It is well established that the clinical manifestations of FD result from a depletion of neurons in the PNS, yet the steps that go awry to produce this deficit have not been determined. The data presented here are the first in vivo studies that identify the stages of PNS development that require IKAP/Elp1. For example, the FD neuronal deficit in the dorsal root ganglia could result from problems with neural crest cell induction, delamination, migration, and/or differentiation. Our study indicates that neural crest cells are induced, delaminate and migrate normally when transfected with IKBKAP shRNAs. This finding is supported by the fact that IKAP is not expressed in neural crest cells; thus the postulate that FD results from disruptions in neural crest motility and/or migration are not supported by investigation of IKAP's function and expression in vivo reported here. Similarly, our data demonstrate that the disease phenotype does not result from a failure in neuronal differentiation, since peripheral neurons differentiate from IKBKAP-shRNA-transfected neural precursors. Rather, our data provide evidence that the deficit in neuronal numbers that marks FD may result from two causes: (1]) peripheral precursor cells precociously differentiating into neurons and (2) neurons prematurely dying. These findings may shed insight on the pathological basis for the FD phenotype.
Using RNAi to investigate the mechanisms underlying the PNS phenotype in FD is an appropriate and useful strategy given the fact that FD tissues express varying levels of both wild type IKBKAP mRNA and truncated mRNA 
and hence FD cells are not truly “null” for IKAP protein. Thus our in vivo
model is more analogous to the human disease than a knock-out mouse model would be, even a conditional knock-out. This is further supported by the fact that the IKBKAP
knock-out mouse is embryonic lethal, dying by E9 
, [40; Lefcort and Tessarollo, unpublished]
A major finding reported here is that neurons expressing reduced levels of IKAP died prematurely in the DRG; the mechanisms mediating that death need to be elucidated. IKAP-depletion has been shown to increase levels of pro-apoptotic genes such as Bax in colon cancer-derived cells and to alter levels of a number of pro-apoptotic genes in FD stem cells 
and may explain the developmental delay and early death observed in IKAP null mice 
. Yeast two hybrid studies have identified binding of IKAP/Elp1 to BAT-3, an apoptosis regulatory gene 
. Our finding of premature differentiation of precursors followed by precocious death is reminiscent of the premature differentiation and subsequent death of DRG neuronal precursors in the neurotrophin-3 (NT-3) knock out mouse 
Our shRNA data suggest that IKAP plays a role in maintaining neural precursor proliferation and preventing their precocious differentiation into neurons. Decreasing IKBKAP mRNA levels both in vivo
and in vitro
led to an increase in neuronal differentiation, yet IKAP levels only become detectable as neural precursors differentiate. Furthermore, overexpressing the cyto-domain of IKAP reduced neuronal differentiation. Without more structure/function studies we can not distinguish whether the ectopically expressed truncated c-terminal IKAP protein acted as a dominant negative or as a gain of function. Johansen et al  showed that overexpression of the N-terminal half of IKAP, either in the presence or absence of the endogenous full length IKAP caused a defect in cerebellar neuronal migration in vitro
. Holmberg et al  showed that overexpression of the c-terminal half of IKAP was sufficient to cause activation of the critical signaling enzyme, JNK. Interestingly, Johansen et al also showed that in IKAP-depleted cells in vitro
, ectopic expression of the N-terminal half of IKAP could not rescue the effects of IKAP depletion. Furthermore, in the IKAP null mouse, Chen et al.  found that overexpression of the N-terminal half of IKAP could not rescue the grave early embryonic lethality induced by total loss of IKAP in mouse.nor could generation of a mouse in which only exon 20 was deleted 
. These last experiments indicate that key functions for IKAP must reside in its C-terminal half, hence the rationale for our experiment. Furthermore, these data would suggest that the level of IKAP is critical and that small alterations in the amount of IKAP in a PNS precursor cell can shift the behavior of that cell between proliferation vs. differentiation. Unfortunately there are no robust markers of chick DRG precursor cells, but our data would suggest that IKAP must begin to be expressed as cells exit the cell cycle and acts as a “break” to prevent precocious differentiation. Decreases in Elp1 in Arabidopsis thaliana
has been shown to cause a decrease in proliferation 
, and FD cells have been demonstrated to be in cell cycle arrest, an effect released by phophatidylserine 
. IKAP-dependent genes include such cell cycle mediators as p57kip and thymidilate synthetase 
. Since the cytoskeleton and cell polarity proteins are so integral to both cell division and cell motility, its not surprising, given the accruing evidence for a role for IKAP in cell polarity, that we found alterations in cell proliferation and cell polarity with disruptions in IKBKAP. As published in previous studies, we too found fewer paxilin-positive focal adhesions with IKAP reduction 
(data not shown). Since paxillin has been shown to mediate the localization of both Crk and syntaxin-2 in the midbody during cytokinesis, IKAP-regulated paxillin might be required for normal cytokinesis 
Our data suggest that IKAP/Elp1 participates in the organization or remodeling of the cytoskeleton during cytokinesis and in branching from somata and axons. ShRNA knock-down of IKBKAP mRNA in fibroblasts also induced an increase in branching from cell bodies 
. Since DRG neurons do not normally extend dendrites, perhaps a normal function for IKAP/Elp1 is to suppress branching in the PNS.
RNAi for IKAP in neuroblastoma cells and in fibroblasts caused the disorganization of microtubules 
. IKAP has also been shown to bind to Filamin A, a protein that organizes the actin cytoskeleton and interacts with cell adhesion pathways 
. Interestingly, X-Linked Periventricular Heterotopia is due to mutations in Filamin A and is marked by an aggregation of neurons along the surface of the ventricular zone. In our IKBKAP shRNA treated spinal cords, we saw an aberrant evagination of cells into the lumen of the neural tube which went on to express neuronal markers – an observation also seen in embryos in which the neural tubes overexpress cadherins 
. IKAP/Elp1 is expressed in the ventricular zone both in the brain 
and in the spinal cord. Thus the integration of signaling between IKAP, filamin A and cadherins may be essential for orchestrating the reorganization of cytoskeleton during the transition from cell cycle withdrawal to neuronal differentiation in the CNS ventricular zone.
There has been a recent surge in interest in interactions between IKAP/Elp1 and Elp3 with evidence that Elp3 mediates histone H3 acetylation, and most recently, acetylation of α-tubulin 
. Acetylation of microtubules leads to stabilization and recruitment of motor proteins that enable transport of proteins and other cellular components 
. Work in c.elegans showed that decreasing Elp1 reduced the velocity of dense core vesicle movement along axons by regulating microtubule acetylation 
. In our study and also in 
we did not find any reductions in tubulin acetylation in the PNS [data not shown], which perhaps points to differences in the function of IKAP/Elp1 in the PNS vs. CNS, and/or to differences in Elp1 and Elp3 interactions in the PNS vs. the CNS.
This study is the first to directly address whether FD results from a problem in cell motility, a hypothesis which is currently popular [e.g. 11]
. Our data do not support a role for IKAP/Elp1 in the motility and migration of neural crest cells, a result not surprising given that we show here that IKAP only becomes detectable as neurons begin to differentiate in the DRG. Our data suggest that IKAP coordinates the intracellular machinery required for the transition from neural precursors to neurons and in the differentiation and survival of neurons in the PNS. Further elucidation of the molecular interactions and intracellular pathways in which IKAP participates will be required to fully understand the cellular mechanisms that result in FD.