The role of the Notch signaling pathway in neurotransmitter phenotype specification in Xenopus laevisMolly J. McDonough and Margaret S. Saha

Acknowledgments • Funding from Howard Hughes Medical Institute Undergraduate Science

Education Program Grant to the College of W&M• NIH Grant 1 R15 NS067566-01 to Margaret S. Saha

5. Future directions

References1. Kabos, P., Kabosova, A., & Neuman, T. Journal of Biological Chemistry, 277(11), 8763-8766.2. Reyes JH et al. (2008). J Neurosci 28:12622–12631.3. Chitnis, A., D. Henrique, et al. (1995). Nature 375(6534): 761-6. 4. Wettstein, D. A., Turner, D. L., & Kintner, C. Development, 124(3), 693-702.

Department of Biology, College of William and Mary, Williamsburg, VA

• Histological analysis of assayed embryos to study the effects of early Notch perturbations on neuronal patterning in later (swimming tadpole) stages

• Assays for markers of apoptosis and markers of neuronal differentiation to determine the molecular mechanisms acting to homeostatically regulate the neuronal population in embryos in which initial perturbations of Notch resulted in an excess or deficiency of differentiated neurons

• Exposures of cell cultures or explants to DAPT, a pharmacological blocker of Notch signaling, at older stages to transiently alter Notch signaling and study the effects of these alterations more specifically on the GABAergic versus glutamatergic fate specification.

4. Conclusions

During primary neurogenesis, neurotransmitter phenotype specification occurs in a nonrandom dispersed pattern, and, due to its role in lateral inhibition, the Notch signaling pathway may serve to regulate this patterning. Previous experiments have shown that blocking downstream effecter genes of Notch can induce a GABAergic (inhibitory) phenotype (1) while by-passing Notch completely by exogenous expression of neural determination genes can induce a glutamatergic (excitatory) phenotype (2). This study aims to explore the effects of perturbing components of the Notch signaling pathway on early neurotransmitter phenotype specification and patterning in vivo.

1. Introduction

Notch signaling in primary neurogenesis

Figure 1. Expression of a proneural gene (e.g. X-NGNR-1) activates the expression of the transmembrane ligand Delta. Delta binds the Notch receptor on adjacent cells causing a conformational change in the Notch receptor. This conformational change induces the cleavage of the Notch intracellular domain (ICD) by presenilin-catalyzed gamma-secretase. ICD then binds to Supressor of Hairless (xSu(H)), and this complex drives the transcription of Hairy/Enhancer of Split transcription factors. These HES transcription factors then repress the expression of proneural genes in the target cell.

xSu(H) DBM low-dose (1.5ng) injection

xSu(H) DBM high-dose (1.5ng) injection

Figure 2. Embryos were unilaterally injected at the two-cell stage with 4.6nl water with 1.5ng or 9.2nl water with 3.0ng X-Notch ICD (3) or xSu(H) DBM (4) sense mRNA. Embryos were co-injected with 0.5 ng GFP sense mRNA to allow for visualization of the injected side at later stages. Vehicle-injected control embryos were injected with 4.6 nl water with 1.0 ng GFP mRNA or 9.2nl water with 2.0ng mRNA. Following normal development, the injected side of the embryo was identified by GFP fluorescence. Embryos were fixed at early neurula through swimming tadpole stages and assayed for the markers listed in Table 1 using whole mount in situ hybridization.

2. Methods

Table 1. Summary of antisense in situ hybridization probes used indicating gene name and type of cell marked by each probe.

Schematic of methods

Stage Injection Gross Defects

Neural Tube Defects

18 control 0/10 0/1022 control 0/24 0/2425 control 0/25 1/2528 control 0/44 0/4432 control 0/38 1/3835 control 0/46 0/4622 GFP 1.0ng 0/39 2/3925 GFP 1.0ng 4/31 2/3132 GFP 1.0ng 3/28 3/2835 GFP 1.0ng 3/33 2/3328 GFP 2.0ng 0/15 2/1522 ICD 1.5ng 3/32 17/3225 ICD 1.5ng 6/36 13/3632 ICD 1.5ng 11/36 8/3618 ICD 3.0ng 2/12 4/1228 ICD 3.0ng 7/28 7/2835 ICD 3.0ng 12/24 4/2418 DBM 1.5ng 0/7 2/722 DBM 1.5ng 0/30 0/3028 DBM 1.5ng 2/13 0/1332 DBM 1.5ng 5/29 12/2935 DBM 1.5ng 3/30 4/3025 DBM 3.0ng 1/28 1/2828 DBM 3.0ng 3/33 5/33

xNICD low-dose (1.5ng) injection

xNICD high-dose (3.0ng) injection

3. ResultsA. Expression of xNBT, xGAD67, and xVGlut1 following perturbations of Notch signaling

Expression of xNBT, xGAD67, and xVGlut1 following over expression of Notch signaling by injection of X-Notch ICD (xNICD)

Whole mount analysis of expression of xNBT, xGAD67, and xVGlut1 using in situ hybridization

Expression of xNBT, xGAD67, and xVGlut1 following inhibition of Notch signaling by injection of a DNA binding mutant of Suppressor of Hairless (xSu(H) DBM)

Figure 3. Embryos were unilaterally injected at the two-cell stage with either 4.6nl water containing 1.5ng xNICD mRNA (A, B, C) or 9.2nl water containing 3.0ng xNICD mRNA (D,E,F). xNICD serves to over express Notch signaling by introducing exogenous NICD (see Figure 1). Embryos were fixed at late neurula through swimming tadpole stages and assayed for xNBT, xGAD67, or xVGlut1 using whole mount in situ hybridization. The graphs in this figure indicate the percentage of embryos in which a decrease in expression for the specified marker was observed on the injected side in comparison to the un-injected side which served as an internal control.

A B C

D E F

A B C

D E F

Figure 5. Embryos were unilaterally injected at the two-cell stage with either 4.6nl water containing 1.5ng xSu(H) DBM mRNA (A, B, C) or 9.2nl water containing 3.0ng xSu(H) DBM mRNA (D, E, F). xSu(H) DBM serves to inhibit the Notch pathway by introducing a DNA binding mutant form of xSu(H) that is able to interact with xNICD but is unable to bind DNA to activate HES transcription. Embryos were fixed at late neurula through swimming tadpole stages and assayed for xNBT, xGAD67, or xVGlut1 using whole mount in situ hybridization. The graphs in this figure indicate the percentage of embryos in which a increase in expression for the specified marker was observed on the injected side in comparison to the un-injected side which served as an internal control.

B. Morphological defects following perturbations of Notch signalingMorphological defects by stage and injection

Table 2. Embryos were analyzed for morphological defects following fixation and in situ hybridization. Abnormal embryos were divided into one of two mutually exclusive categories. “Gross Defects” denotes embryos which failed to develop or that had severely deformed body plans. “Neural Tube Defects” includes embryos with misshapen neural tubes as well as embryos in which the neural tube failed to close properly.

Figure 6. Graphical representation of the data presented in Table 2. Each chart represents the percentage of Gross Defects and Neural Tube Defects (NTDs) observed for each stage studied with each injection dose.

• Early perturbation of components of Notch signaling disrupt patterning of neurotransmitter phenotypes but do not demonstrate an instructive role for Notch signaling in the GABAergic versus glutamatergic fate specification.

• Following initial interruptions of Notch perturbations on neuronal differentiation, homeostatic regulatory mechanisms may be acting to restore a normal neuronal population.

Gene Marker TypexNBT Pan-neuralxVGlut1 Glutamatergic neuronsxGAD GABAergic neurons

Figure 4. Embryos were staged according to Nieuwkoop and Faber (1994) prior to in situ hybridization and were cleared in 2:1 BB:BA for photography. xNICD and xSu(H) DBM embryos were injected on the left lateral side as determined by GFP fluorescence prior to fixation. * indicates embryos that were injected with a high dose of mRNA (3.0ng xNICD or xSu(H) DBM). Scalebar represents 1.5mm.

Sense, capped mRNAs

Unilateral MicroinjectionInjected SideInternal

control

Normal Development

Labeled, antisense mRNA probes

Whole mountin situ hybridization

Fixation