Binhai Zheng

p63 is a p53 homologue required for limb and epidermal morphogenesis

The p53 tumour suppressor is a transcription factor that regulates the progression of the cell through its cycle and cell death (apoptosis) in response to environmental stimuli such as DNA damage and hypoxia. Even though p53 modulates these critical cellular processes, mice that lack p53 are developmentally normal, suggesting that p53-related proteins might compensate for the functions of p53 during embryogenesis. Two p53 homologues, p63 and p73, are known and here we describe the function of p63 in vivo. Mice lacking p63 are born alive but have striking developmental defects. Their limbs are absent or truncated, defects that are caused by a failure of the apical ectodermal ridge to differentiate. The skin of p63-deficient mice does not progress past an early developmental stage: it lacks stratification and does not express differentiation markers. Structures dependent upon epidermal–mesenchymal interactions during embryonic development, such as hair follicles, teeth and mammary glands, are absent in p63-deficient mice. Thus, in contrast to p53, p63 is essential for several aspects of ectodermal differentiation during embryogenesis.

Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock

Mice carrying a null mutation in the Period 1 (mPer1) gene were generated using embryonic stem cell technology. Homozygous mPer1 mutants display a shorter circadian period with reduced precision and stability. Mice deficient in both mPer1 and mPer2 do not express circadian rhythms. While mPER2 regulates clock gene expression at the transcriptional level, mPER1 is dispensable for the rhythmic RNA expression of mPer1 and mPer2 and may instead regulate mPER2 at a posttranscriptional level. Studies of clock-controlled genes (CCGs) reveal a complex pattern of regulation by mPER1 and mPER2, suggesting independent controls by the two proteins over some output pathways. Genes encoding key enzymes in heme biosynthesis are under circadian control and are regulated by mPER1 and mPER2. Together, our studies show that mPER1 and mPER2 have distinct and complementary roles in the mouse clock mechanism.

The mPer2 gene encodes a functional component of the mammalian circadian clock

Circadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological and behavioural processes in a wide range of life forms. In mammals, there is a master circadian clock in the suprachiasmatic nucleus of the anterior hypothalamus. Three putative mammalian homologues (mPer1, mPer2 and mPer3) of the Drosophila circadian clock gene period (per) have been identified,,,,,,. The mPer genes share a conserved PAS domain (a dimerization domain found in Per, Arnt and Sim) and show a circadian expression pattern in the suprachiasmatic nucleus. To assess the in vivo function of mPer2, we generated and characterized a deletion mutation in the PAS domain of the mouse mPer2 gene. Here we show that mice homozygous for this mutation display a shorter circadian period followed by a loss of circadian rhythmicity in constant darkness. The mutation also diminishes the oscillating expression of both mPer1 and mPer2 in the suprachiasmatic nucleus, indicating that mPer2 may regulate mPer1 in vivo. These data provide evidence that an mPer gene functions in the circadian clock, and define mPer2 as a component of the mammalian circadian oscillator.

PTEN deletion enhances the regenerative ability of adult corticospinal neurons

Despite the essential role of the corticospinal tract (CST) in controlling voluntary movements, successful regeneration of large numbers of injured CST axons beyond a spinal cord lesion has never been achieved. We found that PTEN/mTOR are critical for controlling the regenerative capacity of mouse corticospinal neurons. After development, the regrowth potential of CST axons was lost and this was accompanied by a downregulation of mTOR activity in corticospinal neurons. Axonal injury further diminished neuronal mTOR activity in these neurons. Forced upregulation of mTOR activity in corticospinal neurons by conditional deletion of Pten, a negative regulator of mTOR, enhanced compensatory sprouting of uninjured CST axons and enabled successful regeneration of a cohort of injured CST axons past a spinal cord lesion. Furthermore, these regenerating CST axons possessed the ability to reform synapses in spinal segments distal to the injury. Thus, modulating neuronal intrinsic PTEN/mTOR activity represents a potential therapeutic strategy for promoting axon regeneration and functional repair after adult spinal cord injury.

Long-Distance Growth and Connectivity of Neural Stem Cells after Severe Spinal Cord Injury

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Lack of Enhanced Spinal Regeneration in Nogo-Deficient Mice

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.

mPer1 and mPer2 Are Essential for Normal Resetting of the Circadian Clock

Mammalian Per1 and Per2 genes are involved in the mechanism of the circadian clock and are inducible by light. Alight pulse can evoke a change in the onset of wheel-running activity in mice by shifting the onset of activity to earlier times (phase advance) or later times (phase delays) thereby advancing or delaying the clock (clock resetting). To assess the role of mouse Per (mPer) genes in circadian clock resetting, mice carrying mutant mPer1 or mPer2 genes were tested for responses to a light pulse at ZT 14 and ZT 22, respectively. The authors found that mPer1 mutants did not advance and mPer2 mutants did not delay the clock. They conclude that the mammalian Per genes are not only light-responsive components of the circadian oscillator but also are involved in resetting of the circadian clock.

NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1−/−; Ngr2−/−; Ngr3−/−; which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1−/−; Ngr3−/−), but not Ngr1 and Ngr2 (Ngr1−/−; Ngr2−/−), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1−/−; Ngr3−/− mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

The neurite outgrowth inhibitor Nogo A is involved in autoimmune-mediated demyelination

Inhibitors associated with CNS myelin are thought to be important in the failure of axons to regenerate after spinal cord injury and in other neurodegenerative disorders. Here we show that targeting the CNS-specific inhibitor of neurite outgrowth Nogo A by active immunization blunts clinical signs, demyelination and axonal damage associated with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Mice vaccinated against Nogo A produce Nogo-specific antibodies that block the neurite outgrowth inhibitory activity associated with CNS myelin in vitro. Passive immunization with anti-Nogo IgGs also suppresses EAE. Our results identify Nogo A as an important determinant of the development of EAE and suggest that its blockade may help to maintain and/or to restore the neuronal integrity of the CNS after autoimmune insult in diseases such as MS. Our finding that Nogo A is involved in CNS autoimmune demyelination indicates that this molecule may have a far more complex role than has been previously anticipated.

Assessing Spinal Axon Regeneration and Sprouting in Nogo-, MAG-, and OMgp-Deficient Mice

A central hypothesis for the limited capacity for adult central nervous system (CNS) axons to regenerate is the presence of myelin-derived axon growth inhibitors, the role of which, however, remains poorly understood. We have conducted a comprehensive genetic analysis of the three major myelin inhibitors, Nogo, MAG, and OMgp, in injury-induced axonal growth, including compensatory sprouting of uninjured axons and regeneration of injured axons. While deleting any one inhibitor in mice enhanced sprouting of corticospinal or raphespinal serotonergic axons, there was neither associated behavioral improvement nor a synergistic effect of deleting all three inhibitors. Furthermore, triple-mutant mice failed to exhibit enhanced regeneration of either axonal tract after spinal cord injury. Our data indicate that while Nogo, MAG, and OMgp may modulate axon sprouting, they do not play a central role in CNS axon regeneration failure.