Research:  Development and degenerative diseases of sensory neurons, gene regulation

·Requirement for math5 in the differentiation of retinal ganglion cells
·Roles of brn-3b in the development of retinal ganglion cells
·Roles of brn-3c in auditory and vestibular hair cell development
·Defects of the brn-3a-null sensory neurons

The mammalian retina and inner ear are two of the most common places of genetic diseases that cause blindness and deafness due to the degeneration of retinal and inner ear neurons. In order to understand the disease processes, it is very crucial to elucidate the fundamental mechanisms regulating the normal development and maintenance of these neurons at the molecular level. Our research is centered on identifying genes required for neuron differentiation and survival, investigating the genetic pathways involved in these processes, and developing therapies for blindness and deafness via gene therapy and stem cell replacement. We are currently investigating the roles of two classes of transcription factors in the development and survival of mouse retina and inner ear: (a) Math5 and Math1, the basic helix-loop-helix (bHLH) transcription factors homologous to Atonal, a Drosophila proneural transcription factor, and (b) Brn-3 factors, the Class IV POU-domain transcription factors. Using homologous recombination in murine embryonic stem (ES) cells to mutate math5 and brn-3 genes, we have show that the math-class genes regulate the differentiation of neuronal progenitor cells into specific types of neurons and that brn-3 genes are required for the maturation and survival of post-differentiation neurons.

Requirement for math5 in the differentiation of retinal ganglion cells

The bHLH class of proneural factors play an essential role in the differentiation of retinal neurons. The expression of math5, an ortholog of Drosophila atonal, coincides with the differentiation of retinal ganglion cells (RGCs). Using homologous recombination (knockout) approach, we have shown that the deletion of math5 results in the loss of RGCs and an increase of amacrine cells (particularly, the ON-starburst amacrine cells) in mice. Analyses of lacZ (ß-galactosidase) reporter gene expression in the math5-lacZ knockin mice and the expression of early RGC differentiation markers have indicated that the deletion of math5 does not prevent the formation of RGC progenitor cells. However, the null mutation of math5 blocks the initial differentiaton of RGCs and leads to the cell fate conversion of retinal progenitor cells from RGCs into amacrine cells. Furthermore, the loss of math5 abolishes the retinal expression of brn-3b, suggesting that math5 acts upstream of brn-3b in the processes of RGC differentiation.

Roles of brn-3b in the development of retinal ganglion cells

The three members of the Brn-3 family of POU-domain transcription factors are expressed in sensory neurons and a small number of brainstem nuclei. In the developing retina, the expression of brn-3b is first detected at E11.5 in the migrating nascent RGCs, two days prior to the onset of brn-3a and brn-3c expression. In our earlier experiments, we showed that targeted disruption of brn-3b results in the loss of 80% RGCs. Our recent cell lineage studies with brn-3b-lacZ and brn-3b-AP (human placental phosphatase) knockin mice have demonstrated that brn-3b was not required for the initial differentiation or for the migration of RGCs to ganglion cell layer. However, the RGCs lacking brn-3b failed to extend axons properly and consequently, degenerated by programmed cell death before birth. Our results indicate that Brn-3b regulates the activity of genes whose products play essential roles in the formation of axons and/or in maintaining the viability of RGCs.

Roles of brn-3c in auditory and vestibular hair cell development

Compared with the expression of brn-3a and brn-3b in the spiral and vestibular ganglion cells of inner ear, the expression of brn-3c is found in the auditory and vestibular hair cells during embryogenesis and in adult mice. Mice carrying he targeted deletion of brn-3c are deaf and impaired in their balancing abilities. These defects are resulted from the complete degeneration of auditory and vestibular hair cells in brn-3c mutant mice. Mutation of brn-3c has also been implicated in a human form of progressive hearing loss.

Defects of the brn-3a-null sensory neurons

In our earlier studies, we have shown that brn-3a is expressed in primary sensory neurons of the cranial and dorsal root ganglia and in specific neurons in the causal CNS. Mice carrying the brn-3a null mutation died hours after birth due to defective suckling and uncoordinated limb and trunk movement. Phenotypical analysis have shown that the knockout of brn-3a resulted in the loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus. Recently, we have used the brn-3a-lacZ transgenic reporter mice to further study the function of brn-3a. The brn-3a-lacZ expression in the brn-3a knockout mice showed significant axonal growth defects, including excessive and premature branching of major divisions of the trigeminal nerve and a failure to correctly innervate whisker follicles. Such defects precede the programmed cell death of the sensory neurons in brn-3a knockout mice.

Gan, L., Zhang, W., and Klein, W.H. (1990).  Repetitive DNA sequences linked to the sea urchin Spec genes contain transcriptional enhancer-like elements. Dev. Biol.  139, 186-196.

Gan, L., Wessel, G.M., and Klein, W.H. (1990).  Regulatory elements from the related Spec genes of Strongylocentrotus purpuratus  yield different spatial patterns with a lacZ  reporter gene. Dev. Biol.  142, 346-358. (cover photograph).

Klein, W.H., Tomlinson, C.R., Zhang, W., Xiang, M., Kozlowski, M.T., Gan, L., Nguyen, T., and Wessel, G.M. (1990). Ectoderm differentiation and the Spec gene family of sea urchin. In Developmental biology, UCLA Symposium on Molecular and Cellular Biology,  New Series, Vol. 125, 123-134.

Kozlowski, M.T., Gan, L., Venuti, J.M., Sawadogo, M., and Klein, W.H. (1991).  Sea urchin USF: a helix-loop-helix protein active in embryonic ectoderm cells. Dev. Biol.  148, 625-630.

Venuti, J.M., Gan, L., Kozlowski, M.T., and Klein, W.H. (1993).  Developmental potential of muscle cell progenitors and the myogenic factor SUM-1 in the sea urchin embryo. Mech. Dev.  41, 3-14.

Gan, L. and Klein, W.H. (1993).  A positive cis -regulatory element with a bicoid -target site lies within the sea urchin Spec2a enhancer. Dev. Biol.  157,119-132.

Mao, C-A., Gan, L., and Klein, W.H. (1994).  Multiple Otx-binding sites required for expression of the Strongylocentrotus purpuratus  Spec2a gene, Dev. Biol. 165, 229-242.

Gan, L., Mao, C-A., Wikramanayake, A.H., Angerer, L.M., Angerer, R.C., and Klein, W.H. (1995).  An orthodenticle-related protein from Strongylocentrotus purpuratus. Dev. Biol.  167, 517-528.

Mao, C-A., Wikramanayake, A.H., Gan, L., Chuang, C-K., and Klein, W.H. (1996). Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein. Development  122, 1489-1498.

Gan, L., Xiang, M., Zhou, L., Wagner, D.S., Klein, W.H., and Nathans, J. (1996). POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells. Proc. Natl. Acad. Sci.  93, 3920-3925.

Xiang, M.*, Gan, L.*, Zhou, L., Klein, W.H., and Nathans, J. (1996).  Targeted deletion of the mouse POU domain gene Brn-3a causes a selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired sucking. Proc. Natl. Acad. Sci.  93, 11950-11955. (* equal contribution)

Klein, W.H., Mao, C-A., Gan, L., Chuang, C-K., and Wikramanayake, A.H. (1997). Manipulating cell fates in the sea urchin embryo . In Invertebrate Reproduction and Development  31:1-3, 21-29.

Xiang, M., Gan, L., Li, D., Zhou, L., Chen, Z., Wagner, D., O'Malley Jr., B.W., Klein, W., and Nathans, J. (1997). Role of the Brn-3 family of POU-domain genes in the development of the auditory/vestibular, somatosensory, and visual systems. In Cold Spring Harbor Symposium on Quantitative Biology, Vol. LXII, 325-336.

Xiang, M.*, Gan, L.*, Li, D.*, Chen, Z., Zhou, L., O'Malley Jr., B.W., Klein, W, and Nathans, J. (1997). Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development. Proc. Natl. Acad. Sci. 94, 9445-9450. (*equal contribution)

Chen, H., Lun, Y., Ochinnikov, D., Kokubo, H., Oberg, K. C., Pepiclli, C. V., Gan, L., Lee, B., and Johnson, R.J. (1998). Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella symdrome. Nature Genetics 19, 51-55.

Evrard, Y.A., Lun, Y., Aulehla, A., Gan, L. and Johnson, R.L. (1998). Lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394, 377-381.

Wagner, D.S., Gan, L., and Klein, W.H. (1998). Expression of a gene trap reporter construct in a subset of cells in embryonic sites of hematopoiesis: Evidence for alternative rRNA production in hematopoietic cells. Biochem. Biophys. Res. Commun. 250, 674-681.

Lin, X., Gan, L., Klein, W.H., and Wells, D. (1998). Expression and functional analysis of mouse EXT1, a homolog of the human multiple exostoses type 1 gene. Biochem. Biophys. Res. Commun. 248, 738-743.

Vivian, J.L., Gan, L., Olson, E.N., and Klein, W.H. (1999). Threshold levels of myogenin required for viability, skeletal muscle differentiation, and sternum formation revealed through a hupomorphic myogenin allele. Dev. Biol. 208, 44-55.

Gan, L., Wang, S.W., Huang, Z., and Klein, W.H. (1999). POU-domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial cell fate specificatioon or migration. Dev. Biol. 210, 469-480.

Wagner, D.S., Gan, L., and Klein, W.H. (1999). Identification of a differentially expressed RNA helicase by gene trapping. Biochem. Biophys. Res. Commun. 262, 677-684.

Wang, S.W., Gan, L., Martin, S.E., and Klein, W.H. (2000). Abnormal polarization and axon outgrowth in retinal ganglion cells lacking the POU-domain transcription factor Brn-3b. Mol. Cell. Neurosci. 16, 141-156

Lu, J., Chang, P., Richardson, J.A., Gan, L., Weiler, H., Olson, E.N. (2000). The basic helix-loop-helix transcription factor capsulin controls spleen organogenesis. Proc. Natl. Acad. Sci. USA. 97, 9525-9530.

Wang, S.W., Kim, B.S., Ding, K., Wang, H., Sun, D., Johnson, R.L, Klein, W.H. and Gan, L. (2001). Requirement of math5 in the development of retinal ganglion cells. Genes & Dev. 15, 24-29.

Eng, S.R., Gratwick, K., Rhee, J.M., Fedtsova, N., Gan, L., and Turner, E.E. (2001). Defects in sensory axon guidance precede neuronal death in brn-3a-deficient mice. J. Neurosci. 21, 541-549.

People in the Lab

Kan Ding, M.D., Post-doctoral fellow. kan_ding@urmc.rochester.edu

Zhiyong Yang, M.D., graduate student, Neuroscience Program. zhiyong.Yang@mc.rochester.edu.