FGF signaling enhances a sonic hedgehog negative feedback loop at the initiation of spinal cord ventral patterning

A prevalent developmental mechanism for the assignment of cell identities is the production of spatiotemporal concentration gradients of extracellular signaling molecules that are interpreted by the responding cells. One of such signaling systems is the Shh gradient that controls neuronal subtype identity in the ventral spinal cord. Using loss and gain of function approaches in chick and mouse embryos, we show here that the fibroblast growth factor (FGF) signaling pathway is required to restrict the domains of ventral gene expression as neuroepithelial cells become exposed to Shh during caudal extension of the embryo. FGF signaling activates the expression of the Shh receptor and negative pathway regulator Patched 2 (Ptch2) and therefore can enhance a negative feedback loop that restrains the activity of the pathway. Thus, we identify one of the mechanisms by which FGF signaling acts as a modulator of the onset of Shh signaling activity in the context of coordination of ventral patterning and caudal axis extension. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 956–971, 2016     

Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord

The three vertebrate Gli proteins play a central role in mediating Hedgehog (Hh)-dependent cell fate specification in the developing spinal cord; however, their individual contributions to this process have not been fully characterized. In this paper, we have addressed this issue by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. We show that Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. We also show that motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, we provide evidence that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation.     

Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh(-/-), Gli2(-/-) and Gli3(-/-) embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2(-/-) embryos displays ventral patterning defects as severe as those observed in Shh(-/-) embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain.     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.     

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.     

SOX1 links the function of neural patterning and Notch signalling in the ventral spinal cord during the neuron-glial fate switch

During neural development the transition from neurogenesis to gliogenesis, known as the neuron-glial (Ν/G) fate switch, requires the coordinated function of patterning factors, pro-glial factors and Notch signalling. How this process is coordinated in the embryonic spinal cord is poorly understood. Here, we demonstrate that during the N/G fate switch in the ventral spinal cord (vSC) SOX1 links the function of neural patterning and Notch signalling. We show that, SOX1 expression in the vSC is regulated by PAX6, NKX2.2 and Notch signalling in a domain-specific manner. We further show that SOX1 regulates the expression of Hes1 and that loss of Sox1 leads to enhanced production of oligodendrocyte precursors from the pMN. Finally, we show that Notch signalling functions upstream of SOX1 during this fate switch and is independently required for the acquisition of the glial fate perse by regulating Nuclear Factor I A expression in a PAX6/SOX1/HES1/HES5-independent manner. These data integrate functional roles of neural patterning factors, Notch signalling and SOX1 during gliogenesis.     

Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. We show that the regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, we examined the telencephalon in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persist in Shh(-/-) mutants, and that these same markers were expanded in Gli3(-/-) mutants. To understand further the genetic interaction between Shh and Gli3, we examined Shh/Gli3 and Smoothened/Gli3 double homozygous mutants. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon.     

Interconvertibility of induced morphogenetic responses of mouse embryonic somites to notochord and ventral spinal cord

Nine-day mouse somitic mesenchyme normally responds trans-filter to 9-day ventral spinal cord by forming a perichondrium-limited “whole” nodule at a distance from the tissue-filter interface. Chick notochord usually induces a “partial” nodule in which cells in association with the filter are oriented to it. Under proper experimental conditions interconversion of nodule responses is possible—ventral spinal cord yielding “partial” nodules; notochord, “whole” nodules. Conversion of the notochord response occurs under conditions attenuating its inductive effect, while ventral spinal cord induces “partial” nodules under conditions presumably enhancing the availability of inductive substance(s). The notochord does not require a surface for induction of chondrogenesis in vitro, although in vivo the presence of a surface may play a significant morphogenetic role. Notochord appears to be a more “active” inducer than ventral spinal cord.     

miR-196 is an essential early-stage regulator of tail regeneration, upstream of key spinal cord patterning events

Salamanders have the remarkable ability to regenerate many body parts following catastrophic injuries, including a fully functional spinal cord following a tail amputation. The molecular basis for how this process is so exquisitely well-regulated, assuring a faithful replication of missing structures every time, remains poorly understood. Therefore a study of microRNA expression and function during regeneration in the axolotl, Ambystoma mexicanum, was undertaken. Using microarray-based profiling, it was found that 78 highly conserved microRNAs display significant changes in expression levels during the early stages of tail regeneration, as compared to mature tissue. The role of miR-196, which was highly upregulated in the early tail blastema and spinal cord, was then further analyzed. Inhibition of miR-196 expression in this context resulted in a defect in regeneration, yielding abnormally shortened tails with spinal cord defects in formation of the terminal vesicle. A more detailed characterization of this phenotype revealed downstream components of the miR-196 pathway to include key effectors/regulators of tissue patterning within the spinal cord, including BMP4 and Pax7. As such, our dataset establishes miR-196 as an essential regulator of tail regeneration, acting upstream of key BMP4 and Pax7-based patterning events within the spinal cord.