Amaya Miquelajáuregui

Foxp-Mediated Suppression of N-Cadherin Regulates Neuroepithelial Character and Progenitor Maintenance in the CNS

Neuroepithelial attachments at adherens junctions are essential for the self-renewal of neural stem and progenitor cells and the polarized organization of the developing central nervous system. The balance between stem cell maintenance and differentiation depends on the precise assembly and disassembly of these adhesive contacts, but the gene regulatory mechanisms orchestrating this process are not known. Here, we demonstrate that two Forkhead transcription factors, Foxp2 and Foxp4, are progressively expressed upon neural differentiation in the spinal cord. Elevated expression of either Foxp represses the expression of a key component of adherens junctions, N-cadherin, and promotes the detachment of differentiating neurons from the neuroepithelium. Conversely, inactivation of Foxp2 and Foxp4 function in both chick and mouse results in a spectrum of neural tube defects associated with neuroepithelial disorganization and enhanced progenitor maintenance. Together, these data reveal a Foxp-based transcriptional mechanism that regulates the integrity and cytoarchitecture of neuroepithelial progenitors.

LIM-Homeobox Gene Lhx5 Is Required for Normal Development of Cajal–Retzius Cells

Cajal–Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.

Layer 4 Pyramidal Neurons Exhibit Robust Dendritic Spine Plasticity In Vivo after Input Deprivation

Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinal in vivo two-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (∼50%) increase in the rate of dendritic spine loss, acutely (4–8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1.

The Krüppel-like factor 4 controls biosynthesis of thyrotropin-releasing hormone during hypothalamus development

Embryonic neurogenesis is controlled by the activation of specific genetic programs. In the hypothalamus, neuronal thyrotropin-releasing hormone (TRH) populations control important physiological process, including energy homeostasis and autonomic function; however, the genetic program leading to the TRH expression is poorly understood. Here, we show that the Klf4 gene, encoding the transcription factor Krüppel-like factor 4 (Klf4), was expressed in the rat hypothalamus during development and regulated Trh expression. In rat fetal hypothalamic cells Klf4 regulated Trh promoter activity through CACCC and GC motifs present on the Trh gene promoter. Accordingly, hypothalamic Trh expression was down-regulated at embryonic day 15 in the Klf4(-/-) mice resulting in diminished bioactive peptide levels. Although at the neonatal stage the Trh transcript levels of the Klf4(-/-) mice were normal, the reduction in peptide levels persisted. Thus, our data indicate that Klf4 plays a key role in the maturation of TRH expression in hypothalamic neurons.

Slit-Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain.

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5–E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit‐Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

Two-photon excitation microscopy and its applications in neuroscience.

Two-photon excitation (2PE) overcomes many challenges in fluorescence microscopy. Compared to confocal microscopy, 2PE microscopy improves depth penetration, owing to the longer excitation wavelength required and to the ability to collect scattered emission photons as a useful signal. It also minimizes photodamage because lower energy photons are used and because fluorescence is confined to the geometrical focus of the laser spot. 2PE is therefore ideal for high-resolution, deep-tissue, time-lapse imaging of dynamic processes in cell biology. Here, we provide examples of important applications of 2PE for in vivo imaging of neuronal structure and signals; we also describe how it can be combined with optogenetics or photolysis of caged molecules to simultaneously probe and control neuronal activity.

Lhx5 controls mamillary differentiation in the developing hypothalamus of the mouse

Acquisition of specific neuronal identity by individual brain nuclei is a key step in brain development. However, how the mechanisms that confer neuronal identity are integrated with upstream regional specification networks is still mysterious. Expression of Sonic hedgehog (Shh), is required for hypothalamic specification and is later downregulated by Tbx3 to allow for the differentiation of the tubero-mamillary region. In this region, the mamillary body (MBO), is a large neuronal aggregate essential for memory formation. To clarify how MBO identity is acquired after regional specification, we investigated Lhx5, a transcription factor with restricted MBO expression. We first generated a hypomorph allele of Lhx5—in homozygotes, the MBO disappears after initial specification. Intriguingly, in these mutants, Tbx3 was downregulated and the Shh expression domain abnormally extended. Microarray analysis and chromatin immunoprecipitation indicated that Lhx5 appears to be involved in Shh downregulation through Tbx3 and activates several MBO-specific regulator and effector genes. Finally, by tracing the caudal hypothalamic cell lineage we show that, in the Lhx5 mutant, at least some MBO cells are present but lack characteristic marker expression. Our work shows how the Lhx5 locus contributes to integrate regional specification pathways with downstream acquisition of neuronal identity in the MBO.

Origin and Migration of Olfactory Cajal-Retzius Cells

Early telencephalic development involves the migration of diverse cell types that can be identified by specific molecular markers. Most prominent among them are Cajal-Retzius (CR) cells that emanate mainly from the cortical hem and to a lesser extent from rostrolateral, septal and caudo-medial regions. One additional territory proposed to give rise to CR cells that migrate dorsally into the neocortex lies at the ventral pallium, although contradictory results question this notion. With the use of a cell-permeable fluorescent tracer in cultured embryos, we identified novel migratory paths of putative CR cells and other populations that originate from the rostrolateral telencephalon at its olfactory region. Moreover, extensive labeling on the lateral telencephalon along its rostro-caudal extent failed to reveal a dorsally-migrating CR cell population from the ventral pallium at the stages analyzed. Hence, this work reveals a novel olfactory CR cell migration and supports the idea that the ventral pallium, where diverse types of neurons converge, does not actually generate CR cells.

LIM homeobox protein 5 (Lhx5) is essential for mamillary body development

The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.

LIM homeobox protein 5 (Lhx5) is essential for mammillary body development

Themammillary body (MB) comprises several nuclei lying at the posterior end of the hypothalamus that have been implicated in spatial memory function. The MB receives hippocampal input and projects to the thalamus and tegmentum, thus linking together forebrain and midbrain areas. Here we show that the LIM-HD transcription factor Lhx5 is expressed in theMBprimordiumearly in mouse embryogenesis. Targeted inactivation of Lhx5 results in a dramatic reductionofMBareaandaconcomitantabsenceof efferent projections perinatally. The early specification of the MB area appeared severely altered, as shown by the lack of expression of Foxb1, Sim2, and other novel MB markers by in situ hybridization. Interestingly, Lhx5 / embryos expressed Lhx2 and Lhx9 ectopically in this area, suggesting an intricate regulation of gene expression between LIM-HDmembers during mammillary body development.