Neural progenitor cells in the developing dorsal forebrain give rise to excitatory neurons, astrocytes, and oligodendrocytes for the neocortex. While we are starting to gain a better understanding about the mechanisms that direct the formation of neocortical neurons and astrocytes, far less is known about the molecular mechanisms that instruct dorsal forebrain progenitors to make oligodendrocytes. In this study, we show that Sonic hedgehog (Shh) signaling is required in dorsal progenitors for their late embryonic transition to oligodendrogenesis. Using genetic lineage-tracing in mice of both sexes, we demonstrate that most oligodendrocytes in the embryonic neocortex derive from Emx1 dorsal forebrain progenitors. Deletion of the Shh signaling effector specifically in Emx1 progenitors led to significantly decreased oligodendrocyte numbers in the embryonic neocortex. Conversely, knock-out of the Shh antagonist was sufficient to increase neocortical oligodendrogenesis. Using conditional knock-out strategies, we found that Shh ligand is supplied to dorsal progenitors through multiple sources. Loss of from Dlx5/6 interneurons caused a significant reduction in oligodendrocytes in the embryonic neocortex. This phenotype was identical to that observed upon deletion from the entire CNS using, indicating that interneurons migrating into the neocortex from the subpallium are the primary neural source of Shh for dorsal oligodendrogenesis. Additionally, deletion of from migrating interneurons together with the choroid plexus epithelium led to a more severe loss of oligodendrocytes, suggesting that the choroid plexus is an important non-neural source of Shh ligand. Together, our studies demonstrate that the dorsal wave of neocortical oligodendrogenesis occurs earlier than previously appreciated and requires highly regulated Shh signaling from multiple embryonic sources. Most neocortical oligodendrocytes are made by neural progenitors in the dorsal forebrain, but the mechanisms that specify this fate are poorly understood. This study identifies Sonic hedgehog (Shh) signaling as a critical pathway in the transition from neurogenesis to oligodendrogenesis in dorsal forebrain progenitors during late embryonic development. The timing of this neuron-to-glia "switch" coincides with the arrival of migrating interneurons into the dorsal germinal zone, which we identify as a critical source of Shh ligand, which drives oligodendrogenesis. Our data provide evidence for a new model in which Shh signaling increases in the dorsal forebrain late in embryonic development to provide a temporally regulated mechanism that initiates the third wave of neocortical oligodendrogenesis.

This Matters Arising paper is in response to Guo et al (2013) in Neuron. See also the Matters Arising by Eckler et al. published concurrently. Using genetic fate-mapping with Cux2-Cre and Cux2-CreERT2 mice we demonstrated that the neocortical ventricular zone (VZ) contains radial glial cells (RGCs) with restricted fate potentials (Franco et al., 2012). Using the same mouse lines, Guo et al. (2013) concluded that the neocortical VZ does not contain lineage restricted RGCs. We now show that the recombination pattern in Cux2-Cre/CreERT2 mice depends on genetic background and breeding strategies. We provide evidence that Guo et al. likely reached different conclusions because they worked with transgenic sublines with drifted transgene expression patterns. In Cux2-Cre and Cux2-CreERT2 mice that recapitulate the endogenous Cux2 expression pattern, the vast majority of fate-mapped neurons express Satb2 but not Ctip2, confirming that a restricted subset of all neocortical projection neurons belongs to the Cux2 lineage.

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SUMMARYThe eggshell of oviparous lizards is a significant source of calcium for embryos, whereas the eggshell of viviparous lizards, when present, contains little calcium. In view of the potential cost to embryonic nutrition occasioned by the loss of eggshell calcium, the large number of independent origins of viviparity among lizards is surprising. Concomitant evolution of viviparity and calcium placentotrophy would ameliorate the loss of eggshell calcium, but a mechanism linking these events has yet to be discovered. Zootoca vivipara, a lizard with geographic variation in its mode of parity, is an excellent model for studying mechanisms of calcium transport to oviparous and viviparous embryos because each is highly dependent on calcium secreted by the uterus (eggshell or placenta) and ontogenetic patterns of embryonic calcium mobilization are similar. We compared developmental expression of the calcium transport protein calbindin-D 28K in yolk splanchnopleure and chorioallantoic membranes of oviparous and viviparous embryos to test the hypothesis that the mechanism of calcium transport does not differ between modes of parity. We found that the ontogenetic pattern of protein expression is similar between reproductive modes and is correlated with calcium uptake from yolk and either eggshell or placenta. Calbindin-D 28K is localized in the chorionic epithelium of embryos of both reproductive modes. These findings suggest that the embryonic calcium transport machinery is conserved in the transition between reproductive modes and that an adaptation of oviparous embryos for calcium uptake from eggshells functions similarly to transport calcium directly from uterine secretions.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca2+ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the Transient Receptor Potential Melastatin 4, (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 & 100 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150 kD protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

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During neocortical development, neurons are produced by a diverse pool of neural progenitors. A subset of progenitors express the Cux2 gene and are fate-restricted to produce certain neuronal subtypes, but the upstream pathways that specify these progenitor fates remain unknown. To uncover the transcriptional networks that regulate Cux2 expression in the forebrain, we characterized a conserved Cux2 enhancer that we find recapitulates Cux2 expression specifically in the cortical hem. Using a bioinformatic approach, we found several potential transcription factor (TF) binding sites for cortical hem-patterning TFs. We found that the homeobox transcription factor, Lmx1a, can activate the Cux2 enhancer in vitro. Furthermore, we show that multiple Lmx1a binding sites required for enhancer activity in the cortical hem in vivo. Mis-expression of Lmx1a in neocortical progenitors caused an increase in Cux2+-lineage cells. Finally, we compared several conserved human enhancers with cortical hem-restricted activity and found that recurrent Lmx1a binding sites are a top shared feature. Uncovering the network of TFs involved in regulating Cux2 expression will increase our understanding of the mechanisms pivotal in establishing Cux2-lineage fates in the developing forebrain.Summary StatementAnalysis of a cortical hem-specific Cux2 enhancer reveals role for Lmx1a as a critical upstream regulator of Cux2 expression patterns in neural progenitors during early forebrain development.

Yolk is the primary source of calcium for embryonic growth and development for most squamates, irrespective of mode of parity. The calcified eggshell is a secondary source for embryonic calcium in all oviparous eggs, but this structure is lost in viviparous lineages. Virginia striatula is a viviparous snake in which embryos obtain calcium from both yolk and placental transport of uterine calcium secretions. The developmental pattern of embryonic calcium acquisition in V. striatula is similar to that for oviparous snakes. Calbindin-D(28K) is a marker for epithelial calcium transport activity and plasma membrane Ca(2+)-ATPase (PMCA) provides the energy to catalyze the final step in calcium transport. Expression of calbindin-D(28K) and PMCA was measured by immunoblotting in yolk sac splanchnopleure and chorioallantois of a developmental series of V. striatula to test the hypothesis that these proteins mediate calcium transport to embryos. In addition, we compared the expression of calbindin-D(28K) in extraembryonic membranes of V. striatula throughout development to a previously published expression pattern in an oviparous snake to test the hypothesis that the ontogeny of calcium transport function is independent of reproductive mode. Expression of calbindin-D(28K) increased in yolk sac splanchnopleure and chorioallantois coincident with calcium mobilization from yolk and uterine sources and with embryonic growth. The amount of PMCA in the chorioallantois did not change through development suggesting its expression is not rate limiting for calcium transport. The pattern of expression of calbindin-D(28K) and PMCA confirms our initial hypothesis that these proteins mediate embryonic calcium uptake. In addition, the developmental pattern of calbindin-D(28K) expression in V. striatula is similar to that of an oviparous snake, which suggests that calcium transport mechanisms and their regulation are independent of reproductive mode.