Odessa Yabut

miR-125b Promotes Early Germ Layer Specification through Lin28/let-7d and Preferential Differentiation of Mesoderm in Human Embryonic Stem Cells

Unlike other essential organs, the heart does not undergo tissue repair following injury. Human embryonic stem cells (hESCs) grow indefinitely in culture while maintaining the ability to differentiate into many tissues of the body. As such, they provide a unique opportunity to explore the mechanisms that control human tissue development, as well as treat diseases characterized by tissue loss, including heart failure. MicroRNAs are small, non-coding RNAs that are known to play critical roles in the regulation of gene expression. We profiled the expression of microRNAs during hESC differentiation into myocardial precursors and cardiomyocytes (CMs), and determined clusters of human microRNAs that are specifically regulated during this process. We determined that miR-125b overexpression results in upregulation of the early cardiac transcription factors, GATA4 and Nkx2-5, and accelerated progression of hESC-derived myocardial precursors to an embryonic CM phenotype. We used an in silico approach to identify Lin28 as a target of miR-125b, and validated this interaction using miR-125b knockdown. Anti-miR-125b inhibitor experiments also showed that miR-125b controls the expression of miRNA let-7d, likely through the negative regulatory effects of Lin28 on let-7. We then determined that miR-125b overexpression inhibits the expression of Nanog and Oct4 and promotes the onset of Brachyury expression, suggesting that miR-125b controls the early events of human CM differentiation by inhibiting hESC pluripotency and promoting mesodermal differentiation. These studies identified miR-125b as an important regulator of hESC differentiation in general, and the development of hESC-derived mesoderm and cardiac muscle in particular. Manipulation of miR-125b-mediated pathways may provide a novel approach to directing the differentiation of hESC-derived CMs for cell therapy applications.

Suppressor of Fused Is Critical for Maintenance of Neuronal Progenitor Identity during Corticogenesis

Proper lineage progression and diversification of neural progenitor cells (NPCs) ensures the generation of projection neuron (PN) subtypes in the mammalian neocortex. Here, we show that Suppressor of Fused (Sufu) controls PN specification by maintaining the identity of NPCs in the embryonic neocortex. Deletion of Sufu in NPCs of the E10.5 mouse neocortex led to improper specification of progenitors and a reduction in intermediate progenitors (IPs) during corticogenesis. We found that Sufu deletion resulted in unstable Gli2 and Gli3 activity, leading to the ectopic activation of Sonic hedgehog (Shh) signaling. The role of Sufu in maintaining progenitor identity is critical at early stages of corticogenesis, since deletion of Sufu at E13.5 did not cause similar abnormalities. Our studies revealed that Sufu critically modulates Shh signaling at early stages of neurogenesis for proper specification and maintenance of cortical NPCs to ensure the appropriate generation of cortical PN lineages.

Loss of Suppressor of Fused in Mid-Corticogenesis Leads to the Expansion of Intermediate Progenitors

Neural progenitors in the embryonic neocortex must be tightly regulated in order to generate the correct number and projection neuron subtypes necessary for the formation of functional neocortical circuits. In this study, we show that the intracellular protein Suppressor of Fused (Sufu) regulates the proliferation of intermediate progenitor (IP) cells at later stages of corticogenesis to affect the number of Cux1+ upper layer neurons in the postnatal neocortex. This correlates with abnormal levels of the repressor form of Gli3 (Gli3R) and the ectopic expression of Patched 1 (Ptch1), a Sonic Hedgehog (Shh) target gene. These studies reveal that the canonical role of Sufu as an inhibitor of Shh signaling is conserved at later stages of corticogenesis and that Sufu plays a crucial role in regulating neuronal number by controlling the cell cycle dynamics of IP cells in the embryonic neocortex.

The Quintessence of Quiescence

Neurogenic niches in the adult mammalian brain are composed of heterogeneous populations of neural stem cells. In this issue of Neuron, Codega et al. (2014) isolate quiescent neural stem cells from the adult subventricular zone and demonstrate their stem cell characteristics.

Suppressor of Fused controls the proliferation of postnatal neural stem and precursor cells via a Gli3-dependent mechanism

The ventricular-subventricular zone (V-SVZ) of the forebrain is the source of neurogenic stem/precursor cells for adaptive and homeostatic needs throughout the life of most mammals. Here, we report that Suppressor of Fused (SUFU) plays a critical role in the establishment of the V-SVZ at early neonatal stages by controlling the proliferation of distinct subpopulations of stem/precursor cells. Conditional deletion of Sufu in radial glial progenitor cells (RGCs) at E13.5 resulted in a dramatic increase in the proliferation of Sox2+ Type B cells, as well as Gsx2+ ventral forebrain derived transit amplifying precursor cells (TACs). In contrast, we found a significant decrease in Tbr2+ dorsal forebrain derived TACs indicating that innate differences between dorsal and ventral forebrain derived Type B cells influence Sufu function. However, most precursors failed to survive and accumulated in the dorsal V-SVZ, demonstrating that precursors are unable to transition into functional differentiated progenies. These defects were accompanied by reduced Gli3 expression, yet despite reduced Gli3 levels, activation of Sonic hedgehog (Shh) signaling did not occur implying that the Sufu-Gli3 regulatory axis may influence other signaling pathways in the neonatal dorsal V-SVZ. These data suggest that Sufu plays a critical role in controlling Gli3 function in the establishment and survival of functional stem/precursor cells in the postnatal dorsal V-SVZ. SUMMARY STATEMENT Conditional deletion of Sufu cause dramatic expansion of neural stem/precursor cells in the neonatal ventricular-subventricular (SVZ) zone. This defect occurs through a Gli3-dependent mechanism that results in the downregulation of Shh signaling.

The Dorsal Wave of Neocortical Oligodendrogenesis Begins Embryonically and Requires Multiple Sources of Sonic Hedgehog

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 Smo specifically in Emx1+ progenitors led to significantly decreased oligodendrocyte numbers in the embryonic neocortex. Conversely, knock-out of the Shh antagonist Sufu 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 Shh from Dlx5/6+ interneurons caused a significant reduction in oligodendrocytes in the embryonic neocortex. This phenotype was identical to that observed upon Shh deletion from the entire CNS using Nestin-Cre, indicating that interneurons migrating into the neocortex from the subpallium are the primary neural source of Shh for dorsal oligodendrogenesis. Additionally, deletion of Shh 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. SIGNIFICANCE STATEMENT 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.