RIKEN Center for Developmental Biology
2003 Annual Report
Laboratory for Neuronal Differentiation and Regeneration
The early stages of neural network development see the growth of a number of neurons in excess of the number that will ultimately populate any given region. Neurons that have formed synaptic connections establish what is known as a trophic interaction in which the target tissue provides the neuron with chemical signals necessary for its survival and continued function. The supply of such signals is limited, and neurons which fail to compete successfully for these factors undergo apoptosis, a controlled die-off that establishes an appropriate balance in the neural population.
The target-derived chemical signals in the trophic interaction are known as neurotrophic factors. The study of these signaling proteins dates back to the discovery of nerve growth factor (NGF) by Levi-Montalcini and colleagues in the 1950s. Subsequent research has revealed the diversity of neurotrophic factors, which are now thought to include neurotrophins (of which NGF is a member), neuropoietins, fibroblast growth factors, and the transforming growth factor-beta superfamily. Hideki Enomoto is primarily interested in this last group, the GDNF Family Ligands (the GFLs) in particular. This family of neurotrophic factors includes four known members GDNF (for Glial cell line Derived Neurotrophic Factor), Neurturin, Artemin and Persephin. The GFLs signal via receptor complexes formed by the RET receptor tyrosine kinase and one of four co-receptors, GRFƒ¿1-4. In vitro, these four receptors show differential affinities for specific GFLs, with GFRƒ¿1 showing the greatest ability to interact with the range of GFL family members.
GDNF ligand-receptor pairings
Enomoto studied phenotypes of mice in which individual GFL, GFR-ƒ¿ or Ret genes are disrupted and found that, in receptor-ligand interactions in vivo, there are affinitive pairings between GFRƒ¿1-GDNF, GFRƒ¿2-NRTN and GFRƒ¿3-ARTN. The loss of the GDNF-GFRƒ¿1 system had the strongest developmental effects, causing defects in enteric, autonomic, sensory and motor neurons as well as failures in the development of the kidney, and resulting in lethality by the P0 stage of development, birth. Similar neuronal populations are affected by Neurturin or GFRƒ¿2 deficiency, but the phenotypes are less severe, with kidneys forming normally and indefinite effects on sympathetic and motor neural development. Analysis of the consequences of these mutations in developing enteric and parasympathetic neurons suggests that GDNF-GFRƒ¿1 functions in neural precursors to regulate fundamental neurodevelopmental processes such as migration and proliferation, while the later expression of Neurturin-GFRƒ¿2 in target tissues serves to maintain previously established neurons.
Artemin-GFRƒ¿3 signaling seems to operate in a more specific subset of cells, sympathetic neurons, with no apparent function in enteric or central neural development. The sympathetic nervous system arises from the neural crest and follows a stepwise developmental pathway that directs its neurons to establish their appropriate functional roles and positions throughout the body. In a series of signal-directed decisions, sympathetic precursor cells migrate and differentiate into neurons soon after they undergo lineage commitment and restriction to a catecholaminergic (sympathetic neurotransmitter-producing) fate. Enomoto found that Artemin is expressed in blood vessels and attracts growing axons emanating from sympathetic ganglion that expresses RET. This interaction is crucial for sympathetic axons to follow vascular pathways and to reach the final target tissues. The fourth GFL-receptor pairing of Persephin and GFRƒ¿4 is known to be expressed ubiquitously at low levels, but its developmental activity remains obscure.
In related work, members of the Enomoto lab studied the roles of RET and TRKA (neurotrophin receptor) in sympathetic neurogenesis and maintenance. Both RET and TRKA are expressed in developing sympathetic neurons, but the balance between these two receptor types shifts over time, with RET predominating during earlier phases of axon growth and migration, while TRK assumes a more important role in ensuring the perinatal survival and maintenance of these cells.
Studies of RET signaling are of particular interest for their value in biomedical research. Hirschsprung's disease, a congenital disorder afflicting about 1 in 5,000 newborn children in which enteric neuron precursors fail to colonize the distal part of the gut resulting in a loss of bowel motility, chronic constipation and bowel obstruction, is caused by a loss-of-function mutation in the Ret gene. Hyperactivation of RET results in abnormal development of the neural crest, a developmentally transient structure that normally gives rise to neurons, glia, endocrine cells and mesenchymal cells, and has been linked to the oncogenesis of endocrine tumors, such as thyroid carcinoma and pheochromocytoma.
In the future, the Enomoto research team plans to conduct systematic analyses of RET-bearing neurons to achieve a better understanding of the molecular mechanisms involved in RET and GFL-receptor signaling. One immediate focus will be on investigating roles for RET, GDNF and GFRƒ¿1 in the postnatal maintenance of motor, sensory and dopaminergic neurons, using a Cre-Lox conditional knockout strategy. By providing insights into the functions of GFL-specific neurotrophic factor signaling pathways, Enomoto hopes to contribute to the development of stem cell-based therapies for nervous system disorders.
Gianino S, Grider J R, Cresswell J, Enomoto H and Heuckeroth R O. GDNF availability determines enteric neuron number by controlling precursor proliferation. Development 130:2187-98 (2003).
Enomoto H, Crawford P A, Gorodinsky A, Heuckeroth R O, Johnson E M, Jr. and Milbrandt J. RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128:3963-74 (2001).
Enomoto H, Heuckeroth R O, Golden J P, Johnson E M and Milbrandt J. Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin. Development 127:4877-89 (2000).
Baloh R H, Enomoto H, Johnson E M, Jr. and Milbrandt J. The GDNF family ligands and receptors - implications for neural development. Curr Opin Neurobiol 10:103-10 (2000).