Michael R. Stark

Human Neural Crest Stem Cells Derived from Human ESCs and Induced Pluripotent Stem Cells: Induction, Maintenance, and Differentiation into Functional Schwann Cells

The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence‐activated cell sorting (FACS). FACS‐isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro‐expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC‐derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC‐ or iPSC‐derived Schwann cells.

Intrinsic signals regulate the initial steps of myogenesis in vertebrates

In vertebrates, despite the evidence that extrinsic factors induce myogenesis in naive mesoderm, other experiments argue that the initiation of the myogenic program may take place independent of these factors. To resolve this discrepancy, we have re-addressed this issue, using short-term in vivo microsurgery and culture experiments in chick. Our results show that the initial expression of the muscle-specific markers Myf5 and MyoD is regulated in a mesoderm-autonomous fashion. The reception of a Wnt signal is required for MyoD, but not Myf5 expression; however, we show that the source of the Wnt signal is intrinsic to the mesoderm. Gain- and loss-of-function experiments indicate that Wnt5b, which is expressed in the presomitic mesoderm, represents the MyoD-activating cue. Despite Wnt5b expression in the presomitic mesoderm, MyoD is not expressed in this tissue: our experiments demonstrate that this is due to a Bmp inhibitory signal that prevents the premature expression of MyoD before somites form. Our results indicate that myogenesis is a multistep process which is initiated prior to somite formation in a mesoderm-autonomous fashion; as somites form, influences from adjacent tissues are likely to be required for maintenance and patterning of early muscles.

Activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the ophthalmic trigeminal placode.

Vertebrate cranial neurogenic placodes are relatively simple model systems for investigating the control of sensory neurogenesis. The ophthalmic trigeminal (opV) placode, for which the earliest specific marker is the paired domain homeodomain transcription factor Pax3, forms cutaneous sensory neurons in the ophthalmic lobe of the trigeminal ganglion. We previously showed that Pax3 expression in avian opV placode cells correlates with specification and commitment to a Pax3+, cutaneous sensory neuron fate. Pax3 can act as a transcriptional activator or repressor, depending on the cellular context. We show using mouse Splotch(2H) mutants that Pax3 is necessary for the normal neuronal differentiation of opV placode cells. Using an electroporation construct encoding a Pax3-Engrailed fusion protein, which represses Pax3 target genes, we show that activation of Pax3 target genes is required cell-autonomously within chick opV placode cells for expression of the opV placode markers FGFR4 and Ngn2, maintenance of the preplacodal marker Eya2, expression of Pax3 itself (suggesting that Pax3 autoregulates), neuronal differentiation and delamination. Mis-expression of Pax3 in head ectoderm is sufficient to induce FGFR4 and Ngn2 expression, but neurons do not differentiate, suggesting that additional signals are necessary to enable Pax3+ cells to differentiate as neurons. Mis-expression of Pax3 in the Pax2+ otic and epibranchial placodes also downregulates Pax2 and disrupts otic vesicle closure, suggesting that Pax3 is sufficient to alter the identity of these cells. Overall, our results suggest that activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the opV placode.

Signaling mechanisms controlling cranial placode neurogenesis and delamination.

The neurogenic cranial placodes are a unique transient epithelial niche of neural progenitor cells that give rise to multiple derivatives of the peripheral nervous system, particularly, the sensory neurons. Placode neurogenesis occurs throughout an extended period of time with epithelial cells continually recruited as neural progenitor cells. Sensory neuron development in the trigeminal, epibranchial, otic, and olfactory placodes coincides with detachment of these neuroblasts from the encompassing epithelial sheet, leading to delamination and ingression into the mesenchyme where they continue to differentiate as neurons. Multiple signaling pathways are known to direct placodal development. This review defines the signaling pathways working at the finite spatiotemporal period when neuronal selection within the placodes occurs, and neuroblasts concomitantly delaminate from the epithelium. Examining neurogenesis and delamination after initial placodal patterning and specification has revealed a common trend throughout the neurogenic placodes, which suggests that both activated FGF and attenuated Notch signaling activities are required for neurogenesis and changes in epithelial cell adhesion leading to delamination. We also address the varying roles of other pathways such as the Wnt and BMP signaling families during sensory neurogenesis and neuroblast delamination in the differing placodes.

Sensory neuron differentiation is regulated by notch signaling in the trigeminal placode

Trigeminal sensory neurons develop from the neural crest and neurogenic placodes, and have been studied as a principal model of sensory neuron formation. While the Notch pathway has been extensively characterized in central nervous system development and other developmental processes, it has not been well characterized in sensory neurogenesis. Here we studied the functional role of Notch signaling in the trigeminal ophthalmic (opV) placode, a prime model of sensory neurogenesis. To establish a good spatiotemporal description of Notch pathway genes in the chick trigeminal placode, a stage-specific expression analysis was conducted, showing that expression of most Notch pathway genes and effectors are expressed in the placode, with expression primarily being confined to ectodermal cells. Expression was highest at stages of peak neuronal differentiation. To test the function of Notch signaling in opV placode cell differentiation, Notch receptor cleavage was blocked using the gamma-secretase inhibitor, DAPT, or signaling was activated by misexpression of the Notch intracellular domain (NICD). Notch activation resulted in a significant reduction in sensory neurogenesis. Cells remained in the ectoderm and did not differentiate. Expression of the opV specification marker Pax3 was also lost in targeted cells. DAPT exposure resulted in a dramatic increase in neurogenesis without increasing proliferation, where many differentiated cells were found in the mesenchyme and, surprisingly, within the ectoderm. This is the first result clearly showing prolific neuronal differentiation in the ectoderm of the trigeminal placodes after experimental manipulation of a molecular signaling pathway, thus identifying Notch signaling as a primary regulator of the sensory neuron fate in the opV placode.

Expression of Frizzled genes in the developing chick eye

Frizzleds are transmembrane receptors that can transduce signals dependent upon binding of Wnts, a large family of secreted glycoproteins homologous to the Drosophila wingless (wg) gene product and critical for a wide variety of normal and pathological developmental processes. In the nervous system, Wnts and Frizzleds play an important role in anterior-posterior patterning, cell fate decisions, proliferation, and synaptogenesis. However, little is known about the role of Frizzled signaling in the developing eye. We isolated cDNAs for ten chick Frizzleds and analyzed the spatial and temporal expression patterns during eye development in the chick embryo. Frizzled-1 to -9 are specifically expressed in the eye at various stages of development and show a complex and partially overlapping pattern of expression.

Expression of green fluorescent protein in the chicken using in vivo transfection of the piggyBac transposon

The chicken is a well-established model system for studying developmental biology and is recognized as one of the top food production animals in the world. For this reason the chicken is an excellent candidate for transgenic applications, as the technology can be applied to both areas of research. Transgenic technology has not been broadly utilized in the chicken model, however, primarily due to difficulties in targeting germ cells and establishing germ line transmission. Transgenic technologies using non-replicating viral particles have been used in the chick, but are unsuitable for many applications because of size and sequence restraints and low efficiency. To create a more versatile method to target chick germ line stem cells, we utilized the transposable element system piggyBac paired with an in vivo transfection reagent, JetPEI. piggyBac has been previously shown to be highly active in mammalian cells and will transpose into the chicken genome. Here, we show that JetPEI can transfect multiple chick cell types, most notably germline stem cells. We also show that pairing these two reagents is a viable and reproducible method for long-term expression of a transgene in the chicken. Stable expression of the green fluorescent protein (GFP) transgene was seen in multiple tissue types including heart, brain, liver, intestine, kidney and gonad. Combining an in vivo transfection strategy with the PB system provides a simple and flexible method for efficiently producing stable chimeric birds and could be used for production of germ line transgenics.

Microdistribution of tetrodotoxin in two species of blue-ringed octopuses (Hapalochlaena lunulata and Hapalochlaena fasciata) detected by fluorescent immunolabeling

Blue-ringed octopuses (genus Hapalochlaena) possess the potent neurotoxin tetrodotoxin (TTX). We examined the microdistribution of TTX in ten tissues of Hapalochlaena lunulata and Hapalochlaena fasciata by immunolabeling for fluorescent light microscopy (FLM). We visualized TTX throughout the posterior salivary gland, but the toxin was concentrated in cells lining the secretory tubules within the gland. Tetrodotoxin was present just beneath the epidermis of the integument (mantle and arms) and also concentrated in channels running through the dermis. This was suggestive of a TTX transport mechanism in the blood of the octopus, which would also explain the presence of the toxin in the blood-rich brachial hearts, gills, nephridia, and highly vascularized Needhams sac (testes contents). We also present the first report of TTX in any cephalopod outside of the genus Hapalochlaena. A specimen of Octopus bocki from French Polynesia contained a small amount of TTX in the digestive gland.

FGF and Notch signaling in sensory neuron formation: A multifactorial approach to understanding signaling pathway hierarchy

The ophthalmic trigeminal (opV) placode exclusively gives rise to sensory neurons, making it a good model to study the molecular regulation of sensory neurogenesis. A number of signaling pathways including Wnt, PDGF, FGF, and Notch have been shown to be involved in the process of opV placode cell development. However, the regulatory relationships between these signaling pathways in placode cells are still unknown and have been difficult to study experimentally. Using a novel multifactorial approach in chick embryos that allows for inhibition of FGF throughout the tissue or in individual cells, with simultaneous inactivation of Notch signaling, we investigated the potential interaction between the FGF and Notch signaling pathways in trigeminal sensory neurogenesis. This study builds on prior research describing the individual role of FGF signaling or Notch signaling in opV placode development, where blocking FGF signaling resulted in neurogenesis failure, while blocking Notch signaling resulted in enhanced neurogenesis. Reported here, blocking both pathways simultaneously resulted in a reduction in the number of cells delaminating from the opV placode and undergoing sensory neuron differentiation. Further, Notch inhibition alone did not lead to an increase in the number of cells expressing FGFR4 or in the FGFR4 expression domain, but did result in a highly fragmented basal lamina, which was reversed when blocking FGF signaling. Cumulatively, the results presented here do not support a model of Notch/FGF interdependence, rather that FGF and Notch act in parallel to promote sensory neurogenesis.

Vertebrate neurogenic placode development: historical highlights that have shaped our current understanding.

With the flood of published research encountered today, it is important to occasionally reflect upon how we arrived at our current understanding in a particular scientific discipline, thereby positioning new discoveries into proper context with long‐established models. This historical review highlights some of the important scientific contributions in the field of neurogenic placode development. By viewing cumulatively the rich historical data, we can more fully appreciate and apply what has been accomplished. Early descriptive work in fish and experimental approaches in amphibians and chick yielded important conceptual models of placode induction and cellular differentiation. Current efforts to discover genes and their molecular functions continue to expand our understanding of the placodes. Carefully considering the body of work may improve current models and help focus modern experimental design. Developmental Dynamics 243:1167–1175, 2014.