The cortical hem regulates the size and patterning of neocortex.

The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. We examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather we found that hem WNT or BMP signals, or both, have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. Our findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex.

An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool.

MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.

Timing of cortical interneuron migration is influenced by the cortical hem.

Cerebral cortical γ-aminobutyric acid (GABA)ergic interneurons originate from the basal forebrain and migrate into the cortex in 2 phases. First, interneurons cross the boundary between the developing striatum and the cortex to migrate tangentially through the cortical primordium. Second, interneurons migrate radially to their correct neocortical layer position. A previous study demonstrated that mice in which the cortical hem was genetically ablated displayed a massive reduction of Cajal-Retzius (C-R) cells in the neocortical marginal zone (MZ), thereby losing C-R cell-generated reelin in the MZ. Surprisingly, pyramidal cell migration and subsequent layering were almost normal. In contrast, we find that the timing of migration of cortical GABAergic interneurons is abnormal in hem-ablated mice. Migrating interneurons both advance precociously along their tangential path and switch prematurely from tangential to radial migration to invade the cortical plate (CP). We propose that the cortical hem is responsible for establishing cues that control the timing of interneuron migration. In particular, we suggest that loss of a repellant signal from the medial neocortex, which is greatly decreased in size in hem-ablated mice, allows the early advance of interneurons and that reduction of another secreted molecule from C-R cells, the chemokine SDF-1/CXCL12, permits early radial migration into the CP.

Sox6 expression distinguishes dorsally and ventrally biased dopamine neurons in the substantia nigra with distinctive properties and embryonic origins.

Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson's disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6- neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions.

Expression and functional analysis of the Wnt/beta-catenin induced mir-135a-2 locus in embryonic forebrain development.

BACKGROUND: Brain size and patterning are dependent on dosage-sensitive morphogen signaling pathways - yet how these pathways are calibrated remains enigmatic. Recent studies point to a new role for microRNAs in tempering the spatio-temporal range of morphogen functions during development. Here, we investigated the role of miR-135a, derived from the mir-135a-2 locus, in embryonic forebrain development. METHOD: 1. We characterized the expression of miR-135a, and its host gene Rmst, by in situ hybridization (ish). 2. We conditionally ablated, or activated, beta-catenin in the dorsal forebrain to determine if this pathway was necessary and/or sufficient for Rmst/miR-135a expression. 3. We performed bioinformatics analysis to unveil the most predicted pathways targeted by miR-135a. 4. We performed gain and loss of function experiments on mir-135a-2 and analyzed by ish the expression of key markers of cortical hem, choroid plexus, neocortex and hippocampus. RESULTS: 1. miR-135a, embedded in the host long non-coding transcript Rmst, is robustly expressed, and functional, in the medial wall of the embryonic dorsal forebrain, a Wnt and TGFß/BMP-rich domain. 2. Canonical Wnt/beta-catenin signaling is critical for the expression of Rmst and miR-135a, and the cortical hem determinant Lmx1a. 3. Bioinformatics analyses reveal that the Wnt and TGFß/BMP cascades are among the top predicted pathways targeted by miR-135a. 4. Analysis of mir-135a-2 null embryos showed that dorsal forebrain development appeared normal. In contrast, modest mir-135a-2 overexpression, in the early dorsal forebrain, resulted in a phenotype resembling that of mutants with Wnt and TGFß/BMP deficits - a smaller cortical hem and hippocampus primordium associated with a shorter neocortex as well as a less convoluted choroid plexus. Interestingly, late overexpression of mir-135a-2 revealed no change. CONCLUSIONS: All together, our data suggests the existence of a Wnt/miR-135a auto-regulatory loop, which could serve to limit the extent, the duration and/or intensity of the Wnt and, possibly, the TGFß/BMP pathways.