Nubp2 Is Required for Cranial Neural Crest Survival in the Mouse. 1

bioRxiv preprint doi: https://doi.org/10.1101/656652; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Nubp2 is required for cranial neural crest survival in the mouse. 2 Andrew DiStasio1, David Paulding1, Praneet Chatuverdi2, Rolf W. Stottmann1,2,3 # 3 1Divisions of Human Genetics and 2Developmental Biology, Cincinnati Children’s Hospital Medical Center, 4 3Department of Pediatrics, University of Cincinnati, Cincinnati, OH, 45229, USA. 5 6 # Author for correspondence 7 [email protected] 8 9 Keywords: 10 Nubp2, neural crest, ENU mutagenesis, cilia 11 12 13 Summary Statement 14 An ENU screen identifies a novel allele of Nubp2 which is then demonstrated to be required for cranial 15 neural crest survival and proper midfacial development. 16 17 18 19 20 21 bioRxiv preprint doi: https://doi.org/10.1101/656652; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Abstract 23 The N-ethyl-N-nitrosourea (ENU) forward genetic screen is a useful tool for the unbiased discovery of 24 novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a 25 screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy 26 embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, 27 including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense 28 mutation in Nucleotide Binding Protein 2 (Nubp2) to be causative. This finding was confirmed with a 29 complementation analysis between the dorothy allele and a Nubp2 null allele (Nubp2Null). We demonstrate 30 that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the Cytosolic Iron/Sulfur 31 cluster Assembly (CIA) pathway and in the negative regulation of ciliogenesis. Conditional ablation of 32 Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using 33 this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, 34 and that markers of the Shh, Fgf, and Bmp signaling pathways are unaltered. Finally, we show that the 35 phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme. 36 37 38 bioRxiv preprint doi: https://doi.org/10.1101/656652; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 39 Introduction 40 Vertebrate craniofacial development is a dynamic process highly dependent on a fascinating cell 41 population known as the neural crest. Craniofacial neural crest cells (CNCC) are a multipotent and 42 migratory cell type which delaminate from the dorsal aspect of the closing neural tube and travel 43 ventrolaterally into the frontonasal process (FNP) and pharyngeal arches (PAs) where they become the 44 mesenchyme of the emerging facial primordia [1, 2]. On each side of the mouse face, proliferation of the 45 post-migratory neural crest causes medial and lateral nasal processes (MNP, LNP) to swell from the FNP 46 around embryonic day (E) 10, resulting in paired horseshoe-shaped outgrowths with visible depressed 47 nasal pits in the center. The outgrowth of these processes is coupled with that of the maxillary processes 48 (MxPs) to bring the three primordia into contact [3, 4]. This contact and the subsequent fusion of the MNP, 49 LNP, and MxP create the nostrils and upper lip [5]. As development continues past E10.5, further growth 50 brings the paired MNPs into contact at the midface, where they fuse and give rise to the nasal septum, 51 philtrum, and premaxilla. 52 During these early stages of craniofacial development, the CNCC rely on signaling cues from 53 surrounding tissues to regulate their survival and the proliferation required to properly expand the facial 54 primordia. Sonic Hedgehog (SHH) and Fibroblast Growth Factor 8 (FGF8) secreted by the surface 55 ectoderm maintain CNCC survival and induce their proliferation [6-9]. Loss of these signals, or an inability 56 of CNCC to properly transduce ligand binding within the cell, impairs the outgrowth and subsequent fusion 57 of the facial primordia, often ultimately leading to midline defects of the face [10]. The CNCC are also a 58 particularly vulnerable cell population as evidenced by their marked susceptibility to ribosome biogenesis 59 insults, sensitivity to the teratogenic effects of alcohol and many examples of genetic mutations leading to 60 elevated CNCC apoptosis [11-14] [15-17]. 61 Despite significant advances in understanding the signaling pathways and transcriptional networks 62 involved in craniofacial developmental disorders, their etiology remains unclear in many cases. Unbiased 63 forward genetics approaches in model organisms allow discovery of previously unexamined roles for genes 64 and pathways in embryogenesis, providing new insights and angles for investigation [18]. Here we report 65 on a new allele from an N-ethyl-N-nitrosourea (ENU) screen in the mouse that is integral to craniofacial bioRxiv preprint doi: https://doi.org/10.1101/656652; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 66 development. We found the causal mutation to be in the gene nucleotide binding protein 2 (Nubp2). Nubp2 67 is a highly conserved P-loop NTPase first described in early sequencing and mapping of cDNA isolated 68 from E7.5 mouse embryos [19]. In Saccharomyces cerevisiae it was identified as cytosolic Fe-S cluster 69 deficient (Cfd1) in a screen for genes able to convert Iron regulatory protein 1 into c-aconitase [20] [21]. 70 These and subsequent studies in yeast and animal cell lines established that Nubp2 is an integral 71 component of the cytosolic iron-Sulphur (Fe-S) cluster assembly (CIA) pathway where it acts along with 72 its homologous binding partner Nubp1 as a scaffold for transfer of the Fe-S cofactor to non-mitochondrial 73 apoproteins [22-24]. Later studies revealed that knockdown of Nubp1 alone, or of both Nubp1 and Nubp2, 74 led to excessive centrosome duplication in vitro [25]. Further work indicated that silencing of Nubp1 and 75 Nubp2 in cell culture led to supernumerary cilia formation [26]. An ENU screen isolated a mouse allele of 76 Nubp1 with interrupted lung budding and centriole over-duplication [27]. 77 This implication of Nubp2 in regulation of centriole duplication and ciliary dynamics, and the 78 importance of the primary cilia to intercellular signaling, led us to hypothesize dysregulation of ciliogenesis 79 would lead to interruptions in crucial CNCC signaling pathways, resulting in loss of mitogenic and/or 80 survival signals and subsequent midfacial fusion defects. On the contrary, here we demonstrate that loss 81 of Nubp2 from the CNCC does not lead to ciliogenesis or signaling defects, but causes a rapid onset of 82 apoptosis throughout the craniofacial mesenchyme following CNCC migration which underlies the 83 distinctive craniofacial presentation of the Dorothy mutant. This represents the first example of a role for 84 CIA pathway components in midfacial development. 85 Materials and Methods 86 Animal husbandry 87 All animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals 88 (National Institutes of Health), and all animal-associated activity was approved by the Institutional Animal Care and 89 Use Committee of the Cincinnati Children’s Hospital Medical Center (Protocol # IACUC2016-0098). All animals were 90 monitored daily by registered veterinary technicians and additional care provided where necessary. Euthanasia was 91 performed via Isoflurane sedation followed by dislocation of the cervical vertebrae and secondary euthanasia. Mice 92 were housed in ventilated cages provided with automated water supply and Purina 5010 Laboratory Rodent Chow bioRxiv preprint doi: https://doi.org/10.1101/656652; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 ad libitum. During timed mating, noon of the day a copulation plug was detected was considered embryonic day (E) 94 0.5 (E0.5). 95 ENU mutagenesis 96 ENU mutagenesis was performed as previously published [18]. Wildtype C57BL/6J (B6; Jackson Laboratory, Bar 97 Harbor ME) 6- to 8-week-old males were given a fractionated dose of ENU (three weekly dosages of 90-100 mg/kg 98 ENU, Sigma). Mutagenized males were bred with Tg(Etv1-EGFP)B2192 GSat females (FVB/N TaC background) as part 99 of a larger effort to identify potential mutants in cortical neuron patterning [28]. Following a three-generation 100 backcross breeding scheme [18], G3 litters were dissected at late embryonic stages to detect gross developmental 101 anomalies. The allele was propagated with test crosses onto the FVB background (to facilitate mapping if needed) 102 until the causal allele was identified. 103 Variant detection 104 Exome sequencing was carried out in the CCHMC Genetic Variation and Gene Discovery Core Facility (Cincinnati, 105 OH, USA). An exome library was prepared from genomic DNA using the NimbleGen EZ Exome V2 kit (Madison, WI, 106 USA). Sequencing of the resulting exome library was done with an Illumina Hi Seq 2000 (San Diego, CA, USA). The 107 Broad Institute’s GATK algorithm was used for alignment and variant detection. Variants were then filtered as 108 described in Table 1. Mutations were validated by Sanger sequencing. 109 Alleles and genotyping 110 Nubp2Dor (Nubp2 c.626T>A; Nubp2V209D; dorothy) was discovered in an ENU mutagenesis screen as described above 111 and maintained on a mixed background.

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