IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems

IKBKAP shRNAs reduce levels of IKAP protein and IKBKAP mRNA <i>in ovo</i>.

<p>(A) Schematic combines the known partial chicken IKBKAP sequence with the human IKBKAP sequence and indicates the regions of chicken IKBKAP to which shRNAs were designed and the regions used as the immunogens for generation of both antibodies (P1D8 and Santa Cruz H301). The cytoplasmic terminus of the chicken IKAP was used for the CT-IKAP His construct overexpression experiments. QPCR indicated that approximately 50% of the IKBKAP mRNA was reduced following a 24 hr transfection <i>in ovo</i> with the IKBKAP shRNAs, when compared to control levels of IKBKAP (B). In situ hybridization following IKBKAP shRNAs transfection <i>in ovo</i> also showed reduced levels of IKBKAP mRNA (C) in the transfected half of the spinal cord (arrow); sense probe (D). Transfection <i>in ovo</i> with IKBKAP shRNAs reduced levels of IKAP protein (E–H) on the transfected side only (compare the control half to the EGFP+ half of the spinal cord).</p

Reductions in IKAP decreases proliferation of progenitors and increases cell death in the developing neural tube.

<p>Embryos were transfected/electroporated with IKBKAP shRNAs (A,B) or control shRNAs (C,D) at St. 13 and fixed at St. 23 (ca 48 hrs). Reductions in IKAP increase the death of CNS progenitors as indicated by cleaved-caspase 3+ (A–D; vertical line indicates the midline). (E–H) The number of pH3+ cells was determined in the ventricular zone on the transfected and control sides of each embryo and a ratio for each embryo obtained. The graph depicts those ratios from embryos transfected with either control shRNA or IKBKAP shRNA or IKBKAP shRNA and a mouse IKBKAP cDNA. There are significantly fewer pH3+ cells in IKBKAP shRNA transfected embryos (n = 4 embryos, 3300 cells counted) than in control shRNA transfected embryos (n = 3 embryos, 4,331 cells counted; p = 0.004), an effect that is rescued (red bar) with co-transfection with a plasmid encoding full length mouse IKBKAP (E; n = 3 embryos, 2153 cells counted; p = 0.005 by ANOVA). There is no significant difference between the number of pH3+ cells in the rescued embryos vs. the control shRNA transfected embryos (p = 0.103).</p

Reductions in IKAP increase neuronal branching and perturb cell separation.

<p>DRG were dissociated, transfected with IKBKAP shRNA or control scrambled shRNA and cultured for 24 hrs. (A–F). Reductions in IKAP increased the number of neurons in cultures from immature (E5) DRG. (G–L) Reductions in IKBKAP increased branching from neuronal somata/cell bodies (G,H, p = 0.013) and resulted in incomplete cytokinesis (I–L) or separation of nascent sister neurons <i>in vitro</i> (p = 0.01; G–L; 4 experiments, n = 135 control shRNA transfected neurons; n = 116 IKBKAP shRNA transfected neurons) and <i>in vivo</i> (M, N; St. 25).</p

IKAP is expressed in PNS and CNS neurons, but not in neural crest cells.

<p>Chick embryo sections were immunolabeled with commercial anti-IKAP antibody (Santa Cruz H302; red, A–I and with either HNK-1 (A; to label neural crest cells, green) or Tuj-1 (to label neurons; B). IKAP protein (red, A–I) is not expressed in HNK+ neural crest cells (green, A), but is expressed in nascent neurons in the DRG anlagen (A–C, E, arrow) and neural tube (B–C, E; arrowhead), and its expression is maintained through the period of programmed cell death (G–I). IKAP is also expressed in progenitor cells in the ventricular zone (D,J open arrowheads) and in motor neurons (arrowhead) and axons (arrow) (F–H,J,K). IKAP is also strongly expressed on the pre-ganglionic Column of Terni cells (arrow, H). In situ hybridization for IKBKAP confirms the protein expression pattern (J,K).</p

Confirmation of IKAP expression with an anti-chick IKAP antibody.

<p>(A) Generation of a monoclonal antibody to chicken IKAP, P1D8, confirms the expression pattern obtained with anti-human IKAP commercial antibody (H302). Both antibodies demonstrate that the majority of IKAP is in the cytoplasm, primarily perinuclear, and in axons (A, C–E) both <i>in vivo</i> (A, C) and in DRG <i>in vitro</i> (D,E). Incubation with the peptide immunogen used to generate P1D8 completely blocks labeling (compare B to C). Both IKAP antibodies label a band of approximately 140 kDa in lysates of chick embryo spinal cord (F).</p

Reductions in IKAP alter neural tube development and induce ectopic branching of motor axons.

<p>All transfected cells (control shRNA transfected embryos in A, H, I; IKBKAP shRNA in B–G) are EGFP+; right side of spinal cord is transfected in all embryos (asterisk). (A) control shRNA transfected spinal cord develops normally in contrast to (B–D) IKBKAP shRNA transfected spinal cords in which cells at the ventricular zone (VZ) often evaginate into the neural tube lumen rather than migrate laterally away from the VZ as in (A). IKBKAP shRNA-transfected evaginated cells typically express neural markers such as Ben (C: box in B contains the Ben+ evaginated neurons in the VZ that are EGFP+ and magnified in C) and Islet1/2 (D). Spinal interneuron populations are reduced in IKBKAP shRNA-transfected spinal cords including Engrailed+ cells (E) and the dorsal population of Islet1+ cells – compare the left, non-transfected side of the spinal cords to the right, transfected sides (*asterisk) of the spinal cord. (F, I) The ratio of islet 1+ dorsal interneurons on the transfected vs. non-transfected sides of the spinal cords was determined in multiple sections from 2 control shRNA transfected embryos (n = 263 Islet+ cells for the control shRNA transfected side vs. n = 252 Islet+ cells for the non-transfected side) and in 4 IKBKAP shRNA transfected embryos (n = 356 Islet+ cells for the transfected side of the IKBKAP shRNA spinal cord vs. n = 658 Islet+ cells for the non-transfected sides of the IKBKAP shRNA transfected embryos); p = 0.006. (G, H) IKBKAP shRNA transfected motor axons branch abnormally (arrow in G) as they exit the neural tube in the ventral root (compare H to I).; vertical line indicates spinal cord midline.</p