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

Retina-specific loss of Ikbkap/Elp1 causes mitochondrial dysfunction that leads to selective retinal ganglion cell degeneration in a mouse model of familial dysautonomia

Familial dysautonomia (FD) is an autosomal recessive disorder marked by developmental and progressive neuropathies. It is caused by an intronic point-mutation in the IKBKAP/ELP1 gene, which encodes the inhibitor of κB kinase complex-associated protein (IKAP, also called ELP1), a component of the elongator complex. Owing to variation in tissue-specific splicing, the mutation primarily affects the nervous system. One of the most debilitating hallmarks of FD that affects patients' quality of life is progressive blindness. To determine the pathophysiological mechanisms that are triggered by the absence of IKAP in the retina, we generated retina-specific Ikbkap conditional knockout (CKO) mice using Pax6-Cre, which abolished Ikbkap expression in all cell types of the retina. Although sensory and autonomic neuropathies in FD are known to be developmental in origin, the loss of IKAP in the retina did not affect its development, demonstrating that IKAP is not required for retinal development. The loss of IKAP caused progressive degeneration of retinal ganglion cells (RGCs) by 1 month of age. Mitochondrial membrane integrity was breached in RGCs, and later in other retinal neurons. In Ikbkap CKO retinas, mitochondria were depolarized, and complex I function and ATP were significantly reduced. Although mitochondrial impairment was detected in all Ikbkap-deficient retinal neurons, RGCs were the only cell type to degenerate; the survival of other retinal neurons was unaffected. This retina-specific FD model is a useful in vivo model for testing potential therapeutics for mitigating blindness in FD. Moreover, our data indicate that RGCs and mitochondria are promising targets

Nociceptive sensory neurons derive from contralaterally migrating, fate-restricted neural crest cells

Neural crest cells (NCCs) are a transient population of multipotent progenitors that give rise to numerous cell types in the embryo. An unresolved issue is the degree to which the fate of NCCs is specified prior to their emigration from the neural tube. In chick embryos, we identified a subpopulation of NCCs that, upon delamination, crossed the dorsal midline to colonize spatially discrete regions of the contralateral dorsal root ganglia (DRG), where they later gave rise to nearly half of the nociceptor sensory neuron population. Our data indicate that before emigration, this NCC subset is phenotypically distinct, with an intrinsic lineage potential that differs from its temporally synchronized, but ipsilaterally migrating, cohort. These findings not only identify a major source of progenitor cells for the pain- and temperature-sensing afferents, but also reveal a previously unknown migratory pathway for sensory-fated NCCs that requires the capacity to cross the embryonic midline

Loss of Elp1 disrupts trigeminal ganglion neurodevelopment in a model of familial dysautonomia

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Familial dysautonomia (FD) is a sensory and autonomic neuropathy caused by mutations in elongator complex protein 1 (ELP1). FD patients have small trigeminal nerves and impaired facial pain and temperature perception. These signals are relayed by nociceptive neurons in the trigeminal ganglion, a structure that is composed of both neural crest- and placode-derived cells. Mice lacking Elp1 in neural crest derivatives (‘Elp1 CKO’) are born with small trigeminal ganglia, suggesting Elp1 is important for trigeminal ganglion development, yet the function of Elp1 in this context is unknown. We demonstrate that Elp1, expressed in both neural crest- and placode-derived neurons, is not required for initial trigeminal ganglion formation. However, Elp1 CKO trigeminal neurons exhibit abnormal axon outgrowth and deficient target innervation. Developing nociceptors expressing the receptor TrkA undergo early apoptosis in Elp1 CKO, while TrkB- and TrkC-expressing neurons are spared, indicating Elp1 supports the target innervation and survival of trigeminal nociceptors. Furthermore, we demonstrate that specific TrkA deficits in the Elp1 CKO trigeminal ganglion reflect the neural crest lineage of most TrkA neurons versus the placodal lineage of most TrkB and TrkC neurons. Altogether, these findings explain defects in cranial gangliogenesis that may lead to loss of facial pain and temperature sensation in FD.https://doi.org/10.7554/eLife.7145

Dynamic Expression of Neurotrophin Receptors during Sensory Neuron Genesis and Differentiation

AbstractTo identify potential functions for neurotrophins during sensory neuron genesis and differentiation, we determined the temporal and spatial protein expression patterns of neurotrophin receptors throughout the process of sensory neurogenesis in the dorsal root ganglia (DRG). We show that neurotrophin receptors are expressed early, being first detected on subsets of migrating neural crest cells, and that trkC is among the earliest markers of neural lineage specification. In the immature DRG, we find that both trkC and p75NTR are expressed on subsets of dividing progenitor cells in vivo. Furthermore, our data directly reveal distinct patterns of trk receptor expression by individual sensory neurons from the time of their inception with all early arising cells initially being trkC+, some subsets of whom also coexpress either trkA or trkB or both. As sensory neurons innervate their targets and establish their mature identities, the spectrum of trk receptors expressed by individual neurons is altered. The stereotyped trk receptor expression profiles identified here may potentially correspond to distinct lineages of sensory neurons. These data, in conjunction with other studies, argue for multiple functions for neurotrophins during the process of sensory neuron differentiation, including effects on both neural crest and DRG mitotically active progenitor cells, in addition to possibly influencing the establishment of sensory neuron identity

Imaging Cell Migration in Chick Explant Cultures

Migratory cells can travel long distances within the embryo to reach peripheral structures. How cells arrive at precise targets remains unclear, largely because of limitations in the ability to resolve how cells respond to microenvironmental signals and communicate positional information to each other. The avian embryo is a good model system for studying cell migration because the embryos are accessible to tissue transplantation, molecular perturbation, cell labeling, and culture outside of the eg. In particular, several clever culture methods have been developed that allow cell migratory behavior to be analyzed in the intact avian embryo or in tissue explants using optical time-lapse microscopy. Furthermore, advances in cell labeling of avian embryos, using either photoactivation or electroporation of green fluorescent protein (GFP) or GFP variants, allow targeted marking of cell subpopulations at discrete points in development. The combination of new culture methods and targeted cell labeling has significantly enhanced the ability to observe and perturb normal cell movements during important early developmental events, such as cranial neural crest migration, gastrulation, and vasculogenesis. This chapter describes a chick explant culture technique that allows visualization of deep cell movements with single cell resolution in older embryos. This technique provides a means to visualize cell migration both along dorsoventral migratory pathways and along the rostrocaudal axis. It also maintains cell resolution without disruptions caused by vibration from the heartbeat