Michael R. Stark

Background: Circulating plasma ceramides, a class of bioactive sphingolipids, are elevated in metabolic disorders, including obesity. Infants of women with these disorders are at 2‐ to 3‐fold greater risk for developing a neural tube defect (NTD). This study aimed to test the effects of embryonic exposure to C2‐ceramides (C2) during neural tube closure. Preliminary data shows an increase in NTDs in chick embryos after C2 exposure, and addresses potential mechanisms.Results: Cell and embryo models were used to examine redox shifts after ceramide exposure. While undifferentiated P19 cells were resistant to ceramide exposure, neuronally differentiated P19 cells exhibited an oxidizing shift. Consistent with these observations, GSH E _h curves revealed a shift to a more oxidized state in C2 treated embryos without increasing apoptosis or changing Pax3 expression, however cell proliferation was lower. Neural tube defects were observed in 45% of chick embryos exposed to C2, compared to 12% in control embryos.Conclusions: C2 exposure during critical developmental stages increased the frequency of NTDs in the avian model. Increased ROS generation in cell culture, along with the more oxidative GSH E _h profiles of C2 exposed cells and embryos, support a model wherein ceramide affects neural tube closure via altered tissue redox environments.

The chicken embryo is a versatile and effective model for studying the effects of teratogenic compounds during early development. Easy access to the embryo allows for exposure and analysis of toxicant effects during embryogenesis. This chapter will provide detailed protocols for embryonic collection and toxicant exposure techniques, including EC culture and Cornish Pasty methods, LysoTracker staining, glutathione redox potential analysis, and 2′,7′-dichlorodihydrofluorescein diacetate.

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.

Background: In the trigeminal placode, Pax3 is classified as necessary but not sufficient for sensory neuron differentiation. One hypothesis is that different Pax3 isoforms regulate cellular differentiation uniquely. Pax3 is known to sometimes activate and sometimes repress gene transcription, and its activity can be dependent on the isoforms present. Pax3 isoforms had not previously been characterized in chick sensory neurogenesis. Results: Reverse transcriptase‐polymerase chain reaction (PCR) analysis revealed three well‐expressed Pax3 splice variants: full‐length (flPax3), Pax3V1, and Pax3V2. Each was characterized for its effect on neurogenesis by misexpression in placodal ectoderm. The differences observed were more apparent under conditions of enhanced neurogenesis (by means of Notch inhibition), where flPax3 and Pax3V1 caused failed differentiation, while Pax3V2 misexpression resembled the neuronal differentiation seen in controls. Quantitative PCR analysis revealed a progressive increase in Pax3 expression, but no significant change in relative isoform expression. Of interest, Notch inhibition led to a significant increase in Pax3 expression. Conclusions: We can conclude that: (1) flPax3 and Pax3V1 inhibit neuronal differentiation; (2) Pax3V2 is permissive for neuronal differentiation; (3) while absolute levels change over time, relative splice form expression levels are largely maintained in the trigeminal placode domain; and (4) Pax3 expression generally increases in response to Notch inhibition. Developmental Dynamics 243:1249–1261, 2014.

The ophthalmic trigeminal (opV) placode gives rise exclusively to sensory neurons of the peripheral nervous system, providing an advantageous model for understanding neurogenesis. The signaling pathways governing opV placode development have only recently begun to be elucidated. Here, we investigate the fibroblast growth factor receptor‐4 (FGFR4), an opV expressed gene, to examine if and how FGF signaling regulates opV placode development. After inhibiting FGFR4, Pax3+ opV placode cells failed to delaminate from the ectoderm and did not contribute to the opV ganglion. Blocking FGF signaling also led to a loss of the early and late neuronal differentiation markers Ngn2, Islet‐1, NeuN, and Neurofilament. In addition, without FGF signaling, cells that stalled in the ectoderm lost their opV placode‐specific identity by down‐regulating Pax3. We conclude that FGF signaling, through FGFR4, is necessary for delamination and differentiation of opV placode cells. Developmental Dynamics 238:1073–1082, 2009.