Tag: Judith Eisen

Exploring the effect of bacterial signaling pathways on zebrafish neuro-immune development

Presenter: Dana Zaidan – Neuroscience

Faculty Mentor(s): Joseph Bruckner, Judith Eisen

Session: (In-Person) Poster Presentation

The gut microbiota has been linked to human health and development. We found that the gut microbiota is required for normal zebrafish social behavior, but how it influences the brain development required for social behavior is not well understood. We previously identified a population of zebrafish forebrain neurons that are also required for normal social behavior. By raising zebrafish “germ-free”, we found that the microbiota is required for normal forebrain neuronal arborization.

Microglia are brain-resident immune cells that remodel neurons and are excellent candidates for mediating interactions between the microbiota and the brain. We previously discovered that the microbiota promotes forebrain microglial abundance. We also found that neuronal arborization and microglial abundance are restored in germ-free fish after colonization with several different zebrafish-associated bacterial strains, suggesting that the microbiota might influence social neurodevelopment by a mechanism common to many bacteria. One pathway we explored involves a class of host proteins that receive bacterial signals called the Toll-like receptor (TLR) proteins. We also explored if and how proteins present in bacterial cell walls are sensed by host mechanisms in the brain. Identifying the signaling components that link the microbiota and brain development will clarify our understanding of how host-microbe interactions can influence human health.

Do Distinct Types of Progenitors Contribute to the Diversity of Enteric Neurons and Glia?

Presenter: Charlotte Taylor

Mentor: Judith Eisen

Oral Presentation

Major: Biology 

The enteric nervous system (ENS), the largest component of the peripheral nervous system, provides intrinsic innervation of the intestinal tract and modulates gut function. The ENS forms a complex network composed of different neuronal and glial subtypes. Whether these different subtypes arise from distinct progenitors is currently unknown. Developing zebrafish embryos are transparent and genetic manipulations can be used to label progenitor cells and their progeny, thus zebrafish is an excellent model in which to address this question. ENS progenitors express several marker genes, including phox2b, sox10, and ret. Using the zebrafish model, we investigated whether expression of these genes designates distinct ENS progenitor populations. Our co-expression analysis identified three different progenitor subpopulations that express the following marker combinations: phox2b/sox10/ret, phox2b/ret, and phox2b. Our next goal is to test the hypothesis that these subpopulations give rise to distinct neuronal and glial cell types during ENS development. We will use the Cre/loxP lineage tracing system to track progeny of identified progenitor subpopulations. Currently, we are generating BAC constructs that drive expression of Cre recombinase under the control of enteric progenitor specific promoters. We will inject these BAC constructs into a red fluorescent reporter line to permanently label all Cre expressing cells and their progeny and then follow the fate of of these cells in living embryos during ENS development. These results will provide a comprehensive lineage analysis of ENS precursors in vivo and thus offer new insights into ENS development and the developmental potential of individual ENS progenitors.

Do Progenitor Subpopulations Contribute to Zebrafish Enteric Nervous System Development?

Presenter: Charlotte Taylor

Mentors: Judith Eisen and Julia Ganz, Biology

Poster: 62

Major: Biochemistry 

The enteric nervous system (ENS) provides intrinsic intestinal innervation and modulates intestinal function. The ENS forms a complex network of neuronal and glial subtypes. ENS progenitors that give rise to this network express different marker genes, e.g. phox2b, sox10, and ret. Using the zebrafish model, we investigated whether expression of these markers defines distinct ENS progenitor subpopulations. Gene expression revealed subpopulations, the most prominent of which are characterized by the following combinations: phox2b; phox2b/ret; phox2b/sox10; phox2b/ret/sox10. We will now determine whether these distinct progenitors have functional significance for ENS development. We will use the Cre/loxP lineage tracing system to track progeny of distinct progenitor subpopulations and determine if they give rise to different ENS cell types. Using BAC-recombineering, we will generate BAC- constructs that drive Cre recombinase expression under enteric progenitor specific promoters (e.g. ret). To test if BAC enteric progenitor promoter sequences drive expression faithfully, we are generating BAC-constructs that drive expression of green fluorescent proteins (GFP). We are currently analyzing ENS GFP expression of a modified ret BAC. After generating Cre-constructs, we will inject them into a reporter line and identify progeny of labeled progenitors at different times during ENS development. Our results will provide a comprehensive lineage analysis of ENS precursors in vivo and thus offer new insights into ENS development and the potential of individual ENS precursors.

Role of Endothelin Pathway in Enteric Nervous System Development and Hirschsprung Disease

Presenter: Parham Diba

Mentors: Julia Ganz and Judith Eisen, Biology

Poster: 18

Major: Human Physiology 

The enteric nervous system (ENS) is the largest part of the peripheral nervous system, containing about 400–600 million neurons in humans. It comprises a complex network of neurons and glia and controls intestinal functions, such as motility. Hirschsprung disease (HSCR) is a multifactorial congenital disease in which distal intestine is uninnervated and immotile. A variety of signaling pathways, including the endothelin signaling pathway, regulate ENS development during embryonic stages. In mouse, Endothelin3 and endothelin receptor type B regulate ENS development and mutations in these genes are found in some HSCR patients. However, there are still open questions about how the endothelin pathway is involved in ENS development, such as how it affects progenitor migration and neuronal subtype differentiation. To test the role of the endothelin pathway in ENS development, we are generating zebrafish mutants in components of the endothelin pathway using CRISPR/Cas9 genome editing technology. We are currently creating zebrafish mutants in several different endothelin ligands and endothelin converting enzyme 1 and we have generated a mutant in the endothelin receptor gene ednrb1b. We will then analyze the phenotypes of these mutants to learn how ENS progenitor migration and differentiation are affected. Our strategy will enable us to explore the role of endothelin signaling pathway genes in ENS development and to determine if mutations in these genes lead to an HSCR-like phenotype.

Inflammatory Phenotypes Of Zebrafish Enteric Nervous System Mutants

Presenter(s): Lillian Carroll − Biology

Faculty Mentor(s): Judith Eisen, Kristi Hamilton

Oral Session 4S

Research Area: Natural Science

Funding: OURS Program

Intestinal health depends on the microbial community within the dynamic intestinal environment. The enteric nervous system (ENS) innervates the intestine and modulates the microbial community composition. ENS reduction causes Hirschsprung disease (HSCR), resulting in intestinal dysmotility. Many HSCR patients develop potentially life-threatening intestinal inflammation. HSCR is genetically complex, with multiple HSCR genetic loci. The zebrafish is an excellent model in which to study the relationship between inflammation and genes linked to HSCR. Zebrafish with a mutation in the HSCR gene, sox10, have fewer enteric neurons, increased intestinal epithelial cell proliferation, and develop microbiota-dependent intestinal inflammation. Zebrafish with a mutation in another HSCR gene, ret, also have fewer ENS neurons but do not exhibit increased intestinal inflammation. sox10 acts in neural crest cells that form the ENS and ret acts within ENS cells, thus, I hypothesized that the intestinal phenotype of sox10;ret double mutants would be similar to the phenotype of sox10 mutants. To test this hypothesis, I analyzed the phenotypes of double mutants. I used PCR to identify mutants and quantified inflammation by counting intestinal neutrophils and recently-proliferated intestinal epithelial cells, and by determining the intestinal bacterial abundance. Surprisingly, and contrary to my hypothesis, sox10;ret double mutants did not exhibit increased intestinal inflammation or cell proliferation compared to wild-types. These results prompt me to reconsider the potential interactions

of the mutated genes, which will provide insights into the role of the ENS as a crucial regulator of the intestinal microbial community and its function in the maintenance of intestinal health.

Intestinal Phenotypes of Zebrafish Enteric Nervous System Double Mutants

Presenter(s): Lilly Carroll

Faculty Mentor(s): Judith Eisen & Kristi Hamilton

Oral Session 4 S

The enteric nervous system (ENS) innervates the intestine and regulates the dynamic intestinal environment. ENS reduction causes Hirschsprung disease (HSCR), a genetically complex disease that results in intestinal dysmotility and, in many patients, intestinal inflammation. The zebrafish is an excellent model in which to study the relationship between inflammation and genes linked to HSCR. Zebrafish with a mutation in one HSCR gene, sox10, have fewer enteric neurons and develop microbiota-dependent intestinal inflammation. Zebrafish with a mutation in another HSCR gene, ret, also have fewer ENS neurons but do not exhibit increased intestinal inflammation. To investigate the opposing intestinal phenotypes of sox10 and ret mutants, I analyzed intestinal phenotypes of sox10;ret double mutants. Because sox10 acts in neural crest cells that form the ENS and ret acts later, within ENS cells, I hypothesized that intestinal inflammatory phenotypes of sox10;ret double mutants would resemble those of sox10 mutants. To test this hypothesis, I quantified intestinal inflammation in sox10;ret double mutants by counting intestinal neutrophils. Surprisingly, I observed a wild-type (WT) neutrophil abundance phenotype in sox10;ret mutants. This result led me to investigate intestinal enterochromaffin cells, which produce serotonin and express ret but not sox10. I hypothesized that sox10;ret double mutants would exhibit the same decreased enterochromaffin cell phenotype as ret mutants. However, sox10;ret mutants had more enterochromaffin cells that ret mutants and were similar to WT. This result prompts further exploration of the potential interactions of the mutated genes for insights into the role of the ENS in maintenance of intestinal health.

Understanding microbial modulation of neuronal morphology in zebrafish

Presenter(s): Max Grice—Computer Science

Faculty Mentor(s): Judith Eisen, Joseph Bruckner

Session 4: Earning your Stripes

Increasing evidence supports a role for the intestinal microbiota in modulating host neurodevelopment and behavior, including complex social behaviors . Recent research has also linked the microbiota to neurological disorders including autism spectrum disorder (ASD), depression, Alzheimer’s Disease, and Parkinson’s Disease . However, the mechanisms of these interactions between the host-associated microbiota and neurodevelopment remain unclear . Using zebrafish raised in the absence of the microbiota, or germ-free (GF), our group has found that the microbiota modulates zebrafish social behavior . Normal social behavior requires neurons in a region of the brain called the ventral telencephalon (vTel) . Therefore, we hypothesized that the microbiota may modulate social behavior by altering development of vTel neurons, resulting in changes in vTel neuron morphology . To measure morphology of vTel neurons, we combined sparse mosaic labelling and high- resolution confocal microscopy to image individual vTel neurons . We used Imaris software to segment individual neurons and extract morphological measurements and adapted several software packages to warp and register individual neurons to an average reference brain in each condition . We found that vTel neurons from GF fish are significantly more complex than vTel neurons from their conventionally raised siblings . Together, this work suggests that the microbiota may modulate social behavior by restraining complexity of ventral forebrain neurons . Understanding the specific mechanism through which the microbiota normally modulates social behavior will allow us to better understand microbial modulation of neurodevelopment and therefore construct more effective treatments for neurological disorders that may result from dysbiosis of the host-associated microbiota .