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Search Results to Charles G Sagerstrom PhD

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Academic Background

B.A., Macalester College, MN, 1987
Ph.D., Stanford University, CA, 1993

Gene expression in embryogenesis

Formation of the vertebrate central nervous system (CNS) begins early during embryogenesis - at gastrula stages, but extensive refinements continue to take place before the fully functional adult CNS emerges. Neural development is carefully controlled and perturbations of this process give rise to defects ranging from severe developmental abnormalities to mild cognitive impairments. We are studying genes that control early neural development, particularly formation of the caudal CNS (hindbrain and spinal cord) in the zebrafish, using a three-step strategy.

Gene discovery
We are screening for novel genes involved in neural development by several approaches. First, we have used subtractive hybridization to isolate genes expressed specifically in the caudal CNS. Second, we are using ‘expression profiling’ to identify novel genes expressed downstream of paralog group 1 hox genes in the caudal hindbrain. Third, we are undertaking a haploid genetic screen for mutations affecting hindbrain development.

Derive genetic pathways
We next integrate these genes into pathways that drive hindbrain development, using a number of molecular genetic approaches. For instance, we use injection of synthetic mRNA to ectopically activate gene function in wild type embryos, or to rescue defects in various mutant lines, and we use TALEN and CRISPR technology to ‘knock out’ the function of specific genes. In addition, we have recently adapted the Gal4:UAS transgenic system to achieve tissue specific expression of transgenes within the developing hindbrain.

Define biochemical activities
Lastly, we define the function of the various gene products using a variety of biochemical approaches. Since the majority of the genes we identified turn out to act as transcription factors (pbx4, meis3, hoxb1b, hoxb1a, nlz1, nlz2, prdm12), we have used ChIP to assay binding of these factors to target promoters in zebrafish embryos. Similarly, we have used ChIP to detect changes in histone modifications mediated by these transcription factors at specific promoters. Furthermore, several of the hindbrain specific genes we identified appear to act as protein phosphatases and we are currently attempting to identify their substrates both in zebrafish embryos and in cell culture systems.

Research Figure

Zebrafish Image


Wholemount in situ hybridization detects the expressionof four different genes (two in red, two in black) in the formingneurectoderm of the zebrafish. (Dorsal view at 10 hours post fertilization.)

Rotation Projects

Rotation Project Background

During early embryogenesis the primordium of the central nervous system (CNS) consists of an epithelium - the neural plate - that is only a single cell-layer thick. The neural plate subsequently undergoes an extraordinary set of developmental steps to form all structures of the adult CNS. This process raises two separate, but interrelated, questions: 1). How is each neural structure positioned correctly? and 2). How is the differentiation of each structure regulated? We are particularly interested in understanding the earliest steps in these processes and we focus our work on how the caudal CNS (the cerebellum, brainstem and spinal cord) is formed.

We have isolated several genes that are expressed in the neural plate and we use zebrafish embryos to explore the role of these genes in formation of the caudal CNS. We combine in vivo experiments aimed at defining the biological role of each gene with in vitro biochemical experiments aimed at understanding their mechanism of action.

Potential Rotation Projects

  1. Role of Meis and Pbx proteins in controlling histone modifications. Meis and Pbx proteins have been shown to act in complexes together with Hox proteins to regulate transcription. More recently, our ChIP experiments revealed that Meis and Pbx are bound at target promoters several hours prior to the binding of Hox proteins and well before the target gene becomes transcribed. Our preliminary experiments indicate that Meis and Pbx may act to control histone modifications at these early stages of embryogenesis, perhaps to permit subsequent binding of Hox proteins. This project entails the use of ChIP assays to determine whether histone modifications affect Hox binding, as well as to test whether binding of Hox proteins constitutes a 'switch' that initiates transcription of the target gene.
  2. Nucleosome positioning at hox promoters. Nucleosomes are involved in packaging genomic DNA, but also have regulatory functions as they can permit (or prevent) access of various DNA binding factors to genomic DNA. We have used micrococcal nuclease mapping to identify nucleosome occupancy at hox promoters during zebrafish embryogenesis. We find that nucleosomes may shift as hox transcription is initiated. This project involves assaying nucleosome occupancy in response to various stimuli, as well as determining whether shifts in nucleosome occupancy is a cause or an effect of transcription.
  3. Deriving a genetic pathway for the formation of hindbrain rhombomere (r) 4 and 5.  A number of genes are required for formation of r4 and r5, but it is not clear how they fit into a genetic pathway.  This project entails gene misexpression in wild type and mutant zebrafish embryos using mRNA injections and transgenic lines. In addition, it appears that several of these genes may encode protein phosphatases. An additional project therefore centers on identifying likely substrates of these phosphatases.

Search Criteria
  • Embryonic Structures