Familial dysautonomia model reveals Ikbkap deletion + causes apoptosis of Pax3 progenitors and peripheral neurons Lynn Georgea,b,1, Marta Chaverraa,1, Lindsey Wolfea, Julian Thornea, Mattheson Close-Davisa, Amy Eibsa, Vickie Riojasa, Andrea Grindelandc, Miranda Orrc, George A. Carlsonc, and Frances Lefcorta,2 aDepartment of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717; bDepartment of Biological and Physical Sciences, Montana State University Billings, Billings, MT 59101; and cMcLaughlin Research Institute, Great Falls, MT 59405 Edited by Qiufu Ma, Dana-Farber Cancer Institute, Boston, MA, and accepted by the Editorial Board October 7, 2013 (received for review May 8, 2013) Familial dysautonomia (FD) is a devastating developmental and thermoreceptors, mechanoreceptors and proprioceptors. With progressive peripheral neuropathy caused by a mutation in the gene the completion of neural crest migration, multiple steps ensue inhibitor of kappa B kinase complex-associated protein (IKBKAP). that are essential for normal PNS development, including pro- To identify the cellular and molecular mechanisms that cause FD, liferation of discrete sets of neuronal progenitor cells that derive we generated mice in which Ikbkap expression is ablated in the from different waves of migrating neural crest cells, neuronal peripheral nervous system and identify the steps in peripheral differentiation, axonogenesis, target innervation, and circuit nervous system development that are Ikbkap-dependent. We formation. FD could theoretically result from failure in any or show that Ikbkap is not required for trunk neural crest migration several of these key developmental processes. or pathfinding, nor for the formation of dorsal root or sympathetic Insight into the mechanisms that cause FD have been com- ganglia, or the adrenal medulla. Instead, Ikbkap is essential for the plicated by data that implicate functions for IKAP in both the second wave of neurogenesis during which the majority of tropo- nucleus and the cytoplasm. In yeast, the IKAP homolog, Elp1, + myosin-related kinase A (TrkA ) nociceptors and thermoreceptors serves as a scaffold protein within the multisubunit Elongator arise. In its absence, approximately half the normal complement of complex that binds RNA polymerase II and facilitates tran- + – TrkA neurons are lost, which we show is partly due to p53-medi- scription via histone acetylation (7 9). Although studies indicate that Elongator also functions in the cytoplasm to acetylate ated premature differentiation and death of mitotically-active pro- α fi genitors that express the paired-box gene Pax3 and give rise to the -tubulin (10, 11), recent ndings suggest that Elongator may + regulate tubulin acetylation indirectly through tRNA modification majority of TrkA neurons. By the end of sensory development, the – number of TrkC neurons is significantly increased, which may result (12 15). Independent of its role in the Elongator complex, cyto- + from an increase in Runx3 cells. Furthermore, our data demon- solic IKAP has also been shown to regulate actin cytoskeletal strate that TrkA+ (but not TrkC+) sensory and sympathetic neurons organization and cell migration, which has prompted the sugges- tion that FD results from a failure in neural crest cell migration undergo exacerbated Caspase 3-mediated programmed cell death in (16–19). Mice that are completely null for Ikbkap die early in the absence of Ikbkap and that this death is not due to a reduction embryogenesis [by embryonic day (E) 10.5] with failure in neu- in nerve growth factor synthesis. In summary, these data suggest rulation and vasculogenesis, precluding their usefulness for ana- that FD does not result from a failure in trunk neural crest migra- lyzing the aspects of PNS development that are most impacted in tion, but rather from a critical function for Ikbkap in TrkA progen- + FD (20, 21). Although a mouse hypomorphic Ikbkap model was itors and TrkA neurons. recently generated that recapitulates many of the phenotypic ereditary sensory and autonomic neuropathies (HSANs) are Significance Ha group of five phenotypically diverse but overlapping dis- orders of the peripheral nervous system (PNS) that result from Familial dysautonomia (FD) is a devastating developmental pe- mutations in 12 distinct genes (1). HSAN type 3, or familial dys- ripheral autonomic and sensory neuropathy caused by a muta- autonomia (FD) (also called Riley–Day syndrome), results from tion in the gene inhibitor of kappa B kinase complex-associated an intronic mutation (IVS20 + 6T > C; 99.5% of patients) in protein (IKBKAP). It is marked by tachycardia, blood pressure a gene called inhibitor of kappa B kinase complex-associated lability, autonomic vomiting “crises,” and decreased pain and protein or IKBKAP, causing mis-splicing and subsequent tissue- temperature sensation. FD is progressive, and affected individ- specific reductions in IKAP protein (2, 3). FD is marked by “ ” uals commonly die during early adulthood. To identify the cel- tachycardia, blood pressure lability, autonomic vomiting crises, lular and molecular mechanisms that cause FD, we generated decreased pain and temperature sensation, and commonly death a mouse model for the disease in which Ikbkap expression is during early adulthood (4). The function of IKAP in the nervous ablated in the neural crest lineage. This study is a mechanistic system is unclear, nor is it understood why deletions in this broadly analysis of the cellular events that go awry in the developing expressed gene primarily devastate the PNS. The earliest pathol- peripheral nervous system in FD and identifies essential func- ogy study, performed on a 2-y-old child with FD, showed that tions of IKAP protein in the peripheral nervous system. ∼90% of cells in the dorsal root and sympathetic ganglia (SG) were missing (5). To identify IKAP’s function in the developing Author contributions: L.G., M.C., M.O., G.A.C., and F.L. designed research; L.G., M.C., L.W., PNS, we first need to establish the steps in which it is essential. J.T., M.C.-D., A.E., V.R., and A.G. performed research; L.G. and G.A.C. contributed new The vertebrate PNS derives primarily from the neural crest, reagents/analytic tools; L.G., M.C., L.W., G.A.C., and F.L. analyzed data; and L.G. and F.L. a multipotent, heterogeneous cell population that delaminates wrote the paper. from the neural tube and migrates throughout the embryo (6). The authors declare no conflict of interest. Those neural crest cells that stop laterally to the neural tube give This article is a PNAS Direct Submission. Q.M. is a guest editor invited by the Editorial Board. rise to the chain of sensory dorsal root ganglia (DRG), whereas 1L.G. and M.C. contributed equally to this study. those that migrate further ventrally give rise to the vertebral 2To whom correspondence should be addressed. E-mail: [email protected]. chain of SG. Within the DRG, neural crest cells generate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. heterogeneous neuronal subpopulations including nociceptors, 1073/pnas.1308596110/-/DCSupplemental. 18698–18703 | PNAS | November 12, 2013 | vol. 110 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1308596110 Downloaded by guest on September 29, 2021 hallmarks of FD (22), the data presented here comprise a mech- was reduced by nearly 70% in Ikbkap CKO embryos compared anistic analysis of the molecular and cellular events that go awry with controls (Fig. 2 A–C). The total number of DRG neurons in during sensory neurogenesis in the absence of Ikbkap. CKO mice was also reduced by one-third compared with controls (Fig. 2 D and E and Table S1). To determine whether pro- Results grammed cell death contributed to this reduction in neuronal + Generation of a Neural Crest-Specific Mouse Model for FD. To de- number, we quantified the number of cleaved-Caspase 3 cells in termine the steps in PNS development that fail in FD, we used both the SCG [wild type (WT), 6.8 ± 2.20; CKO, 13.3 ± 3.70; P < a Wnt1-Cre transgene to generate mice in which Ikbkap is se- 0.001] and DRG (Table S1) at E17.5 and found a significantly lectively deleted in the neural crest lineage and in discrete increased number of apoptotic cells in both ganglia types in CKO regions of the CNS (Fig. 1 A and B) (23). Mice homozygous for embryos relative to controls. a floxed allele of Ikbkap were crossed to mice heterozygous for We next determined whether neuronal deficits in Ikbkap CKO both the floxed Ikbkap allele and for a Wnt1-Cre transgene (Fig. mice were specific to particular subpopulations of DRG neurons, S1). Ikbkap conditional knockout (CKO) embryos, which ex- because a hallmark of FD is decreased pain and temperature pressed Wnt1-Cre and were homozygous for the IkbkapLoxP allele sensation. Pain- and temperature-receptive neurons express the (Wnt1-Cre;IkbkapLoxP/LoxP), were generated at the expected 3-to-1 neurotrophin receptor TrkA, whereas proprioceptors and many Mendelian ratio (255 expressing Ikbkap to 83 CKO; P > 0.95), as mechanoreceptors express TrkC (26, 27). Our data demonstrate determined by PCR-based genotyping (Fig. S1 and SI Materials that the TrkA subpopulation was reduced by one half at E17.5 and Methods). Decreased expression of IKAP protein in CKO (Fig. 2 F and G and Table S1). Nociceptors synthesize and re- embryos was confirmed via Western blot and immunostaining for lease the neuropeptide substance P and autopsy studies on FD IKAP in the DRG (Fig. 1 C–E). Although CKO pups were born patients show a severe reduction in substance P staining in the alive, they died within 24 h of birth. This early death was not at- spinal cord dorsal horn (28). To further determine the fate of the tributable to an inability to suckle as they were often found with nociceptive subpopulation of DRG neurons in the absence of milk in their stomachs.