In a recent study published in Nature cardiovascular researchresearchers from California used multimodal single-cell analysis in mice to investigate the mechanisms by which maternal diabetes contributes to congenital malformations in the fetus.
They found that during embryonic development, maternal diabetes alters the epigenomic landscape in cardiac and craniofacial progenitors, leading to developmental defects.
Stady: Multimodal single-cell analyzes reveal the epigenetic and transcriptional basis of birth defects in maternal diabetes. Image credit: com. adriaticfoto/ shutterstock.com
background
Congenital cardiac and craniofacial disabilities often occur together due to shared progenitors and cross-signaling between these cells. There are many genetic and environmental factors that can affect congenital disabilities in offspring.
Pregestational diabetes mellitus (PGDM) is one such factor associated with a fivefold higher incidence of these defects. Likewise, exposure to retinoic acid (RA) can negatively affect the cardiac and craniofacial regions.
Although the mechanisms linking hyperglycemia to congenital disabilities are not fully understood, oxidative stress and metabolic changes leading to epigenomic alterations are implicated.
To answer this question, the researchers in this study used single-cell analysis during mouse development to study cell-specific epigenomic changes and potential mechanisms that lead to congenital disabilities associated with maternal diabetes.
About the study
The present study used the following mouse strains: wild-type C57BL/6J, RARE–hsp68LacZ, B6.Cg–Gt(ROSA)26Sortm6(CAG–ZsGreen1)Hze/J (Ai6), Mef2c–AHF–Cre, and Alx3. -Cre. To understand how PGDM affects fetal cardiac and pharyngeal development, diabetes was induced in female mice with streptozotocin (STZ) given intraperitoneally for five days.
Females with hyperglycemia (>250 mg/dL) were mated with untreated males, and the resulting embryos were extracted at embryonic day 10.5. Vehicle-treated (VEH) mice were used as controls. The shape of the heart was imaged using micro-CT.
Furthermore, single-cell RNA sequencing (scRNA-seq) and single-cell transposase-accessible chromatin sequencing (scATAC-seq) assay were performed on the cardiac and pharyngeal regions of dissected embryos.
Peak calling was performed for each scATAC-seq array, and differentially accessible chromatin regions (DARs) were identified. Various bioinformatics tools were used at different stages of the analysis, including but not limited to Seurat, Gene Co-Expression Network Analysis (WGCNA), Cell Ranger, and ArchR.
In addition, in situ hybridization experiments, luciferase assay, and X-gal staining were performed on tissues. No samples were excluded in the statistical analysis, and three independent embryos were analyzed in gene expression experiments.
results
Compared with controls, fetuses from STZ-treated mice showed an increased frequency of defects in the cone-trunk ventricular septum, atrial septum, outflow tract (OFT), neural tube, skull, and face. scATAC-seq analysis indicates that PGDM affects the distribution of cell populations in cardiac and craniofacial defects.
A significant increase in the number of cardiopharyngeal mesoderm progenitors was observed, along with a decrease in neural crest derivatives and differentiated cardiomyocytes. A total of 4324 DARs were identified, mainly in cardiac and mesopharyngeal progenitors (48.8%) and neural crest-derived cells (48.5%).
The results suggest that PGDM induces cell-specific changes in embryonic chromatin accessibility. Transcriptional and epigenomic changes were observed in pharyngeal arch 2 (PA2) and Six2-high PA4/PA6 cells, suggesting a less differentiated state.
Furthermore, multimodal analysis of neural crest cells at single-cell resolution showed that a relatively undifferentiated subset of PA2 neural crest cells are genetically modified by PGDM. This leads to transcriptional dysregulation of genes involved in patterning, cell migration, and cellular differentiation.
Examination of regulatory elements within these specific cell types revealed dysregulation of RA and downstream homeobox signaling (hawks) Genes in AHF2 (abbreviation for anterior heart field 2) and PA2. This aberrant RA activity led to dysregulation of patterning and loss of cell-specific transcriptional signatures.
In addition, maternal diabetes has been found to cause genetic changes. It led to increased “lag” in cardiac progenitors that express a protein called Alx3 (short for aristalis-like homeobox 3), especially in the newly identified AHF2 subset, affecting anterior-posterior (AP) patterning.
According to the study, Alx3-positive AHF2 cells contribute to specific defects in the OFT and atrium. Under hyperglycemic conditions, AHF2 cells transition toward a skeletal-like state with increased HoXP1 (homeobox gene B1), suggesting a possible mechanism for the abnormal AP pattern in heart development.
Conclusion
In conclusion, the study shows that although all fetal cells experience similar environmental exposure in the form of PGDM and associated hyperglycemia, only a small subset of cells show epigenomic vulnerability, which may explain congenital craniofacial and cardiac disabilities in Offspring.
It demonstrates the utility of multimodal single-cell analysis as a tool to study the influence of environmental factors on embryonic development. More research needs to be done to determine why only certain types of cells show higher sensitivity to environmental influences than others.
In addition, given the high incidence of PGDM globally, it will be important to further investigate the mechanisms underlying the epigenomic and transcriptional changes associated with the condition to help implement potential therapeutic approaches during fetal development.
