Determination of key events in mouse hepatocyte maturation at the single-cell level.

Hepatocytes, the liver's predominant cells, perform numerous essential biological functions. However, crucial events and regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) to explore the precise hepatocyte development process in mice. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct proliferation rates. Hepatic zonation is gradually formed during hepatocyte maturation. Hepatocytes or their nuclei with distinct ploidies exhibit zonation preferences in distribution and asynchrony in maturation. Moreover, by combining gene regulatory network analysis with in vivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates the comprehensive transcriptomic profiles of hepatocyte maturation but also presents a paradigm to identify genes that function in the development of hepatocyte maturation and zonation by combining genetic manipulation and measurement of coordinates in a single-cell developmental trajectory.

Sequential progenitor states mark the generation of pancreatic endocrine lineages in mice and humans.

The pancreatic islet contains multiple hormone+ endocrine lineages (α, ß, δ, PP and ε cells), but the developmental processes that underlie endocrinogenesis are poorly understood. Here, we generated novel mouse lines and combined them with various genetic tools to enrich all types of hormone+ cells for well-based deep single-cell RNA sequencing (scRNA-seq), and gene coexpression networks were extracted from the generated data for the optimization of high-throughput droplet-based scRNA-seq analyses. These analyses defined an entire endocrinogenesis pathway in which different states of endocrine progenitor (EP) cells sequentially differentiate into specific endocrine lineages in mice. Subpopulations of the EP cells at the final stage (EP4early and EP4late) show different potentials for distinct endocrine lineages. ε cells and an intermediate cell population were identified as distinct progenitors that independently generate both α and PP cells. Single-cell analyses were also performed to delineate the human pancreatic endocrinogenesis process. Although the developmental trajectory of pancreatic lineages is generally conserved between humans and mice, clear interspecies differences, including differences in the proportions of cell types and the regulatory networks associated with the differentiation of specific lineages, have been detected. Our findings support a model in which sequential transient progenitor cell states determine the differentiation of multiple cell lineages and provide a blueprint for directing the generation of pancreatic islets in vitro.

Single-cell patterning and axis characterization in the murine and human definitive endoderm.

Defining the precise regionalization of specified definitive endoderm progenitors is critical for understanding the mechanisms underlying the generation and regeneration of respiratory and digestive organs, yet the patterning of endoderm progenitors remains unresolved, particularly in humans. We performed single-cell RNA sequencing on endoderm cells during the early somitogenesis stages in mice and humans. We developed molecular criteria to define four major endoderm regions (foregut, lip of anterior intestinal portal, midgut, and hindgut) and their developmental pathways. We identified the cell subpopulations in each region and their spatial distributions and characterized key molecular features along the body axes. Dorsal and ventral pancreatic progenitors appear to originate from the midgut population and follow distinct pathways to develop into an identical cell type. Finally, we described the generally conserved endoderm patterning in humans and clear differences in dorsal cell distribution between species. Our study comprehensively defines single-cell endoderm patterning and provides novel insights into the spatiotemporal process that drives establishment of early endoderm domains.

IDH1-R132H Suppresses Glioblastoma Malignancy through FAT1-ROS-HIF-1α Signaling.

BACKGROUND: Glioblastoma (GBM) is one of the most deadly primary malignant brain tumors in adults. R132H mutation of isocitrate dehydrogenase 1 (IDH1) predicts a better prognosis of GBM. IDH1-R132H is associated with increased hypoxia-inducible factor-1α (HIF-1α) expression in GBM tumors. However, the molecular mechanism underlying IDH1-R132H-HIF-1α signaling in GBM is still unclear. AIM: We aimed to investigate the molecular pathway of IDH1-R132H-HIF-1α in the regulation of GBM. MATERIALS AND METHODS: U87 and U251 GBM cells and xenograft tumor mice were used. RESULTS: We found that overexpression of IDH1-R132H decreased cell proliferation, increased apoptosis, decreased migration and invasion, enhanced temozolomide (TMZ)-induced cytotoxicity, and reduced tumor growth in xenograft mice. Overexpression of IDH1-R132H increased the expression of HIF-1α and downregulation of HIF-1α suppressed IDH1-R132H-induced effect on GBM. Reactive oxygen species (ROS) level was increased by IDH1-R132H over expression and the use of antioxidant inhibited IDH1-R132H-induced increase of HIF-1α expression. FAT Atypical Cadherin 1 (FAT1) expression was increased by IDH1-R132H over expression. Knockdown of FAT1 blocked IDH1-R132H-induced reduction of tumor growth in xenograft mice. Down regulation of FAT1 decreased HIF-1α expression and inhibited IDH1-R132H-induced increase of ROS level. CONCLUSIONS: Our findings provide new insights into IDH1-R132H-regulated downstream signaling in GBM and highlight the importance of IDH1-R132H-FAT1-ROS-HIF-1α signaling pathway in potential therapeutic intervention of GBM.

Characterizing pancreatic ß-cell heterogeneity in the streptozotocin model by single-cell transcriptomic analysis.

OBJECTIVES: The streptozotocin (STZ) model is widely used in diabetes research. However, the cellular and molecular states of pancreatic endocrine cells in this model remain unclear. This study explored the molecular characteristics of islet cells treated with STZ and re-evaluated ß-cell dysfunction and regeneration in the STZ model. METHODS: We performed single-cell RNA sequencing of pancreatic endocrine cells from STZ-treated mice. High-quality sequencing data from 2,999 cells were used to identify clusters via Louvain clustering analysis. Principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), force-directed layout (FDL), and differential expression analysis were used to define the heterogeneity and transcriptomic changes in islet cells. In addition, qPCR and immunofluorescence staining were used to confirm findings from the sequencing data. RESULTS: Untreated ß-cells were divided into two populations at the transcriptomic level, a large high-Glut2 expression (Glut2high) population and a small low-Glut2 expression (Glut2low) population. At the transcriptomic level, Glut2low ß-cells in adult mice did not represent a developmentally immature state, although a fraction of genes associated with ß-cell maturation and function were downregulated in Glut2low cells. After a single high-dose STZ treatment, most Glut2high cells were killed, but Glut2low cells survived and over time changed to a distinct cell state. We did not observe conversion of Glut2low to Glut2high ß-cells up to 9 months after STZ treatment. In addition, we did not detect transcriptomic changes in the non-ß endocrine cells or a direct trans-differentiation pathway from the α-cell lineage to the ß-cell lineage in the STZ model. CONCLUSIONS: We identified the heterogeneity of ß-cells in both physiological and pathological conditions. However, we did not observe conversion of Glut2low to Glut2high ß-cells, transcriptomic changes in the non-ß endocrine cells, or direct trans-differentiation from the α-cell lineage to the ß-cell lineage in the STZ model. Our results clearly define the states of islet cells treated with STZ and allow us to re-evaluate the STZ model widely used in diabetes studies.

Mechanisms and Therapeutic Effects of Human Olfactory Mucosa-Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis in Mice / 中山大学学报(医学科学版)

@#【Objective】 To study the mechanisms and therapeutic effects of human olfactory mucosa-derived mesenchymal stem cells（OMSC）on experimental autoimmune encephalomyelitis（EAE）in mice.【Methods】Under local anesthesia by using nasal endoscopy，olfactory epithelia of healthy donors were obtained，digested and cultured up to the 5th passage. OMSC were identified，differentiated and stained. EAE models were induced in C57 female mice by myelin oligodendrocyte glycoprotein（MOG35- 55）and pertussis toxin（PT）. Neurological function was documented daily. On day 16 after immunization（peak of incidence），the mice were divided randomly into two groups and treated with OMSC and PBS via tail vein injection respectively. On day 24 after immunization ，blood was collected from angular vein and levels of IL-10，IL-17，IFN-γ and IL-6 were determined by cytometric beads array（CBA）. The size of the spinal cord lesion in mice was observed and measured by using HE and LFB staining. Peripheral blood lymphocytes（PBL）of healthy donors were obtained and then co-cultured with OMSC. The proportions of CD4+ T cells secreting IFN- γ（Th1 cells）in lymphocyte group and co-culture group were compared after 2 days of cultivation. Adding IDO or COX pathway inhibitor to co- culture group and cultivating for 2 days，we observed and compared the proportions of Th1 cells in lymphocyte group，co-culture group and inhibitor treatment group respectively.【Results】OMSC exhibited certain mesenchymal stem cell-like characteristics with respect to expression of stem cell surface markers and multilineage differentiation potentials. After induced by MOG35- 55 and PT，EAE models showed different levels of neurological damage. Compared with those in PBS treatment group，in OMSC treatment group，the severity of neural dysfunction in mice was significantly reduced（P =0.002），the level of IFN-γ in serum was lower（P = 0.032），but no significant differences in the levels of IL-10，IL-17 and IL-6 were found between two groups. HE and LFB staining revealed that the inflammatory infiltration and demyelinating areas in OMSC treatment group were less than those in PBS treatment group. The proportion of Th1 cells was lower in co-culture group than that in lymphocyte group（P = 0.001），higher in IDO inhibitor group than that in co-culture group（P = 0.01），but no significant difference was found between IDO inhibitor group and lymphocyte group or between COX inhibitor group and co-culture group.【Conclusions】OMSC may regulate the proportion of Th1 lymphocytes through IDO pathway so as to inhibit the demyelinating injuries of EAE in mice. This study provides a new idea for the clinical treatment of multiple sclerosis.

Defining multistep cell fate decision pathways during pancreatic development at single-cell resolution.

The generation of terminally differentiated cell lineages during organogenesis requires multiple, coordinated cell fate choice steps. However, this process has not been clearly delineated, especially in complex solid organs such as the pancreas. Here, we performed single-cell RNA-sequencing in pancreatic cells sorted from multiple genetically modified reporter mouse strains at embryonic stages E9.5-E17.5. We deciphered the developmental trajectories and regulatory strategies of the exocrine and endocrine pancreatic lineages as well as intermediate progenitor populations along the developmental pathways. Notably, we discovered previously undefined programs representing the earliest events in islet α- and ß-cell lineage allocation as well as the developmental pathway of the "first wave" of α-cell generation. Furthermore, we demonstrated that repressing ERK pathway activity is essential for inducing both α- and ß-lineage differentiation. This study provides key insights into the regulatory mechanisms underlying cell fate choice and stepwise cell fate commitment and can be used as a resource to guide the induction of functional islet lineage cells from stem cells in vitro.

The contributions of mesoderm-derived cells in liver development.

The liver is an indispensable organ for metabolism and drug detoxification. The liver consists of endoderm-derived hepatobiliary lineages and various mesoderm-derived cells, and interacts with the surrounding tissues and organs through the ventral mesentery. Liver development, from hepatic specification to liver maturation, requires close interactions with mesoderm-derived cells, such as mesothelial cells, hepatic stellate cells, mesenchymal cells, liver sinusoidal endothelial cells and hematopoietic cells. These cells affect liver development through precise signaling events and even direct physical contact. Through the use of new techniques, emerging studies have recently led to a deeper understanding of liver development and its related mechanisms, especially the roles of mesodermal cells in liver development. Based on these developments, the current protocols for in vitro hepatocyte-like cell induction and liver-like tissue construction have been optimized and are of great importance for the treatment of liver diseases. Here, we review the roles of mesoderm-derived cells in the processes of liver development, hepatocyte-like cell induction and liver-like tissue construction.

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting ß cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic ß cells from embryonic, neonatal and postnatal pancreases.

Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs.

The pancreas of vertebrates is separately derived from both the dorsal and ventral endodermal domains. However, the difference between these two programs has been unclear. Here, using a pancreatic determination gene, Pdx1, driven GFP transgenic mouse strain, we identified Pdx1-GFP highly expressing cells (Pdx1high) and Pdx1-GFP lowly expressing cells (Pdx1low) in both embryonic dorsal Pdx1-expressing region (DPR) and ventral Pdx1-expressing region (VPR). We analyzed the transcriptomes of single Pdx1low and Pdx1high cells from the DPR and VPR. In the VPR, Pdx1low cells have an intermediate progenitor identity and can generate hepatoblasts, extrahepatobiliary cells, and Pdx1high pancreatic progenitor cells. In the DPR, Pdx1high cells are directly specified as pancreatic progenitors, whereas Pdx1low cells are precocious endocrine cells. Therefore, our study defines distinct road maps for dorsal and ventral pancreatic progenitor specification. The findings provide guidance for optimization of current ß-cell induction protocols by following the in vivo dorsal pancreatic specification program.

Dynamics of chromatin marks and the role of JMJD3 during pancreatic endocrine cell fate commitment.

Pancreatic endocrine lineages are derived from pancreatic progenitors that undergo a cell fate transition requiring a switch from low to high Ngn3 expression. However, the underlying chromatin regulatory mechanisms are unclear. Here, we performed epigenomic analysis of gene regulatory loci featuring histone marks in cells with low or high level of Ngn3 expression. In combination with transcriptomic analysis, we discovered that in Ngn3-high cells, the removal of H3K27me3 was associated with the activation of key transcription factors and the establishment of primed and active enhancers. Deletion of Jmjd3, a histone demethylase for H3K27me3, at the pancreatic progenitor stage impaired the efficiency of endocrine cell fate transition and thereafter islet formation. Curiously, single-cell RNA-seq revealed that the transcriptome and developmental pathway of Ngn3-high cells were not affected by the deletion of Jmjd3 Our study indicates sequential chromatin events and identifies a crucial role for Jmjd3 in regulating the efficiency of the transition from Ngn3-low to Ngn3-high cells.

Deciphering Pancreatic Islet ß Cell and α Cell Maturation Pathways and Characteristic Features at the Single-Cell Level.

Pancreatic ß and α cells play essential roles in maintaining glucose homeostasis. However, the mechanisms by which these distinct cell populations are generated, expand, and mature during pancreas development remain unclear. In this study, we addressed this critical question by performing a single-cell transcriptomic analysis of mouse ß and α cells sorted from fetal to adult stages. We discovered that ß and α cells use different regulatory strategies for their maturation and that cell proliferation peaks at different developmental times. However, the quiescent and proliferative cells in both the ß lineage and α lineage are synchronous in their maturation states. The heterogeneity of juvenile ß cells reflects distinct cell-cycling phases, origins, and maturation states, whereas adult ß cells are relatively homogeneous at the transcriptomic level. These analyses provide not only a high-resolution roadmap for islet lineage development but also insights into the mechanisms of cellular heterogeneity, cell number expansion, and maturation of both ß and α cells.

Analysis on population health in Minhang District of Shanghai within circumstance of universal health / 复旦学报（医学版）

Objective To evaluate the population health for disease prevention and control in Shanghai Minhang District of Shanghai the data of mortality from 1993 to 2015 and communicable diseases from 2002 to 2015.Methods We adopted descriptive epidemiological method to analyze the trends of average life expectancy (ALE),specific death rate and causes of death cis-position from 1993 to 2015,and the incident rates of communicable diseases,incidence trends in Minhang District from 2002 to 2015.Results Overall,the ALE of population in Minhang District increased 11.80 years from 1993 to 2015 (from 71.78 years in 1993 to 83.58 years in 2015),including the ALE of male population increased 14.03 years (from 67.43 years in 1993 to 81.37 years in 2015) and the ALE of female population elevated 9.67 years (from 76.22 years in 1993 to 85.89 years in 2015).In 2015,Crude death rate (CDR) was 755.35/105,which was 21.45％ higher than in 1993 and 2.71％ higher than in 2014,respectively.In the same year,standardized mortality rate (SMR) was 196.07/105,which was 54.17％ lower than in 1993 and 0.51％ lower than in 2014.The top five leading causes of death were circulatory system diseases,tumors,respiratory diseases,endocrine and metabolic diseases,and injury and poisoning,which contributed 91.33％ of the population death.From 2002 to 2015,a total of 23 kinds of notifiable infectious diseases were reported in Minhang District,including 62 845 cumulative cases and 152 cases died.The total reported incidence rate of communicable diseases sharply elevated by 291.98％ during 14 years (Z=10 943.83,P＜0.001),and it increased after standardized.The top five communicable diseases were hand foot and mouth disease (HFMD),scarlet fever,syphilis,tuberculosis and hepatitis B in 2015.Conclusions Over the years,Minhang District of Shanghai comprehensive implemented "health in all policies" by integrating the resources of all levels of regional healthcare institutions.The ALE of the residents was at a high level.The control and prevention of chronic non-communicable diseases and major communicable diseases will continue to be the priority of public health.

Analysis on cause of death in residents of Minhang District of Shanghai from 1996 to 2015 / 复旦学报（医学版）

Objective To analyze changes and trends of the mortality and causes of death in the residents of Minhang District of Shanghai from 1996 to 2015.Methods Crude death rates (CDR) and age-standardized death rates (ADR) were calculated,respectively.Joinpoint regression was used to analyze the trends in the leading causes of death.Permutation test was used to find whether the joinpoints were statistically significant based on P value＜0.05.Results The elderly population in Minhang District accounted for 18.07％ of the total population in 2015,which increased by 73.89％ than it in 1996.The CDRs of all causes for resent 20 years gradually increased with the annual percentage change (APC) of 0.62％ (P＜0.05),but decreased significantly after standardization (APC =-3.73％,P＜0.05).In 2015,the top five causes of death were circulatory disease;neoplasms;respiratory disease;endocrine,nutritional and metabolic diseases;injury and poisoning in the total population,males and females registered in Minhang District.ADRs of circulatory disease,neoplasms,respiratory disease and injury and poisoning decreased to-3.16％,-1.86％,-8.03％ and -4.96 ％,respectively (P＜0.05).ADRs of endocrine,nutritional and metabolic diseases significantly increased during 1996 to 2001 (APC=16.58％,P＜0.05) and thereafter remained stable.Conclusions The issue of population aging in Minhang District is getting worse,and chronic non-communicable diseases and injury and poisoning can be identified as major public health concerns at present.

Analysis on population health in Minhang District of Shanghai within circumstance of universal health / 复旦学报（医学版）

Objective To evaluate the population health for disease prevention and control in Shanghai Minhang District of Shanghai the data of mortality from 1993 to 2015 and communicable diseases from 2002 to 2015.Methods We adopted descriptive epidemiological method to analyze the trends of average life expectancy (ALE),specific death rate and causes of death cis-position from 1993 to 2015,and the incident rates of communicable diseases,incidence trends in Minhang District from 2002 to 2015.Results Overall,the ALE of population in Minhang District increased 11.80 years from 1993 to 2015 (from 71.78 years in 1993 to 83.58 years in 2015),including the ALE of male population increased 14.03 years (from 67.43 years in 1993 to 81.37 years in 2015) and the ALE of female population elevated 9.67 years (from 76.22 years in 1993 to 85.89 years in 2015).In 2015,Crude death rate (CDR) was 755.35/105,which was 21.45％ higher than in 1993 and 2.71％ higher than in 2014,respectively.In the same year,standardized mortality rate (SMR) was 196.07/105,which was 54.17％ lower than in 1993 and 0.51％ lower than in 2014.The top five leading causes of death were circulatory system diseases,tumors,respiratory diseases,endocrine and metabolic diseases,and injury and poisoning,which contributed 91.33％ of the population death.From 2002 to 2015,a total of 23 kinds of notifiable infectious diseases were reported in Minhang District,including 62 845 cumulative cases and 152 cases died.The total reported incidence rate of communicable diseases sharply elevated by 291.98％ during 14 years (Z=10 943.83,P＜0.001),and it increased after standardized.The top five communicable diseases were hand foot and mouth disease (HFMD),scarlet fever,syphilis,tuberculosis and hepatitis B in 2015.Conclusions Over the years,Minhang District of Shanghai comprehensive implemented "health in all policies" by integrating the resources of all levels of regional healthcare institutions.The ALE of the residents was at a high level.The control and prevention of chronic non-communicable diseases and major communicable diseases will continue to be the priority of public health.

Analysis on cause of death in residents of Minhang District of Shanghai from 1996 to 2015 / 复旦学报（医学版）

Objective To analyze changes and trends of the mortality and causes of death in the residents of Minhang District of Shanghai from 1996 to 2015.Methods Crude death rates (CDR) and age-standardized death rates (ADR) were calculated,respectively.Joinpoint regression was used to analyze the trends in the leading causes of death.Permutation test was used to find whether the joinpoints were statistically significant based on P value＜0.05.Results The elderly population in Minhang District accounted for 18.07％ of the total population in 2015,which increased by 73.89％ than it in 1996.The CDRs of all causes for resent 20 years gradually increased with the annual percentage change (APC) of 0.62％ (P＜0.05),but decreased significantly after standardization (APC =-3.73％,P＜0.05).In 2015,the top five causes of death were circulatory disease;neoplasms;respiratory disease;endocrine,nutritional and metabolic diseases;injury and poisoning in the total population,males and females registered in Minhang District.ADRs of circulatory disease,neoplasms,respiratory disease and injury and poisoning decreased to-3.16％,-1.86％,-8.03％ and -4.96 ％,respectively (P＜0.05).ADRs of endocrine,nutritional and metabolic diseases significantly increased during 1996 to 2001 (APC=16.58％,P＜0.05) and thereafter remained stable.Conclusions The issue of population aging in Minhang District is getting worse,and chronic non-communicable diseases and injury and poisoning can be identified as major public health concerns at present.

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification.

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells.

[Therapeutic effect of ketogenic diet for refractory epilepsy in children: a prospective observational study].

OBJECTIVE: To study the clinical efficiency, electroencephalogram (EEG) changes and cognitive improvements of ketogenic diet (KD) in children with refractory epilepsy. METHODS: Twenty pediatric patients (7-61 months in age) with refractory epilepsy were recruited between August 2012 and August 2013. KD therapy was performed on all participants for at least 3 months based on a fasting initiation protocol with the lipid-to-nonlipid ratio being gradually increased to 4 : 1. Seizure frequency, type and degree were recorded before and during KD therapy. A 24 hours video-electroencephalogram (V-EEG) examination and Gesell Developmental Scale assessment were performed prior to KD therapy, and 3, 6, 9 months after KD therapy. RESULTS: Six patients became seizure free after KD therapy, with a complete control rate of 30%. Seizure frequency reduction occurred in 13 (65%) patients, EEG improvement in 8 (40%) patients, and improvement in Gesell Developmental Scales (gross motor and adaptability in particular) in 6 (30%) patients. The KD therapy-related side effects were mild. CONCLUSIONS: KD therapy is safety and effective in reducing seizure frequency and improving EEG and cognitive function in children with refractory epilepsy.

HDA18 affects cell fate in Arabidopsis root epidermis via histone acetylation at four kinase genes.

The differentiation of hair (H) and non-hair (N) cells in the Arabidopsis thaliana root epidermis is dependent on positional relationships with underlying cortical cells. We previously found that histone acetylation relays positional information and that a mutant altered in the histone deacetylase gene family member HISTONE DEACETYLASE 18 (HDA18) exhibits altered H and N epidermal cell patterning. Here, we report that HDA18 has in vitro histone deacetylase activity and that both mutation and overexpression of HDA18 led to cells at the N position having H fate. The HDA18 protein physically interacted with histones related to a specific group of kinase genes, which are demonstrated in this study to be components of a positional information relay system. Both down- and upregulation of HDA18 increased transcription of the targeted kinase genes. Interestingly, the acetylation levels of histone 3 lysine 9 (H3K9), histone 3 lysine 14 (H3K14) and histone 3 lysine 18 (H3K18) at the kinase genes were differentially affected by down- or upregulation of HDA18, which explains why the transcription levels of the four HDA18-target kinase genes increased in all lines with altered HDA18 expression. Our results reveal the surprisingly complex mechanism by which HDA18 affects cellular patterning in Arabidopsis root epidermis.