth roots and severe incisor defects. Here we show that Nfib-deficient mice possess unique defects in lung maturation and exhibit callosal agenesis and forebrain defects that are similar to, but more severe than, those seen in Nfia-deficient animals. In addition, loss of Nfib results in defects in basilar pons formation and hippocampus development that are not seen in Nfia-deficient mice. Heterozygous Nfib-deficient animals also exhibit callosal agenesis and delayed lung maturation, indicating haploinsufficiency at the Nfib locus. The similarity in brain defects in Nfia- and Nfib-deficient animals suggests that these two genes may cooperate in late fetal forebrain development, while Nfib is essential for late fetal lung maturation and development of the pons.

Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

Mechanisms regulating the development of the corpus callosum and its agenesis in mouse and human

The development of the corpus callosum depends on a large number of different cellular and molecular mechanisms. These include the formation of midline glial populations, and the expression of specific molecules required to guide callosal axons as they cross the midline. An additional mechanism used by callosal axons from neurons in the neocortex is to grow within the pathway formed by pioneering axons derived from neurons in the cingulate cortex. Data in humans and in mice suggest the possibility that different mechanisms may regulate the development of the corpus callosum across its rostrocaudal and dorsoventral axes. The complex developmental processes required for formation of the corpus callosum may provide some insight into why such a large number of human congenital syndromes are associated with agenesis of this structure.

Abnormal Development of Forebrain Midline Glia and Commissural Projections in Nfia Knock-Out Mice

Nuclear factor I (NFI) genes are expressed in multiple organs throughout development (Chaudhry et al., 1997; for review, seeGronostajski, 2000). All four NFI genes are expressed in embryonic mouse brain, with Nfia, Nfib, andNfix being expressed highly in developing cortex (Chaudhry et al., 1997). Disruption of the Nfia gene causes agenesis of the corpus callosum (ACC), hydrocephalus, and reduced GFAP expression (das Neves et al., 1999). Three midline structures, the glial wedge, glia within the indusium griseum, and the glial sling are involved in development of the corpus callosum (Silver et al., 1982; Silver and Ogawa, 1983; Shu and Richards, 2001). BecauseNfia−/−mice show glial abnormalities and ACC, we asked whether defects in midline glial structures occur inNfia− / − mice. NFI-A protein is expressed in all three midline populations. InNfia− / − , mice sling cells are generated but migrate abnormally into the septum and do not form a sling. Glia within the indusium griseum and the glial wedge are greatly reduced or absent and consequently Slit2 expression is also reduced. Although callosal axons approach the midline, they fail to cross and extend aberrantly into the septum. The hippocampal commissure is absent or reduced, whereas the ipsilaterally projecting perforating axons (Hankin and Silver, 1988; Shu et al., 2001) appear relatively normal. These results support an essential role for midline glia in callosum development and a role for Nfia in the formation of midline glial structures.

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm−3), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-d-aspartate (4-methoxy-7-nitroindolinyl-d-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and d-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

The transcription factor Nfix is essential for normal brain development

BackgroundThe Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice.ResultsHere we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.ConclusionThese data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.

Neuropilin 1-Sema Signaling Regulates Crossing of Cingulate Pioneering Axons during Development of the Corpus Callosum

Pioneer axons from the cingulate cortex initiate corpus callosum (CC) development, yet nothing is known about the molecules that regulate their guidance. We demonstrate that neuropilin 1 (Npn1) plays an integral role in the development of the CC. Npn1 is localized to axons of cingulate neurons as they cross the midline, and multiple class 3 semaphorins (Semas) are expressed around the developing CC, implicating these guidance molecules in the regulation of Npn1-expressing axons emanating from the cingulate cortex. Furthermore, axons from the cingulate cortex display guidance errors in Npn1(Sema-) mice, a knockin mouse line in which Npn1 is unable to bind Semas. Analysis of mice deficient in the transcription factor Emx2 demonstrated that the cingulate cortex of these mice was significantly reduced in comparison to wild-type controls at E17 and that the CC was absent in rostral sections. Expression of Npn1 was absent in rostral sections of Emx2 mutants, suggesting that Npn1-expressing cingulate pioneers are required for CC formation. These data highlight a central role for Npn1 in the development of projections from the cingulate cortex and further illustrate the importance of these pioneer axons in the formation of the CC.

Mapping postnatal mouse brain development with diffusion tensor microimaging

While mouse brain development has been extensively studied using histology, quantitative characterization of morphological changes is still a challenging task. This paper presents how developing brain structures can be quantitatively characterized with magnetic resonance diffusion tensor microimaging coupled with techniques of computational anatomy. High resolution diffusion tensor images of ex vivo postnatal mouse brains provide excellent contrasts to reveal the evolutions of mouse forebrain structures. Using anatomical landmarks defined on diffusion tensor images, tissue level growth patterns of mouse brains were quantified. The results demonstrate the use of these techniques to three-dimensionally and quantitatively characterize brain growth.

Olfactory Bulb Short Axon Cell Release of GABA and Dopamine Produces a Temporally Biphasic Inhibition–Excitation Response in External Tufted Cells

Evidence for coexpression of two or more classic neurotransmitters in neurons has increased, but less is known about cotransmission. Ventral tegmental area (VTA) neurons corelease dopamine (DA), the excitatory transmitter glutamate, and the inhibitory transmitter GABA onto target cells in the striatum. Olfactory bulb (OB) short axon cells (SACs) form interglomerular connections and coexpress markers for DA and GABA. Using an optogenetic approach, we provide evidence that mouse OB SACs release both GABA and DA onto external tufted cells (ETCs) in other glomeruli. Optical activation of channelrhodopsin specifically expressed in DAergic SACs produced a GABAA receptor-mediated monosynaptic inhibitory response, followed by DA–D1-like receptor-mediated excitatory response in ETCs. The GABAA receptor-mediated hyperpolarization activates Ih current in ETCs; synaptically released DA increases Ih, which enhances postinhibitory rebound spiking. Thus, the opposing actions of synaptically released GABA and DA are functionally integrated by Ih to generate an inhibition-to-excitation “switch” in ETCs. Consistent with the established role of Ih in ETC burst firing, we show that endogenous DA release increases ETC spontaneous bursting frequency. ETCs transmit sensory signals to mitral/tufted output neurons and drive intraglomerular inhibition to shape glomerulus output to downstream olfactory networks. GABA and DA cotransmission from SACs to ETCs may play a key role in regulating output coding across the glomerular array.

Nuclear Factor I Gene Expression in the Developing Forebrain

Three members of the Nuclear Factor I (Nfi) gene family of transcription factors; Nfia, Nfib, and Nfix are highly expressed in the developing mouse brain. Nfia and Nfib knockout mice display profound defects in the development of midline glial populations and the development of forebrain commissures (das Neves et al. [ 1999 ] Proc Natl Acad Sci U S A 96:11946–11951; Shu et al. [ 2003 ] J Neurosci 23:203–212; Steele‐Perkins et al. [ 2005 ] Mol Cell Biol 25:685–698). These findings suggest that Nfi genes may regulate the substrate over which the commissural axons grow to reach targets in the contralateral hemisphere. However, these genes are also expressed in the cerebral cortex and, thus, it is important to assess whether this expression correlates with a cell‐autonomous role in cortical development. Here we detail the protein expression of NFIA and NFIB during embryonic and postnatal mouse forebrain development. We find that both NFIA and NFIB are expressed in the deep cortical layers and subplate prenatally and display dynamic expression patterns postnatally. Both genes are also highly expressed in the developing hippocampus and in the diencephalon. We also find that principally neither NFIA nor NFIB are expressed in callosally projecting neurons postnatally, emphasizing the role for midline glial cell populations in commissure formation. However, a large proportion of laterally projecting neurons express both NFIA and NFIB, indicating a possible cell‐autonomous role for these transcription factors in corticospinal neuron development. Collectively, these data suggest that, in addition to regulating the formation of axon guidance substrates, these genes also have cell‐autonomous roles in cortical development. J. Comp. Neurol. 508:385–401, 2008.