SUMMARY Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-sosked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.
Birth defects of the heart and face are common, and most have no known genetic cause, suggesting a role for environmental factors. Maternal fever during the first trimester is an environmental risk factor linked to these defects. Neural crest cells are precursor populations essential to the development of both at-risk tissues. We report that two heat-activated transient receptor potential (TRP) ion channels, TRPV1 and TRPV4, were present in neural crest cells during critical windows of heart and face development. TRPV1 antagonists protected against the development of hyperthermia-induced defects in chick embryos. Treatment with chemical agonists of TRPV1 or TRPV4 replicated hyperthermia-induced birth defects in chick and zebrafish embryos. To test whether transient TRPV channel permeability in neural crest cells was sufficient to induce these defects, we engineered iron-binding modifications to TRPV1 and TRPV4 that enabled remote and noninvasive activation of these channels in specific cellular locations and at specific developmental times in chick embryos with radio-frequency electromagnetic fields. Transient stimulation of radio frequency-controlled TRP channels in neural crest cells replicated fever-associated defects in developing chick embryos. Our data provide a previously undescribed mechanism for congenital defects, whereby hyperthermia activates ion channels that negatively affect fetal development.
Sonic hedgehog signaling in the secondary heart field has a clear role in cardiac arterial pole development. In the absence of hedgehog signaling, proliferation is reduced in secondary heart field progenitors, and embryos predominantly develop pulmonary atresia. While it is expected that proliferation in the secondary heart field would be increased with elevated hedgehog signaling, this idea has never been tested. We hypothesized that up-regulating hedgehog signaling would increase secondary heart field proliferation, which would lead to arterial pole defects. In culture, secondary heart field explants proliferated up to 6-fold more in response to the hedgehog signaling agonist SAG, while myocardial differentiation and migration were unaffected. Treatment of chick embryos with SAG at HH14, just before the peak in secondary heart field proliferation, resulted unexpectedly in stenosis of both the aortic and pulmonary outlets. We examined proliferation in the secondary heart field and found that SAG-treated embryos exhibited a much milder increase in proliferation than was indicated by the in vitro experiments. To determine the source of other signaling factors that could modulate increased hedgehog signaling, we co-cultured secondary heart field explants with isolated pharyngeal endoderm or outflow tract and found that outflow tract co-cultures prevented SAG-induced proliferation. BMP2 is made and secreted by the outflow tract myocardium. To determine whether BMP signaling could prevent SAG-induced proliferation, we treated explants with SAG and BMP2 and found that BMP2 inhibited SAG-induced proliferation. In vivo, SAG-treated embryos showed up-regulated BMP2 expression and signaling. Together, these results indicate that BMP signaling from the outflow tract modulates hedgehog-induced proliferation in the secondary heart field.
Magnetogenetics promises remote control of neurons, but its validity is questioned due to controversies surrounding the underlying mechanisms and deficits in reproducibility. Recent studies discovered that ferritin, used in Magnetogenetics, transduces radiofrequency (RF) magnetic fields into biochemical signals (reactive oxygen species and oxidized lipids). Magnetic stimulation of ferritin-tethered TRPV channels induces Ca2+ responses and modulates behavior but electrophysiological studies indicate that a particular channel, Magneto2.0, is ineffective in affecting the neuronal bioelectrical properties. We investigated this problem using the Magnetogenetic technique FeRIC. We resolved the electromagnetic interference caused by RF in patch-clamp recordings and supported the data with voltage imaging experiments. In neurons expressing TRPV4FeRIC, RF depolarizes the membrane potential and increases the spiking frequency. In neurons expressing the chloride-permeable TMEM16AFeRIC, RF hyperpolarizes the membrane potential and decreases the spiking frequency. Our study reveals the control of neuronal bioelectrical properties with Magnetogenetics that is non-instantaneous, long-lasting, and moderate, but effective and comparable to that induced by endogenous signaling molecules.
BACKGROUND/PURPOSE: Therapeutic hypothermia (TH) initiated within the fi rst 6 hours after birth, is proven to decrease brain tissue injury in moderate to severe neonatal encephalopathy (NE) and improve 18-24 month neurological outcomes. Recently, a correlation between the resistive index (RI) of the anterior cerebral artery on head ultrasound (HUS) performed after rewarming and severity of injury on MRI was reported. It remains unknown whether early RI anomalies (before rewarming) may be associated with severity of injury on MRI and as such, might have prognostic value for infants too unstable to undergo an MRI within the optimal time window. The aim of this study was to evaluate: (i) the association between a low anterior cerebral artery RI on HUS obtained during active TH and the severity of brain injury on MRI, and to examine the association between the RI value and outcomes at 48 months of age.
Fetal and/or neonatal brain development, both normal and abnormal RESULTS: Multiple oxysterols were identifi ed in human maternal breast milk that induced oligodendrocyte production from NSCs in vitro. We found that Gli2 is functionally required for oxysterol-induced oligodendrogenesis. Following neonatal WMI in vivo, 20HC treatment increased numbers of mature OLs, improved myelination and rescued motor defi cits in mice. Lineage tracing experiments showed that 20HC-mediated recovery of OL defi cit is mediated in part through 20HCinduced SVZ-derived oligodendrogenesis in vivo. Additional recovery may be due to the impact of 20HC on oligodendrocyte progenitor cell maturation.
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