A cell atlas of human adrenal cortex development and disease

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but the mechanisms underlying human adrenal gland development are not fully understood. Here, we combined single-cell and bulk RNA-sequencing, spatial transcriptomics, immunohistochemistry and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with cell division in the outer definitive zone (DZ). Steroidogenic pathways favor androgen synthesis in the central fetal zone (FZ), but DZ capacity to synthesize cortisol and aldosterone develops with time. Core transcriptional regulators were identified, with a role for HOPX in the DZ. Potential ligand- receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g. IGF2, PEG3). These findings reveal new aspects of human adrenal development, and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders and neonatal stress.


Introduction 1 2
The mature, adult adrenal glands are essential endocrine organs that consist of an outer 3 cortex and a central medulla. The adrenal cortex has three layers that synthesize and 4 release key groups of steroid hormones [1][2][3][4] . Mineralocorticoids (e.g., aldosterone) are 5 released from the outer zona glomerulosa and are needed for salt retention and blood 6 pressure maintenance. Glucocorticoids (e.g. cortisol) are released predominantly from the 7 zona fasciculata and are needed for wellbeing and glucose regulation. Weak androgens 8 (e.g., dehydroepiandrosterone) are released from the inner zona reticularis and influence 9 adrenarche in mid-childhood, with potential effects on health in adult women [5][6][7] . In 10 contrast, the central adrenal medulla is neuroectodermal in origin and releases epinephrine 11 (adrenaline) and norepinephrine (noradrenaline) 8 . Thus, the adrenal glands play an essential 12 role in the acute stress response, many aspects of physiological homeostasis, and long-term 13 wellbeing. 14 15 Disruption of adrenal gland function (known as primary adrenal insufficiency, PAI) leads to 16 glucocorticoid insufficiency, often combined with mineralocorticoid insufficiency [9][10][11] . PAI can 17 present at various ages with symptoms such as malaise, weight loss, hyperpigmentation and 18 hypotension, and can be fatal if not diagnosed and treated appropriately 9 . Although 19 autoimmune destruction of the adrenal gland (sometimes referred to as "Addison disease") 20 is the most common cause of PAI in adolescents and adults, around 30 different single gene 21 disorders have now been identified that result in PAI through diverse processes such as 22 impaired development (hypoplasia), blocks in steroid biosynthesis (congenital adrenal 23 hyperplasia, CAH), adrenocorticotropic hormone (ACTH) resistance ('familial glucocorticoid 24 deficiency', FGD), and metabolic conditions 10,12,13 . PAI often presents soon after birth, or 25 more gradually in childhood or even adulthood. Individuals with PAI require lifelong adrenal 26 steroid hormone replacement, with management sometimes modified based on the 27 underlying cause 9,14 . 28

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In humans, the adrenal cortex develops from bilateral condensations of intermediate 30 mesoderm, known as the "adrenogonadal primordium", at approximately 4 weeks post 31 conception (wpc) (6 weeks gestation) [15][16][17][18] . These structures are in close proximity to the 32 developing kidneys, and give rise to both the adrenal gland and gonad (testis, ovary) 15,19 . 1 The adrenal cortex and gonad share several distinct functional pathways, such as the ability 2 to synthesize steroid hormones and regulation by the nuclear receptor, NR5A1 (also known 3 as steroidogenic factor-1, SF-1) [20][21][22][23] . In contrast, the adrenal medulla is ectodermal in origin 4 and arises from Schwann cell precursor cells that migrate into the adrenal gland and 5 differentiate into sympathoblastic and chromaffin cells 8,24 . These cells ultimately coalesce 6 centrally to form the adrenal medulla. 7 8 Although insights into adrenal development and function are being obtained from studies in 9 model systems (e.g., mice, zebrafish) 25-29 , the adrenal cortex in humans and higher primates 10 has distinct structural and functional components 30 . Most notable is the development of a 11 large fetal zone (FZ), which is capable of synthesizing and releasing substantial amounts of 12 the weak androgen, dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S. DHEA is 13 converted to estrogens by the placenta, which enter the maternal circulation during 14 pregnancy 16 . The FZ regresses in the first few months of postnatal life 30,31 . The teleological 15 function of the FZ is not known although DHEA may have a role in neurodevelopment 32 . 16 Mice have an X-zone that regresses with sexual maturity (males) orpregnancy (females), but 17 similarities with the human FZ are somewhat limited [33][34][35] . Furthermore, cortisol is the 18 primary glucocorticoid synthesized by the adrenal gland in humans whereas rodents 19 generate higher concentrations of corticosterone 3 . 20 21 In recent years, a limited number of studies of human adrenal development or fetal adrenal 22 steroidogenesis have been undertaken using gene expression approaches or focussed RT-23 PCR/immunohistochemistry (IHC) of steroid pathways 18,20,[36][37][38][39][40] . However, few data currently 24 exist for detailed transcriptomic analysis of the human adrenal cortex at a single cell level 25 during the critical first half of gestation (to 20wpc) or transcriptomics linked to 26 developmental anatomy. We therefore developed a multimodal approach to investigate 27 human adrenal cortex development in detail. In order to study the key biological events involved in human adrenal development, we 5 integrated single-cell RNA-sequencing (scRNA-seq), bulk RNA-seq, spatial transcriptomics, 6 immunohistochemistry (IHC) and micro-focus computed tomography (micro-CT) imaging 7 across a critical developmental time-frame between 6 to 20wpc (Fig. 1a, Supplementary  8 Data 1). 9 10 During this period, the adrenal gland undergoes rapid growth, and specific morphological 11 changes such as the development of a deep sulcus and marked increases in vascularization 12 transcriptomic profile compared to control tissues. This transcriptome is more similar to 16 kidney at early stages of development (7wpc) but becomes increasingly distinct with age 17 (Fig. 1e, Supplementary Fig. 2). A subset of highly differentially expressed adrenal-specific 18 genes was identified, including known genes (e.g. MC2R, STAR, CYP11A1) as well as several 19 genes not previously identified as differentially-expressed in adrenal development 20 In order to define specific cell populations within the adrenal gland in more detail, single cell 23 mRNA transcriptome analysis (scRNA-seq) was undertaken at four timepoints (6wpc, 24 6wpc+6days (d), 8wpc+5d, 19wpc) (Fig. 1a, g) 24 . This analysis clearly identified a cluster of 25 adrenal cortex cells, with strong enrichment for genes involved in steroidogenesis (Fig. 1g, 26 and Projection (UMAP), Fig. 3c). Across all stages, cells of the FZ region showed high 24 expression of genes encoding the key enzymes needed for DHEA synthesis (STAR, CYP11A1, 25 CYP17A1, POR, CYB5A) as well as of SULT2A1 (encoding sulfotransferase 2A1), which is 26 required for sulfation of DHEA to DHEA-S and protects the developing fetus from androgen 27 exposure (Fig. 3d). As expected, HSD3B2 expression was low in the FZ during development, 28 resulting in the likely shuttling of steroid precursors (e.g. pregnenolone) into the androgen 29 pathway. The high expression of STAR and CYP11A1 in the FZ cluster was mirrored by high 30 expression of the ACTH receptor and its accessory protein (MRAP) suggesting not only that 31 the FZ has the capacity to be highly biosynthetically active but also that FZ DHEA synthesis 32 may be ACTH-dependent. Of note, enzymes proposed to be involved in the "backdoor" 1 pathway of androgen synthesis 40 were not strongly expressed, although several components 2 of the pathway needed for 11-oxygenation of androgens 47 were (Supplementary Figs. 7-9). 3 4 As 3β-hydroxysteroid dehydrogenase type 2/HSD3B2 is effectively a "gatekeeper" to 5 glucocorticoid and mineralocorticoid biosynthesis (Fig. 3d), we investigated HSD3B2 6 expression across time-series data. Although a potential transiently higher expression was 7 seen at 8wpc in bulk RNA-seq data ( Supplementary Fig. 6), as reported previously 37,39 , 8 single-cell transcriptomic data showed overall greater increase in HSD3B2 across time, with 9 the highest levels in the DZ cluster at 19wpc (Fig. 3e). A similar graded increase in CYP21A2 10 (encoding 21-hydroxylase) and CYP11B1 (encoding 11β-hydroxylase type 1) was seen (Fig.  11 3e). Single-cell gene co-expression analysis revealed a distinct subset of cells that 12 proportionately co-expressed HSD3B2 and CYP21A2 in early developmental stages, although 13 by 19wpc it appeared that CYP21A2 expression occurred in a greater number of cells, and 14 that expression of HSD3B2 (and its protein) was the likely rate-limiting factor (Fig. 3f). 15 Expression of CYP21A2 and CYP11B1 was linked ( Supplementary Fig. 10). Taken together, 16 these data suggest that there is an increase in gene expression of the enzymatic machinery 17 needed for glucocorticoid synthesis across time. It is also debated at what stage the developing fetal adrenal gland can synthesize 20 mineralocorticoids, such as aldosterone 38,39 . Here, CYP11B2 (encoding 11β-hydroxylase type 21 2/aldosterone synthase) is a key enzyme in the final stages of aldosterone synthesis, as well 22 as HSD3B2, which is needed to allow precursors into this pathway (Fig. 3d). In our scRNA-23 seq data, CYP11B2 expression was low in early stages but increased by 19wpc in a sub-24 population of cells in the DZ (Fig. 3d, e). Again, single-cell co-expression analysis suggested 25 that HSD3B2 and CYP11B2 are often linked (Fig. 3g)  In order to study "core" transcriptional regulators of human adrenal cortex, we first 31 identified genes that were differentially-expressed in the cortex cluster compared to non-32 cortex clusters at each scRNA-seq stage (log2FC>0.25, padj<0.05), and compared these 1 genes to the Animal Transcription Factor Database (TFDB) 48 (Supplementary Data 4). At each 2 developmental time point studied, transcription factors represented between 1.8-2.4% of all 3 differentially expressed cortex genes ( Supplementary Fig. 11a). By intersecting these 4 analyses, 17 "core" transcriptional regulators were identified that were common to all 5 datasets (Fig. 4a-c, Supplementary Fig. 11, Supplementary Fig. 12). These factors were all 6 present in bulk RNA-seq analysis of adrenal gland samples compared to control tissues 7 (log2FC>1.5, padj<0.05), suggesting that they had a strong degree of adrenal specificity 8 ( Supplementary Fig. 11b, c; Supplementary Data 4). 9 10 Two key transcription factors that are well-established regulators of adrenal development 11 are the orphan nuclear receptors, NR0B1 (DAX-1) and NR5A1 (SF-1) 15,20,22,23 . Disruption of 12 NR0B1 causes X-linked adrenal hypoplasia, which is one of the most common causes of PAI 13 in children (boys) 13,49 . NR5A1 is a master-regulator of adrenal and reproductive 14 development and function, and more severe disruption is also associated with PAI in 15 humans 23,50 . Many studies have suggested that NR0B1 and NR5A1 can be functional 16 partners, but data about expression in human development are still limited 15,20,33,51 . Cluster 17 analysis in scRNA-seq datasets as well as spatial transcriptomic analysis showed that 18 expression of NR0B1 and NR5A1 occurs extensively throughout the fetal adrenal gland and 19 that co-expression occurs in a subset of cells ( Fig. 4c-e, Supplementary Fig. 11c). Taken 20 together, these data support the key role that NR0B1 and NR5A1 play in transcription 21 regulation and specification of human adrenal development. 22

HOPX is a novel definitive zone factor 24 25
Although most of the "core" transcription factors identified showed expression throughout 26 the adrenal cortex (i.e., DZ and FZ), one adrenal-enriched gene that was expressed very 27 strongly in the DZ compared to the FZ was HOPX (Fig. 5a, b). HOPX is an atypical 28 homeodomain protein (also known as Hop homeobox/"homeobox-only protein') that lacks 29 direct DNA-binding capacity but interacts with transcriptional regulators to maintain 30 quiescence in specific embryonic and adult stem cell populations, and to control cell 31 proliferation during organogenesis 52,53 . HOPX also acts as a tumor suppressor, and reduced 1 HOPX expression is associated with several cancers 52,53 . 2 3 In our scRNA-seq dataset, HOPX was consistently one of the most differentially-expressed 4 markers of the DZ compared to the FZ in all ages studied (Supplementary Fig. 13; 5 Supplementary Data 3). This highly-specific enrichment of HOPX in the DZ was confirmed by 6 spatial transcriptomic analysis, which showed a strong "ring" of HOPX DZ expression at 7 7wpc+4d, with a peripheral ring of weaker expression likely representing mesenchymal cells 8 (Fig. 5c). This finding was validated by immunohistochemistry, which showed that HOPX 9 defined the outer border of the DZ at the interface of the peripheral mesenchyme at late 10 6wpc (Fig. 5d). Furthermore, serial IHC analyses showed that HOPX was expressed in the 11 outer DZ across time (late 6wpc-20wpc), marking the boundary between the developing 12 adrenal gland and the mesenchyme (early) or subcapsular region of cells (later) (Fig. 5e, 13 Supplementary Fig. 14). 14 15 As expected given its role in the DZ, HOPX co-localized in clusters with the DZ marker NOV in 16 scRNA-seq analysis, especially during early stages of development (Fig. 5f). However, by 17 19wpc, HOPX expression was relatively reduced (Fig.4b, Fig. 5f) and localized within a zona 18 glomerulosa-like cluster that also expressed HSD3B2, CYP11B2, and the orphan nuclear As the adrenal gland forms within a region of mesoderm/mesenchyme (Fig. 5c-e), more 1 detailed analyses of potential ligand-receptor signaling interactions were undertaken using a 2 combined adrenal cortex-mesenchyme scRNA-seq data. Notably, a potential transcriptomic 3 "bridge" between the mesenchyme and cortex was identified in the merged adrenal 4 dataset, particularly in the 6wpc+5d sample (Fig. 1g, Fig. 6a). A trajectory of cells undergoing 5 differentiation from the mesenchyme to cortex was also observed (Fig. 6b). Rspo3/Rspondin3 has been proposed previously to be a key ligand released by subcapsular 22 cells in both mouse and human adrenal development, with potential interactions with Lgr5 23 and Znrf3 18,55,56 . Using spatial transcriptomics, RSPO3 expression was found to be expressed 24 in the mesenchyme, including in an outer layer around the early adrenal gland (7wpc+4d), 25 whereas LGR4 was expressed more centrally in the fetal zone region (Fig. 7g, j). Strong LGR5 26 and LGR6 expression or interactions were not seen ( Supplementary Fig. 16). Thus, although 27 several signalling systems have been proposed in adrenal development from data in the 28 mouse 25,27,57 , our unsupervised analysis of ligand-receptor interactions support the roles of 29 IGF2, DLK1 and RSPO3/Rspondin3 as major components in human adrenal development, 30 and suggest that CXCL12 may also influence potential mesenchyme-adrenal interactions. 31 Imprinted genes are enriched in the human fetal adrenal gland 1 2 IGF2 and DLK are both imprinted genes, and it is well recognized that imprinted genes play a 3 key role in many aspects of fetal and placental growth in humans 58 . Paternally-expressed 4 (maternally-imprinted) genes are frequently linked to growth promotion, whereas 5 maternally-expressed (paternally-imprinted) genes are associated with growth restriction. 6 To address the potential role of imprinted genes in the developing fetal adrenal gland in 7 more detail, differential expression was initially studied using bulk-RNAseq data (adrenal 8 versus control, log2FC>1.5 padj<0.05). We found that 15 out of 84 (17.9%) well-established, 9 non-placental-specific human imprinted genes 59 were differentially expressed in the adrenal  Fig. 18). This finding contrasts with HOPX, which is predominantly 31 expressed in the fetal adrenal but not in the adult organ. 32 1 We also analyzed developmental expression of genes known to be monogenic causes of PAI 2 ( Fig. 8b) 10 . Most key transcription factors (e.g. NR5A1, NR0B1), components of 3 steroidogenesis (e.g. STAR, CYP11A1, CYP21A2) and genes involved in ACTH-signalling (e.g., 4 MC2R, MRAP) showed high specificity for expression in the fetal adrenal cortex cluster (Fig.  5 8b). However, many genes linked to oxidative stress processes or metabolic function were 6 more ubiquitously expressed (e.g., NNT, AAAS, SGPL1, ABCD1) 60-63 (Fig. 8b, c). In addition, 7 out of those genes associated with multisystem growth restriction phenotypes (e.g. It is already established that the human adrenal gland undergoes marked growth 9 throughout gestation, and at birth is approximately the same weight as in adult life 37,38 . 10 Much of this growth is due to the expansion of the large FZ, which is only found in humans 11 and higher primates. Here, we document changes in growth and morphology up to 20 wpc. 12 Using scRNA-seq analysis of cycling cells, coupled with IHC markers of cell division (KI-67), 13 we show that there is rapid cell division during the late embryonic/early fetal stage, and that 14 the majority of dividing cells are located in the outer DZ region. A potential trajectory of cell 15 differentiation from the DZ to FZ was seen during early adrenal development 25,27,43-46 . 16 Imprinted genes, such as IGF2 and DLK1, play a key role in adrenal growth 36,58,67-70 . Here, we 17 demonstrate strong expression of paternally-expressed growth-promoting genes, especially 18 in the FZ region, consistent with the rapid growth seen during this stage of development. Although the main role of the adult adrenal cortex is the biosynthesis and release of steroid 1 hormones (mineralocorticoids, glucocorticoids, androgens), the extent to which these 2 hormones can be generated in the fetal adrenal gland remains to be fully elucidated. Recent 3 studies have looked at expression of key components of these pathways, or attempted to 4 measure the major steroid hormones and their metabolites directly 38,39 . Here, we show that 5 the FZ has the transcriptomic machinery to secrete large amounts of adrenal androgens, 6 such as DHEA(S). Precursors are shunted into this pathway due to the lack of HSD3B2 7 (encoding 3β-hydroxysteroid dehydrogenase type 2). Expression of the adrenocorticotropin 8 (ACTH) receptor (MC2R) and its accessory protein (MRAP) increased with age, and showed 9 strong expression in the FZ region. This finding is in keeping with ACTH-dependent 10 stimulation of androgens in fetal adrenal cell or tissue cultures, suggesting the FZ androgen 11 biosynthesis has the capacity to be ACTH-driven 79-81 . 12

13
In contrast, glucocorticoid biosynthesis (e.g. cortisol) requires HSD3B2 expression. 14 Consistent with two previous reports 37,39 , we detected a potential transient increase in 15 HSD3B2 at around 8.5 wpc, but expression was more consistent by 19wpc in DZ cells that 16 often co-expressed CYP21A2 (encoding 21-hydroxylase) and CYP11B1 (encoding 11 β-17 hydroxylase). Very limited expression of the genes required for mineralocorticoid 18 biosynthesis (e.g. aldosterone) was seen early on, but a small proportion of DZ cells did 19 express CYP11B2 together with other relevant enzymes by 19wpc. This finding is consistent 20 with a lack of aldosterone synthesis in the first half of gestation, although increases in 21 CYP11B2 expression towards the end of the second trimester suggest the capacity for 22 aldosterone synthesis is being established 38,39 . Of note, pre-term babies often have 23 hypotension and salt-loss, which may in part be due to immature development of 24 mineralocorticoid biosynthesis, as well as relative mineralocorticoid resistance. 25 Understanding the dynamic transcriptomic and physiological changes around this time is 26 key. 27

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Two key transcription factors (TFs) that regulate fetal adrenal development are the nuclear 29 receptors NR0B1 (DAX-1) and NR5A1 (SF-1) 15,20,22,23,82 . These genes encode two important 30 nuclear receptors within a "core" set of 17 transcription factors identified, that were 31 consistently differentially expressed in the adrenal cortex across time. Another transcription 32 regulator identified that was remarkable for its consistent differential expression in the DZ 1 compared to the FZ was HOPX 52 . HOPX is an atypical homeobox factor that lacks direct DNA 2 binding and likely interacts with transcriptional regulators 52 , so is not universally classified as 3 a TF. Nevertheless, HOPX is emerging as a key embryonic and adult stem cell marker 4 involved in stem cell maintenance/quiescence 83,84 and controlled tissue differentiation 52 . 5 HOPX is reported to play a role in the development of mesoderm progenitor 6 cells/hematopoietic stem cells 84,85 , osteogenic cells, neuronal tissue 83 , cardiomyoblasts 86 , 7 intestinal crypt/colonic cells 53,87 , skin 88 , alveolar epithelial cells (Type I) 89 and endometrium. 8 HOPX can influence tissue repair and regeneration 87 , and reduced HOPX expression 9 (through promoter methylation) is associated with several cancers (e.g. colon 53 , breast 90 , 10 thyroid, pancreas 91 ) and metastasis risk (e.g. nasopharyngeal 92 ), suggesting HOPX acts as a 11 tumor suppressor. Interactions with WNT signalling 86 , activated SMAD 86 and CXCL12 84 have 12 also been proposed. Our findings support a recent report of HOPX expression in early 13 human adrenal development 18  and has been shown to be expressed in the subcapsular region of cells in the developing 10 mouse adrenal gland 55 , as well as in subcapsular cells in the 8wpc human adrenal gland 18 , 11 and potentially mediates a gradient of WNT signalling involved in adrenal zonation. 12 Although interactions with LGR5 have been suggested 55 , we identified LGR4 as the most 13 likely expressed putative cortex receptor. A potential role for CXCL12 (mesenchymal ligand) 14 and CXCR4 (adrenal cortex receptor) was also identified. Other signaling systems proposed 15 from mouse models (eg Shh/Gli) were not found to be strongly expressed in the developing 16 human adrenal gland at this stage. Taken together, these data suggest the Rspondin3-driven 17 WNT signalling has a key role in human adrenal development, as well as in mice. detailed data. Also, whilst scRNA-seq and spatial transcriptomic platforms provide 25 significant new insight, the ability to obtain increased sequencing reads per cell, more cells 26 sequenced per sample, or greater spatial resolution is always improving and will help 27 address some of the hypotheses generated here in the future. Understanding anatomical 28 and physiological relations during development will be key going forward, at gene 29 transcription, RNA expression and protein levels, and integrating detailed histology and 30 imaging with basic cell biology will be crucial, as we have attempted to do here. 31 In summary, this study highlights the unique developmental complexities of human fetal 1 adrenal gland development up unto mid-gestation, and provides an integrated 2 transcriptomic roadmap with potential long-term consequences for human health and 3 disease.  The age of embryos up to 8wpc was calculated based on Carnegie staging, whereas in older 11 fetuses the age was estimated from foot length and knee-heel length in relation to standard 12 growth data. Samples were karyotyped by G-banding or quantitative PCR (chromosomes 13, The 17wpc adrenal gland studied (10% formalin) was immersed in 1.25% potassium tri-27 iodide (I2KI) at room temperature for 48 hours, then rinsed, dried and wax embedded 115 . 28 Once hardened, excess wax was trimmed in order to preserve tissue shape, to reduce 29 dehydration and movement artefact, and to optimize contact with the X-ray beam source.  HiSeq 4000). A processed single cell matrix was generated as described before 24 27 with minor modifications. Unless specified, cycling cells were discarded from the analysis. 28 The R package Seurat (v4.0.2) 119 was used for processing the single cell matrix. Briefly, the 29 count matrix was normalized and 2000 highly variable genes chosen. After gene scaling, 30 dimensionality reduction was performed using the first 75 principal components (PCs). The 31 FindClusters and RunUMAP functions were used to identify clusters and to allow UMAP 32 visualization. The clustree package in R 120 was used to select the resolution parameter for 1 clustering. Differentially-expressed genes between clusters were calculated using the 2 FindAllMarkers or FindMarkers functions using the parameters 'min.pct=0.25 and 3 logfc.threshold=0.25' (Wilcoxon Rank Sum test). Internal functions in Seurat (FeaturePlot, 4 RidgePlot) were used to visualize marker expression. The FeatureScatter function was used 5 to generate plots for pair of genes. The dittoSeq Bioconductor package 121 was used to 6 generate barplots, heatmaps and dotplots. RNA velocity on selected fetal adrenal samples 7 was calculated using velocyto and plotted using the velocyto.R package in R as described 8 before 24 . Adrenal cortex sample integration was performed using datasets normalized with Statistical analysis for bulk-and single-cell RNA-seq data is described above within packages 16 of differential expression analysis, with adjustments for multiple corrections. Chi-square 17 analysis was performed GraphPad (Prism). In all analyses, a p-value or adjusted p-value less 18 than 0.05 was taken as significant.