My laboratory studies the molecular mechanisms that govern mammalian development, using the mouse as a model. We use a combination of biochemical, molecular and genetic approaches to identify and characterize signaling molecules and pathways that control the development and maintenance of the musculoskeletal and hypothalamic systems.

The muskuloskeletal system:
The musculoskeletal system provides the mechanical support for our posture and movement. How it arises during embryogenesis pertains to the basic problem of embryonic induction. How the components of this system are repaired after injury and maintained throughout life is of biological and clinical significance. We study how this system is generated and maintained.

      The origin and induction: The musculoskeletal system of the trunk originates from a common embryonic structure called the somite. Somites are segmented mesodermal units flanking both sides of the spinal cord. Their reiterated pattern is the basis for the repeated organization of the trunk. Under the inductive influence of adjacent tissues, cells within the somite give rise to muscles and bones. We have developed a 3-dimensional culture system that allows characterization of crucial long-range and contact-dependent cellular interactions that induce early skeletal and muscle fates. Our efforts toward designing new methods and assays to track somite development have enabled us to make novel observations.

      Induction of the axial skeletal progenitors: We have identified the Hedgehog (Hh) proteins responsible for inducing the early skeletal fate. Hh largely utilizes evolutionarily conserved downstream mediators for inductive signaling. In addition, we also found a vertebrate-specific cell surface Hh binding protein Gas1. Gas1 mutants display skeletal defects related to or due to altered Hh signaling. Mechanistically, Gas1 helps transform the Hh diffusion gradient into its observed signaling activity gradient. This unexpected mechanism provides a new vision of Hh signaling pathway initiation and has direct implications for the long-range action of Hh.

      Induction of the early embryonic muscle: Conversely, the Wnt family of proteins plays a key role in inducing the dermis/muscle dual potential progenitors. Combining our in vitro assay with microarrays analyses, we have uncovered previously unknown effectors and target genes of Wnt. Using an ex vivo whole embryo culture system coupled with somite-specific gene delivery, we discovered an unconventional pathway for Wnt signaling via the adenylyl cyclase/protein kinase A/Creb cascade that selectively activates myogenic transcriptional determinants Myf5 and MyoD.

      Embryonic and adult muscle stem cells: Somites not only supply cells for embryonic muscles, but also contain muscle progenitors. The proliferative capacity of these progenitors depends upon the transcription factors Pax3 and Pax7. Both genes are activated by Wnt. Using inducible cell lineage tracing, we have found that early Pax7-expressing somitic cells directly give rise to adult muscle stem cells, i.e. the satellite cells. Lineage tracing of Pax7-expressing adult satellite cells indicates that they are indeed a stem cell source for muscle regeneration. Conditional inactivation of Pax7 at different developmental time points reveals that Pax7 is required for the proliferative properties of muscle progenitors up to 3 weeks after birth when they transition into quiescence. After this transition is made, however, both Pax3 and Pax7 are completely dispensable. Our finding of an age-dependent cell-intrinsic change in the genetic requirement for muscle stem cells cautions against inferring adult stem cell biology from embryonic studies, and has direct implications for the use of stem cells from hosts of different ages in transplantation-based therapies.

The neuroendocrine hypothalamus:
The hypothalamus is an essential brain center that maintains multiple physiological homeostatic processes by modulating pituitary hormone secretions. Two centers (nuclei) of the hypothalamus, the paraventricular and supraoptic nuclei (PVN and SON), contain various hormone-producing neurons. Studies of these hormones have been instrumental to our understanding of endocrine homeostasis, including the maintenance of bodily fluid osmolarity and the balance of energy metabolism. Despite the importance of PVN and SON function, molecules that control their embryonic specification, neuronal differentiation, nucleus formation, and neuronal connections are largely unknown.
During early embryogenesis, the PVN and SON have a common origin but segregate from each other to diversify their inputs from other brain regions. After their segregation, the PVN and SON project axons to other homeostasis centers, including the pituitary. Through a series of genetic analyses, we have constructed a hierarchical map composed of five transcription factors that define the key steps of proliferation, survival, differentiation, and hormone expression of the PVN and SON neurons during development. Sitting at the top of this genetic cascade is the Sim1 gene, which also controls both the segregation and axonal projections of the PVN and SON. We have therefore expanded our work to investigate neuronal migration cues. We found that a Sim1 downstream gene, PlexinC1 (a receptor for neuronal guidance signals), mediates PVN and SON segregation.

We found that heterozygosity at the Sim1 locus causes the animal to develop morbid obesity due to over-feeding. Sim1 heterozygous mice exhibit compromised PVN structure and function, supporting a role for PVN in maintaining energy homeostasis via the regulation of feeding. Interestingly, people haploid for the SIM1 gene also clinically present with childhood obesity. Thus, our approach directed toward studying embryonic development of the PVN and SON has directly led us to identify a genetic contributor to feeding regulation and obesity in rodents and humans. Therefore, our basic developmental studies of the PVN and SON suggest a new avenue for finding genetic determinants of homeostasis regulation.

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Fan, C.-M. and Maniatis, T. (1989). Two different virus-inducible elements are required for beta-interferon gene regulation. EMBO J. 8, 101-110.

Lenardo, M.J., Fan, C.-M., Maniatis, T. and Baltimore, D. (1989). The involvement of NF-kappa-B in beta-interferon gene regulation reveals its role as widely inducible mediator of signal transduction. Cell 57, 287-294.

Fan, C.-M. and Maniatis, T. (1990). A DNA binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence. Genes & Development 4, 29-42.

Fan, C.-M. and Maniatis, T. (1991). The p50 subunit of NF-kappa-B is generated by processing of p105 through an ATP-dependent pathway. Nature 354, 395-398.

Maniatis, T., Whittemore, L.-A., Du, W., Fan, C.-M., Keller, A., Palombella, V.J. Thanos, D.N. (1992). Positive and Negative Control of Human Interferon- Gene Expression. Harvey Lecture 44, 1193-1220. Cold Spring Harbor Press.

Fan, C.-M. and Tessier-Lavigne, M. (1994). Patterning of Mammalian Somites by the Surface Ectoderm and the Notochord: Evidence for Sclerotome Induction by a Hedgehog Homolog. Cell 79, 1175-1186.

Fan, C.-M., Porter, J., Chiang, C., Chang, D.T., Beachy, P.A., and Tessier-Lavigne, M. (1995). Long-Range Sclerotome Induction by Sonic Hedgehog: Direct Role of the Amino-Terminal Cleavage Product and Modulation by the cyclic-AMP Signaling Pathway. Cell 81, 457-465.

Muenke, M., Bone, L., Mitchell, H., Hart, I., Walton, K., Hall-Johnson, K., Ippel. E., Dietz-Band, J., Karloy, K., Fan, C.-M., Tessier-Lavigne, M., and Patterson, D. (1995). Physical Mapping of the Holoprosencephaly Critical Region in 21q22.3, Exclusion of SIM2 as a Candidate Gene for HPE, and Mapping of SIM2 to a Region of Chromosome 21 Important for Down Syndrome. The American Journal of Human Genetics 57, 1074-1079

Pourquie, O., Fan, C.-M., Coltey, M., Hirsinger, E., Watanabe, Y., Breant, C., Francis-West, P., Brickell, P., Tessier-Lavigne, M., and Le Douarin, N.M. (1996). Lateral and Axial Signals involved in Avian Somite Patterning : Role of the BMP4 Protein. Cell 84, 461-471.

Fan, C.-M., Kuwana, E., Bulfone, A., Fletcher, C. F., Copeland, N. G., Jenkins, N. A., Crews, S., Salvador, M., Puelles, L., Rubenstein, J. L. R., and Tessier-Lavigne, M. (1996). Expression Patterns of Two Murine Homologs of Drosophila single-minded Suggest Possible Roles in Embryonic Patterning and in the Pathogenesis of Down Syndrome. Molecular and Cellular Neuroscience 7, 1-16.

Mastick, G.S., Fan, C.-M., Tessier-Lavigne, M., Serbedzija, G.N., MaMahon, A.P., Easter, S.S., Jr. (1996). Early Deletion of Neuromeres in Wnt-1-/- Mutant Mice: Evaluation by Morphological and Molecular Markers. The Journal of Comparative Neurology 374, 246-258.

Probst, M.R., Fan, C.-M.,Tessier-Lavigne, M., and Hankinson, O. (1997). Two Murine Homologs of the Drosophila single-minded Protein that Interact with the Mouse Aryl Hydrocarbon Receptor Nuclear Translocator Protein. Journal of Biological Chemistry 272, 4451-4457.

Michaud, J. and Fan, C.-M. (1997). Single-minded - Two Genes, Three Chromosomes. Genome Research 7, 569-571.

Fan, C.-M., Lee, C.S., and Tessier-Lavigne, M. (1997) A Role for WNT Proteins in Induction of Dermomyotome. Developmental Biology 191, 160-165.

McMahon, J., Takada, S., Zimmerman, L.B., Fan, C.-M., Harland, R.M., and McMahon, A.P. (1998) Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes & Development 12, 1438-1452.

Michaud, J., Rosenquist, T., May, N.R., and C.-M. Fan. (1998). Development of neuroendocrine lineages requires the BHLH-PAS transcription factor SIM1. Genes & Development 12, 3264-3275.

Lee, C.S., Buttitta, L., May, N.R., Kispert, A., and C.-M. Fan. (2000). SHH-N upregulates Sfrp2 in the somitic mesoderm to mediate its competitive action with WNT1 and WNT4. Development 127, 109-118.

Michaud, J.L., De Rossi, C., May, N.R., Holdener, B., and C.-M. Fan. (2000). ARNT2 acts as the dimerization partner of SIM1 for the development of the hypothalamus. Mechanism of Development 90, 253-261.

Epstein, D.J., Martinu, L., Michaud, J.L., Losos, K.M., Fan, C.-M., and A.-L. Joyner. (2000). Members of the bHLH-PAS family regulate Shh transcription in forebrain regions of the mouse CNS.
Development 127, 4701-4709.

Lee, S.C. and C.-M. Fan. (2001). Embryonic expression patterns of the mouse and chick Gas1 genes. Mechanism of Development 101, 293-297.

Lee, C.S., May, N.R., and C.-M. Fan. (2001). Transdifferentiation of the ventral retinal pigmented epithelium to neural retina in the Graowth Arrest Specific Gene 1 mutant. Developmental Biology 236, 17-29.

Liu, Y., May, N.R., C.-M. Fan. (2001). Growth Arrest Specific Gene 1 is a positive growth regulator for the cerebellum. Developmental Biology 236, 30-45.

Lee, C.S., Buttitta, L., and C.-M. Fan. (2001). Evidence that the WNT-inducible growth arrest specific gene 1 encodes an antagonist of sonic hedgehog signaling in the somite. Proc. Natl. Acad. Sci. USA 98, 11347-11352.

Michaud, J.L., Boucher, F., Melnyk, A., Gauthier, F, Goshu, E., Levy, E., Mitchell, G.A., Himms-Hagen, J., and C.-M. Fan. (2001). Human Molecular Genetics 10, 1465-1473.

Goshu, E. Jin, H., Fasnacht, R., Spenski, M., Michaud, J.L., and C.-M. Fan. (2002). Sim2 mutants have developmental defects not overlapping with those of Sim1 mutants. Mol.Cell. Biol. 22, 4147-4157.

Liu, Y., Liu, C., Yamada, Y., and C.-M. Fan. (2002). Growth arrest specific gene 1 acts as a region-specific mediator of the Fgf10/Fgf8 regulatory loop in the limb. Development 129, 5289-5300.

Buttitta, L., Tanaka, T., Chen, A., Ko, M., and C.-M. Fan. (2003) Microarray analysis reveals novel WNT targets in the somites. Developmental Biology 258, 91-104.

Liu C, Goshu E, Wells A, Fan CM. (2003) Identification of the downstream targets of SIM1 and ARNT2, a pair of transcription factors essential for neuroendocrine cell differentiation. J Biol Chem. 278:44857-67.

Buttitta L, Mo R, Hui CC, Fan CM. (2003) Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction. Development 130:6233-43.

Goshu E, Jin H, Lovejoy J, Marion JF, Michaud JL, Fan CM. (2004) Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus. Mol Endocrinol. 18:1251-62. 

Chen AE, Ginty DD, Fan CM. (2005) Protein kinase A signalling via CREB controls myogenesis induced by Wnt proteins. Nature 433: 208-9.

Chen AE, Fan CM. (2005) Targeting gene expression in the mouse somite: adenovirus-mediated gene delivery and whole embryo culture. Genesis 42:71-6.

Martinelli DE, Fan CM (2007) Gas1 extends the range of Hedgehog action by facilitating its signaling. Genes Dev 2007 May 15;21(10):1231-43.

Xu C, Fan CM (2007) Allocation of paraventricular and supraoptic neurons requires Sim1 function: a role for a Sim1 downstream gene PlexinC1. Mol Endocrinol 2007 May; 21(5):1234-45.

Maisa Seppala, Michael J. Depew, David C. Martinelli, Chen-Ming Fan, Paul T. Sharpe and Martyn T. Cobourne (2007) Gas1 is a modifier for holoprosencephaly and genetically interacts with sonic hedgehog. J Clin Invest 2007 Jun;117(6):1575-84.

Martinelli DE, Fan CM (2007) The Role of Gas1 in Embryonic Development and its Implications for Human Disease. Cell Cycle 2007 Aug 13; 6(21).

Friedman ER, Fan CM. (2007) Separate necdin domains bind ARNT2 and HIF1alpha and repress transcription. Biochem Biophys Res Commun 363, 113-118.

Xu, C. and Fan, C.-M. (2007) Allocation of paraventricular and supraoptic neurons requires Sim1 function: a role for a Sim1 downstream gene PlexinC1. Molecular Endocrinology 21, 1234-1245.

Martinelli, D.C. and Fan, C.-M. (2007) Gas1 extends the range of Hedgehog action by facilitating its signaling. Genes & Development 15, 1231-1243.

Seppala, M., Depew, M.J., Martinelli, D.C., Fan, C.-M., Sharpe P.T., and Cobourne M.T. (2007) Gas1 is a modifier for holoprosencephaly and genetically interacts with sonic hedgehog. Journal of Clinical Investigation 117, 1575-1584.

Martinelli, D.C. and Fan, C.M. (2007) The role of Gas1 in embryonic development and its implications for human disease. Cell Cycle 6, 2650-2655.

Friedman, E.R. and Fan, C.-M. (2007) Separate Necdin domains bind ARNT2 and HIF1a and repress transcription. Biochemical Biophysical Research Communications 63, 113-118.

Xu, C. and Fan, C.-M. (2008) Neuropilin, Plexin, and Semaphorin family members are expressed in the developing hypothalamus. Gene expression patterns 8, 502-507.

Zhang, Y., Narayan, S., Geiman, E., Lanuza, G. M., Velasquez, T., Shanks, B., Akay, T., Dyck, J., Pearson, K., Gosgnach, S., Fan, C.-M., and Goulding, M. (2008) V3 spinal neurons establish a robust and balanced locomotor rhythm during walking Neuron 60, 84-95.

Ohazama, A., Haycraft, C.J., Seppala, M., Blackburn, J., Cobourne, M., Ghafoor, S., Martinelli, D., Fan, C.-M., Peterkova, R., Lesot, H., Yoder, B.K., and Sharpe, P.T. (2009) Primary cilia regulate Shh activity in the control of molar tooth number. Development 136, 897-903.

Martinelli, D.C. and Fan, C.-M. (2009) A sonic hedgehog missense mutation associated with holoprocensephaly causes defective binding to Gas1. Journal of Biological Chemistry 284, 19169-19172.

Lepper, C., Conway, S, and Fan, C.-M. (2009) Adult muscle stem cells have distinct genetic requirement from embryonic and neonatal progenitors. Nature 460, 627-631.

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