Seth  Blackshaw, PhD Associate Professor Telephone Number:   443-287-5609 Fax Number:   410-614-9568 The Solomon H. Snyder Department of Neuroscience Johns Hopkins University School of Medicine 725 North Wolfe St. Baltimore, MD 21205 Room:  329 Broadway Research Building sblack@jhmi.edu

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Molecular basis of cell specification in vertebrate retina and hypothalamus

The vertebrate central nervous system is an amazingly complex structure composed of distinct subtypes of neurons and glia.  Proper development of these cell types is critical in the regulation of physiology and behavior. Despite this, surprisingly little is known about how this amazing range of cell types is specified in development.  What are the genetic programs that allow this assortment of cell types to be specified during development? To elucidate the molecular mechanisms that regulate this development, we have selected the mouse retina and hypothalamus as model systems to ask this question. The retina initially emerges as a lateral extension of the hypothalamus prior to the onset of neurogenesis, although the two organs later give rise to essentially non-overlapping cell types.  Each structure offers unique advantages for our studies.

The retina is perhaps the best-characterized region of the central nervous system, and provides an excellent system to identify the novel molecular mechanisms that regulate neuronal cell fate.  The retina is comprised of seven major cell types, each identified by unambiguous morphology and molecular markers, and changes in their differentiation are easily measured. Defects in the differentiation of retinal photoreceptors often result in blindness.  We are interested in developing a detailed understanding of the molecular mechanisms that guide photoreceptor development in progenitor/in these different types of cells, which may in turn provide insight into the development of therapies for photoreceptor dystrophies.

The mammalian hypothalamus is a critical central physiological regulatory center.  It is comprised of many different cell types that are organized into discrete nuclei, each of which have been shown to be critical for the regulation of behaviors ranging from the sleep-wake cycle to appetite to the care of offspring, although the identity and connectivity of the precise cell types that mediate these effects is largely unknown.  Identification of genes that selectively control the differentiation of individual hypothalamic neuronal subtypes thus provides an opportunity to determine their contribution to these behaviors. 

As a means of identifying genes that control cell specification in retinal and hypothalamus, we have comprehensively profiled gene expression in both these tissues from the start to the end of neurogenesis using both microarray and SAGE analysis, and have used high-throughput in situ hybridization to characterize the cellular expression patterns of over 1800 differentially expressed transcripts in both tissues.   Projects currently underway in the lab include:

1. Functional analysis of candidate regulators of cell specification and survival in retina.

We are conducting functional analysis of genes identified in our screen that are selectively expressed in the four main retinal cell types that differentiate postnatally in the mouse – specifically rod photoreceptors, bipolar neurons, amacrine cells and Muller glia. These genes include transcription factors, regulators of signal transduction, and also putative noncoding RNAs.  In the course of this work, we have identified the E3 SUMO ligase and transcriptional coregulator Pias3 as a global regulator of both rod and long-wavelength cone photoreceptor differentiation.  We have also identified the orphan nuclear hormone receptor ERRb as essential for rod photoreceptor terminal differentiation and survival, and have identified implicated the long noncoding RNAs Six3OS and RNCR2 as important regulators of the differentiation of other retinal cell types.   

We employ a variety of high-throughput approaches to identify cellular targets of these factors.  Working with the lab of Heng Zhu in the Department of Pharmacology, we have generated a microarray consisting of over 17,000 non-redundant recombinant human proteins.    We have developed this not only to identify cellular targets of proteins and RNAs of interest, but also as a tool for rapidly characterizing the target sequences of DNA binding proteins.  Using DNA probes derived from predicted transcriptional regulatory elements, we have greatly expanded our knowledge of the binding selectivity of known transcription factors.  Unexpectedly, we have also shown that many proteins not annotated as transcription factors, are also sequence-specific DNA binding proteins. One such example is the protein kinase MAPK1. We are now extending these studies, using bioinformatic approaches to study the putative regulatory regions of genes specifically expressed in photoreceptors, bipolar neurons, and Muller glia, and are now characterizing DNA binding proteins that specifically bind to these DNA elements. 

2. Regulation of hypothalamic cell fate specification and function.

Our work has identified both a large number of unique molecular markers that demarcate different hypothalamic nuclei and genes that may be involved in spatial patterning of hypothalamic neuroepithelium.  This provides us both with an extensive list of candidate genes for regulating hypothalamic differentiation, and a set of molecular markers to interpret the effects of altering the function of candidate genes.  We are using a combination of in utero electroporation and conditional knockout mice to investigate the cellular and behavioral phenotypes induced following the gain or loss of function of genes of interest.  We are particularly interested in the development of hypothalamic tanycytes, a radial glial cell type that may play a key role in regulating multiple physiological pathways.