A mouse model of Zhu-Tokita-Takenouchi-Kim syndrome reveals indispensable SON functions in organ development and hematopoiesis

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. To obtain a more comprehensive understanding of the changes within HSPCs at the molecular level, we employed single-cell RNA-sequencing (scRNAseq) using both WT and Son+/− bone marrow HSPCs isolated and sorted based on their Lin− cKit+ (LK) cell markers (24,355 total LK cells from 2 WT mice; 31,232 total LK cells from 2 Son+/− mice. The linage-marker-negative (Lin-) c-kit+ population containing hematopoietic stem and progenitor cells was sorted from dissociated mouse bone marrow cells by flow cytometry. Sorted single-cell suspension, 10x barcoded gel beads and oil (10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1 PN 1000268) were loaded into 10x Chromium™ Single Cell Chip to capture single cells into nanoliter-scale oil droplets by 10xChromium X Controller and generate Gel Bead-In-EMulsions (GEMs). cDNA from single cells were synthesized and barcoded by incubation of the GEMs in a thermocycler machine and then purified from GEMs by DynaBeads (10xGenomics PN 2000048). cDNA was pre-amplified by PCR to generate sufficient mass for sequencing library construction. The single-cell cDNA libraries were constructed by following 10xGenomics 3’ NextGem 3.1 version kit instructions. The final constructed 3’-biased single-cell libraries were sequenced by the Illumina Novaseq6000 machine, targeting 50,000 read pairs/cell. We identified hematopoietic abnormalities in Son+/− mice, including leukocytopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing genes required for lymphoid and B-cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction. The linage-marker-negative (Lin-) c-kit+ population containing hematopoietic stem and progenitor cells was sorted from dissociated mouse bone marrow cells by flow cytometry. Sorted single cell suspension, 10x barcoded gel beads, and oil (all were included in 10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1 PN 1000268) were loaded into 10x Chromium™ Single Cell Chip to capture single cells into nanoliter-scale oil droplets by 10xChromium X Controller and generate Gel Bead-In-EMulsions (GEMs). cDNA from single cells were synthesized and barcoded by incubation of the GEMs in a thermocycler machine and then purified from GEMs by DynaBeads (10xGenomics PN 2000048). cDNA was pre-amplified by PCR to generate sufficient mass for sequencing library construction. The single cell cDNA libraries were constructed by following 10xGenomics 3’ NextGem 3.1 version kit instruction, briefly by following steps of cDNA fragmentation, end repair & A-tailing, and size selection by SPRIselect beads (Beckman Coulter, PN B23318), adaptor ligation, and sample index PCR amplification, and then SPRIselect beads size selection again. The final constructed 3'-biased single cell libraries were sequenced by Illumina Novaseq6000 machine, targeting 50,000 read pairs/cell. The sequencing cycles consisted of 28bp for read 1, 90bp for read 2, and 10bp for i7 and i5.罕见病在生物医学研究中占比偏低，导致社会对其认知不足。朱-外间-竹内-金（Zhu-Tokita-Takenouchi-Kim, ZTTK）综合征是一种罕见病，由引发SON基因杂合性功能丧失的遗传变异所导致。尽管ZTTK综合征患者存在多种临床症状，但模式生物的匮乏阻碍了我们对SON基因及该复杂综合征的理解。本研究构建了Son单倍体不足（Son+/-）小鼠作为ZTTK综合征的模式生物，并揭示了Son基因在器官发育与造血过程中的不可或缺的作用。Son+/-小鼠可重现ZTTK综合征的临床症状，包括生长迟缓、认知障碍、骨骼异常以及肾发育不全。 为了更全面地解析造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）的分子层面变化，我们针对野生型（Wild Type, WT）与Son+/-小鼠的骨髓HSPCs开展单细胞RNA测序（single-cell RNA-sequencing, scRNAseq），这些细胞通过Lin−cKit+（LK）细胞标记物进行分离分选：2只WT小鼠共获取24355个LK细胞，2只Son+/-小鼠共获取31232个LK细胞。我们通过流式细胞术从解离的小鼠骨髓细胞中分选出表达谱系标记阴性（Lin−）、c-Kit阳性的细胞群，该细胞群包含HSPCs。将分选得到的单细胞悬液、10x条形码标记的凝胶微珠与油液（均包含于10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1，货号PN 1000268）上样至10x Chromium™ 单细胞芯片，通过10xChromium X控制器将单细胞捕获至纳升级油滴中，生成凝胶微珠包裹的乳液（Gel Bead-In-EMulsions, GEMs）。通过在热循环仪中孵育GEMs，完成单细胞cDNA的合成与条形码标记，随后通过DynaBeads（10xGenomics，货号PN 2000048）从GEMs中纯化得到cDNA。通过PCR对cDNA进行预扩增，以获得足够构建测序文库的模板量。按照10xGenomics 3' NextGem 3.1版本试剂盒说明书构建单细胞cDNA文库。最终构建的3'端偏好性单细胞文库通过Illumina NovaSeq6000测序仪进行测序，预设每个细胞的测序读对数为50000。 本研究在Son+/-小鼠中发现了造血异常，包括白细胞减少症与免疫球蛋白缺乏症，这与人类患者中观察到的表型一致。对造血干祖细胞的表面标记物分析与单细胞转录组分析显示，Son单倍体不足会使细胞命运偏向髓系细胞谱系，并通过降低淋巴系与B细胞谱系特化所需基因的表达，损害淋巴系细胞的发育。此外，Son单倍体不足会导致红细胞系基因的异常激活以及红细胞成熟障碍。上述研究结果凸显了Son基因完整剂量在多个器官中的重要性。本研究构建的小鼠模型可为该罕见病及与SON功能异常相关的其他疾病提供极具价值的研究工具。 我们通过流式细胞术从解离的小鼠骨髓细胞中分选出表达谱系标记阴性（Lin−）、c-Kit阳性的细胞群，该细胞群包含HSPCs。将分选得到的单细胞悬液、10x条形码标记的凝胶微珠与油液（均包含于10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1，货号PN 1000268）上样至10x Chromium™ 单细胞芯片，通过10xChromium X控制器将单细胞捕获至纳升级油滴中，生成GEMs。通过在热循环仪中孵育GEMs，完成单细胞cDNA的合成与条形码标记，随后通过DynaBeads（10xGenomics，货号PN 2000048）从GEMs中纯化得到cDNA。通过PCR对cDNA进行预扩增，以获得足够构建测序文库的模板量。按照10xGenomics 3' NextGem 3.1版本试剂盒说明书构建单细胞cDNA文库，具体步骤包括cDNA片段化、末端修复与A尾加尾、通过SPRIselect磁珠（Beckman Coulter，货号PN B23318）进行片段大小筛选、接头连接以及样本索引PCR扩增，随后再次通过SPRIselect磁珠进行片段大小筛选。最终构建的3'端偏好性单细胞文库通过Illumina NovaSeq6000测序仪进行测序，预设每个细胞的测序读对数为50000。测序循环参数为：read 1为28bp，read 2为90bp，i7与i5索引各为10bp。