bioRxiv preprint doi: https://doi.org/10.1101/761064; this version posted September 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 ADNP promotes neural differentiation by modulating Wnt/β-catenin 2 signaling 3 Xiaoyun Sun1, Xixia Peng1, Yuqing Cao1, Yan Zhou2 & Yuhua Sun1* 4 5 Abstract 6 ADNP (Activity Dependent Neuroprotective Protein) is proposed as a neuroprotective 7 protein whose aberrant expression has been frequently linked to neural developmental 8 disorders, including the Helsmoortel-Van der Aa syndrome. However, its role in 9 neural development and pathology remains unclear. Using mESC (mouse embryonic 10 stem cell) directional neural differentiation as a model, we show that ADNP is 11 required for ESC neural induction and neuronal differentiation by maintaining Wnt 12 signaling. Mechanistically, ADNP functions to maintain the proper protein levels of 13 β-Catenin through binding to its armadillo domain which prevents its association with 14 key components of the degradation complex: Axin and APC. Loss of ADNP promotes 15 the formation of the degradation complex and hyperphosphorylation of β-Catenin by 16 GSK3β and subsequent degradation via ubiquitin-proteasome pathway, resulting in 17 down-regulation of key neuroectoderm developmental genes. We further show that 18 ADNP plays key role in cerebellar neuron formation. Finally, adnp gene disruption in 19 zebrafish embryos recapitulates key features of the mouse phenotype, including the 20 reduced Wnt signaling, defective embryonic cerebral neuron formation and the 21 massive neuron death. Thus, our work provides important insights into the role of 22 ADNP in neural development and the pathology of the Helsmoortel-Van der Aa 23 syndrome caused by ADNP gene mutation. 24 bioRxiv preprint doi: https://doi.org/10.1101/761064; this version posted September 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 25 Introduction 26 ADNP was first described as a neural protective protein and has been implicated in 27 various neural developmental disorders and cancers1. This protein contains nine zinc 28 fingers, a homeobox domain and a PxLxP motif, suggesting that it functions as a 29 transcription factor. The exact roles of ADNP remain unclear, but an increasing 30 number of studies have shown that it functions as a key chromatin regulator by 31 interacting with chromatin remodelers or regulators. For instances, ADNP interacts 32 with core sub-units of SWI/SNF chromatin remodeling complex such as BRG1 and 33 BAF2502. By association with the chromatin regulator HP1, ADNP localizes to 34 pericentromeric heterochromatin regions where it silences major satellite repeat 35 elements3. Recently, ADNP was shown to form a triplex with CHD4 and HP1 to 36 control the expression of lineage specifying genes in embryonic stem cells4. 37 Mouse Adnp mRNA is abundantly expressed during early gestation and reaches 38 its maximum expression on E9.5. Adnp-/- mice are early embryonic lethal, displaying 39 severe defects in neural tube closure and brain formation5. Of note, E8.5 Adnp mutant 40 embryos exhibited ectopic expression of pluripotency gene Pou5f1 and reduced 41 expression of neural developmental gene Pax6 in the anterior neural plate. These 42 observations strongly suggested that ADNP plays essential roles during neurogenesis 43 of mouse embryos. 44 De novo mutations in ADNP gene have recently been linked to neural 45 developmental disorders, including the Helsmoortel-Van der Aa syndrome6. Patients 46 display multiple symptoms, usually manifesting in early childhood with features such 47 as intellectual disability, facial dysmorphism, motor dysfunction and developmental bioRxiv preprint doi: https://doi.org/10.1101/761064; this version posted September 9, 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. 48 delay, which share many features with another neural developmental disorder: autism 49 spectrum disorder (ASD). In fact, ADNP has been proposed as one of the most 50 frequent ASD-associated genes as it is mutated in at least 0.17% of ASD cases7. In a 51 mouse model, haploinsufficiency of Adnp leads to defective neuronal/glial 52 formation and compromised cognitive function8. Due to the significant relevance of 53 ADNP to neural developmental disorders, it is of great interests and importance in the 54 field to elucidate the pathogenic mechanism by ADNP gene mutation6. 55 In this work, we hypothesized that loss of ADNP leads to neural developmental 56 defects. We took use of ES cell directional neural differentiation as a model system to 57 investigate the role of ADNP in embryonic neural development. We showed that 58 ADNP is required for proper neural induction by modulating Wnt/β-catenin signaling. 59 Mechanistically, ADNP functions to maintain protein levels of β-Catenin through 60 binding to its armadillo domain which prevents its interaction with key components of 61 degradation complex: Axin and APC. Loss of ADNP leads to hyperphosphorylation of 62 β-Catenin by GSK3β and subsequent degradation via ubiquitin-proteasome pathway, 63 resulting in down-regulation of neuroectoderm developmental genes. Small 64 molecule-mediated activation of Wnt signaling can rescue the defects by loss of 65 ADNP. This work provides important insights into the role of ADNP in neural 66 development which would be useful for understanding the pathology of the 67 Helsmoortel-Van der Aa syndrome caused by ADNP mutation. 68 . 69 70 bioRxiv preprint doi: https://doi.org/10.1101/761064; this version posted September 9, 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. 71 Results 72 Generation and characterization of Adnp-/- ESCs. To understand the molecular 73 function of ADNP, we generated Adnp mutant ESCs by using CRISPR/Cas9 74 technology. gRNAs were designed to target the 3' end of exon 4 of mouse Adnp gene 75 (Fig. 1a). We have successfully generated 2 Adnp mutant alleles (-4 and -5 bp), 76 revealed by DNA genotyping around the CRISPR targeting site (Fig. 1b). ADNP 77 protein was hardly detectable in Adnp-/- ESCs by Western blot using ADNP 78 antibodies from different resources (Fig. 1c), which strongly indicated that the mutant 79 alleles are functional nulls. 80 In the traditional self-renewal medium containing LIF-KSR plus FBS, the newly 81 established Adnp-/- ESC colonies overall exhibited typical ESC-like morphology (Fig. 82 1d). To understand how loss of Adnp affected the global gene expression, we 83 performed RNA-sequencing (RNA-seq) experiments for control and early passaged 84 Adnp-/- ESCs. Our RNA-seq analysis showed that the primitive endoderm genes such 85 as Gata6, Gata4, Sox17, Sox7 and Sparc were slightly up-regulated in the absence of 86 ADNP. However, loss of ADNP effects on pluripotency-related, mesodermal and 87 neuroectodermal genes were subtle. Our qRT-PCR and immunofluorescence assay 88 confirmed the RNA-seq results (Fig. 1e; Supplementary Fig. 1a). Consistently, 89 Adnp-/- ESCs can maintain self-renewal capacity for many generations before 90 eventually adopting a flatten morphology and exhibiting reduced alkaline phosphotase 91 activity (Supplementary Fig. 1b). Thus, our results indicated that acute ADNP 92 depletion in ESCs does not result in sudden and complete loss of self-renewal, while bioRxiv preprint doi: https://doi.org/10.1101/761064; this version posted September 9, 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 prolonged ADNP depletion causes loss of ESC phenotype in the LIF/KSR medium. 94 To confirm that the observed phenotypes were not due to the possible off-target 95 effect of the CRISPR/Cas9 technology, we also reduced Adnp transcript levels in 96 ESCs by shRNA knockdown approach. The infection of lentivirus made from each of 97 the two individual shRNAs led to about 70-80% reduction of Adnp mRNA levels (Fig. 98 1c; Supplementary Fig. 1c). Adnp knockdown ESCs in the LIF-KSR medium 99 exhibited similar morphological characteristics and gene expression profile to Adnp-/- 100 ESCs, and could be passaged for many generations without obvious morphological 101 change (Supplementary Fig. 1d,e). 102 103 ADNP promotes ES cell directional neural differentiation. Next, we examined the 104 pluripotency of Adnp-/- ESCs by performing the classical embryoid bodies (EBs) 105 formation assay. Control ESCs were capable of forming large smooth spheroid EBs, 106 whereas Adnp-/- ESCs formed smaller rough disorganized structures (Fig. 1f). To 107 understand how loss of Adnp affected the global gene expression, we performed RNA 108 sequencing (RNA-seq) using day 6 EBs derived from control and Adnp-/- ESCs. A 109 total of 2,004 differentially expressed genes (DEGs) (log2 fold change>1.5 and p≤ 110 0.05) were identified based on two RNA-seq replicates. Of which, an average of 111 1,088 genes were significantly up-regulated and 916 genes were down-regulated. 112 Extra-embryonic endodermal genes such as Gata6, Gata4, Sox17, Sox7 and Foxa2 113 were substantially up-regulated, while pluripotency genes such as Nanog and Sox2, 114 and neural-ectodermal genes such as Pax6, Olig2, Sox1, Nestin and Wnt1 were bioRxiv preprint doi: https://doi.org/10.1101/761064; this version posted September 9, 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. 115 significantly down-regulated (Fig. 1g). Our qRT-PCR assay of selected genes 116 confirmed the RNA-seq results (Fig. 1h). Thus, based on the ESC-derived embryoid 117 body (EB) model, ADNP is required for the neuroectodermal differentiation of 118 mESCs, which was in line with the recent report4.