Human embryonic development begins with fertilized eggs, passes through pre-implantation embryo development, the formation of gastrulation and organogenesis, and finally the newborn is born. Human embryonic development from a single cell to trillions of cells, lasted 280 days. The entire process of gene expression is regulated by a variety of factors, many mechanisms of which are not clear.

 

In order to analyze the gene expression regulation network at all stages of human fetal development, since 2010, Fuchou Tang’s group from the Biomedical Pioneering Innovation Center of Peking University and Jie Qiao’s group from the Third Hospital of Peking University have been working closely together to systematically carry out researches on the gene expression regulation mechanism for the development of human reproductive cells (including pre-implantation embryonic cells and post-implantation embryonic reproductive cells) (Figure 1).

Figure 1 An overview of the cooperation between Fuchou Tang’s group and Jie Qiao’s group

 

First, using high throughput single-cell RNA sequencing technology developed by Fuchou Tang’s group, the Fuchou Tang’s group and the Jie Qiao’s group dissected the developmental maps of human reproductive cells, including the developmental maps of pre-implantation embryos (Yan et al., Nature And Molecular Biology, 2013; Dang et al., Genome Biology, 2016) and primordial germ cells(Guo et al., Cell, Cell, 2015; Li et al., Cell Stem Cell, 2017), conducting an in-depth study of epigenetic regulation mechanisms at different levels of DNA methylation, chromatin state, etc. (Guo et al., Nature, 2014; Guo et al., Cell Research, 2017; Zhu et al., Nature Genetics, 2018; Li et al., Nature Cell Biology, 2018). The two teams also worked with the Zhao Xiaoyang from Southern Medical University of Guangdong to complete the cell fate transformation and gene expression mapping during spermatogenesis in adult male humans (Wang et al., Cell Stem Cell, 2018).

 

Since then, the two teams have conducted a comprehensive study of the developmental landscape of human reproductive cells and various important organs of non-reproductive lines, with the ultimate goal to dissect a high-precision developmental landscape of all major human fetal organs at all critical developmental stages. In 2018, they collaborated with Xiaoqun Wang, a group from the Institute of Biophysics of the Chinese Academy of Sciences, to map the first single cell map of the development of the human prefrontal cortex(Zhong et al., Nature, 2018). They also conducted a single-cell transcriptome study of all major brain regions of the cerebral cortex in human embryos, revealing important characteristics of regionalized gene expression and neuronal maturation of the cerebral cortex (Fan et al., Cell Research, 2018). The above work has laid an important foundation for mapping the complete developmental landscape of human brain. At the same time, the two teams studied the gene expression maps of the four main endodermal organs in the digestive tract (esophagus, stomach, small intestine and large intestine), further analyzing the development, maturation path and key biological characteristics of human large intestine from the fetus to the adult(Gao et al.Nature, Cell Cell Biology, 2018). In addition, the two teams studied the development of human mesodermal organs, such as kidney and heart. For the kidney, they studied the process of cell differentiation and development of complete kidney units(Wang et al., Cell Reports, 2018). For the heart, they revealed the location-specific characteristics of key signaling pathways during cardiac development and the complex signaling interaction between cardiomyocytes and non-cardiomyocytes, providing important clues for the study of cardiac developmental regeneration (Cui et al., Cell Reports, 2018).

 

On July 3, 2019, a new work was added to the developmental atlas of the major human organs. Fuchou Tang’s and Jie Qiao’s groups published a paper titled "Dissecting the transcriptome landscape of the human fetal neural retina and retinal pigment epithelium by single-cell RNA-seq analysis" on Plos Biology, which revealed the development of the retina from the ectoderm.

The eye is the window to the mind, and the retina can convert the outside light into a nerve signal, which is an important part of the eye's function to sense the light. The inner layer of the retina is the nerve layer (mainly comprised of retinal ganglion cells (RGCs); interneurons such as horizontal cells (HCs), amacrine cells (ACs), and bipolar cells (BCs); and rod and cone photoreceptor cells (PCs), as well as glial cells and Müller glia cells), and the outer layer is the pigment epithelium layer (as shown in Figure 2). At present, a large number of mouse-model based studies have revealed the development of the retina. However, the molecular mechanism of the development of the human fetal neural retina and retinal pigment epithelium as well as the cellular interactions between these two layers are not yet clear. Fuchou Tang’s and Jie Qiao’s groups sequenced more than 2,400 cells from the neural retina(NR) and retinal pigment epithelium(RPE) of human embryos(ranging from 5-24 weeks). The main findings are:

Figure 2 The schematic diagram of human retina

 

The differences of molecular basis between the NR tissue and the RPE tissue during the developmental stages

The study sampled cells from the neural retina and retinal pigment epithelium separately. The transcriptome analysis (shown in Figure 3) showed the neural retina expressed genes associated with the development of the nervous system and retinal pigment epithelium expressed genes associated with retinol metabolism. With the development of the embryo, both tissues have gone from a proliferative state to a mature state. Both are involved in the visual perception of the retina in the latter stages of development, suggesting that they may have functional interactions in the later stages of development.

Figure 3 The transcriptome features of the neural retina and retinal pigment epithelium

 

Then, They revealed markers of human retinal cells and dissected the temporal order of the generation of retinal cells in vivo(shown in Figure 4). In humans, RGCs were already present at 5 W and peaked at 8 W. HCs were detected at 7 W and peaked at 9 W. Subsequently, ACs peaked at 17 W, followed by PCs. BCs and Müller glia cells were observed last.

Figure 4 The temporal order of the generation of human retinal cells. AC, amacrine cell; BC, bipolar cell; HC, horizontal cell; NR, neural retina; PC, photoreceptor cell; RGC, retinal ganglion cell; RPC, retinal progenitor cell; RPE, retinal pigment epithelium.

 

The expression patterns of transcription factors in the human NR and RPE

An important biological issue in the study of retinal development is the mechanism of cell fate determination regulated by transcription factors. This study provides a map of key transcription factors. These transcription factors are highly active in retinal neural cells and pigment epithelial cells, and their target genes are involved in important events of retinal development (Figure 5). For example, transcription factor RAX2 is primarily active in photoreceptor cells, and with the development of the retina, the expression of some RAX2 target genes increases. These genes participate in the detection of visual perception and light stimulation, which is consistent with the function of photoreceptor cells. MITF has high activity only in pigment epithelial cells, and its target genes are involved in the transport of amino acids and neural tube closure at an early stage, and in the cation transport at the late stages. Throughout the development, a portion of MITF's target genes are continuously expressed, and these genes are involved in pigmentation and cell motion regulation. The above events in which MITF target genes are involved are consistent with the function of retinal pigment epithelial cells.

Figure 5 The expression patterns of representative transcription factors’ target genes across developmental stages (left) and the gene ontology analysis of these genes (right).

 

Clues about interactions of RPE cells with photoreceptors

The study provides many clues about the interaction of RPE cells with photoreceptor cells (Figure 6). In the adult retina, RPE cells and photoreceptor cells participate in the visual cycle and light transduction pathway. During the development of human embryos, the expression of rhodopsin was first detected at 24W, while the visual cycle genes in RPE cells have already existed at 13W. Light transduction was reported to begin with photon absorption by rhodopsin. These results indicated that visual cycle might begin before vision started. The study also detected the expression of pairs of receptors and ligand genes in RPE cells and photoreceptor cells, which also provides important clues to the study of the interaction of the two cells.

Figure 6 Interactions of RPE cells with photoreceptors

 

Profiling of inherited retinal disease–related genes in human fetal retinal cells

The study mapped the expression of inherited retinal disease–related genes in human fetal retinal cells. The results show that most disease-related genes are enriched in photoreceptor cells, bipolar cells, and pigment epithelial cells. In Chinese patients, USH2A, EYS and CRB1 are reported to contribute the most to inherited retinal dystrophy. In the human fetal retina, USH2A is expressed mainly in photoreceptor cells. EYS is highly expressed in bipolar and photoreceptor cells, and CRB1 is concentrated in Müller glia cells and retinal progenitor cells. Retinal progenitor cells are critical to retinal formation, and this study found that retinal progenitor cells also expresss CLRN1, KCNJ13 and KIF11.Mutations of inherited retinal disease–related genes in retinal progenitor cells may be important clues to the process of inherited retinal diseases.

 

In summary, this study mapped a high-precision human retinal single-cell transcriptome, and analyzed the developmental events in which cell-type specific transcription factors and their target genes were involved. Fuchou Tang’s and Jie Qiao’s groups explored the ways in which the neural retina layer interacted with the retinal pigment epithelium layer during development, and also  provided a map of disease-related genes for human fetal retinal cells. The results provide insightful clues for future studies on not only the development of the human NR and RPE, but also in the treatment of human inherited retinal disease.

 

Yuqiong Hu, postdoctoral fellow from Biomedical Pioneering Innovation Center of Peking University, Xiaoye Wang from the Third Hospital of Peking University, Boqiang Hu Ph.D., Ph.D. Candidates   Yuno Mao and Yidong Chen from Biomedical Pioneering Innovation Center of Peking University, were the co-authors of the paper. Professor Fuchou Tang and Professor Jie Qiao are corresponding authors. The work is supported by grants from the National Basic Research Program of China, the National Natural Science Foundation of China, Biomedical Pioneering Innovation Center of Peking University and Peking-Tsinghua Center for Life Sciences.