Plant-on-chip: Core morphogenesis processes in the tiny plant Wolffia australiana.

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.

Single-cell RNA sequencing reveals abnormal fluctuations in human eight-cell embryos associated with blastocyst formation failure.

Infertility has become a global health issue, with the number of people suffering from the disease increasing year by year, and ART offering great promise for infertility treatment. However, the regulation of early embryonic development is complicated and a series of processes takes place, including the maternal-to-zygotic transition. In addition, developmental arrest is frequently observed during human early embryonic development. In this study, we performed single-cell RNA sequencing on a biopsied blastomere from human eight-cell embryos and tracked the developmental potential of the remaining cells. To compare the sequencing results between different eight-cell embryos, we have combined the research data of this project with the data previously shared in the database and found that cells from the same embryo showed a higher correlation. Additionally, the transcriptome of embryos with blastocyst formation failure was significantly different from developed embryos, and the gene expression as well as cell signaling pathways related to embryonic development were also altered. In particular, the expression of some maternal and zygotic genes in the failed blastocyst formation group was significantly altered: the overall expression level of maternal genes was significantly higher in the failed blastocyst than the developed blastocyst group. In general, these findings provide clues for the causes of human embryonic arrest after the eight-cell stage, and they also provide new ideas for improving the success rate of ART in clinical practice.

Single-cell analyses identify distinct and intermediate states of zebrafish pancreatic islet development.

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA.

Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.

Comparison of the method establishing embryonic stem cell lines from five different mouse strains.

We compared the characteristics of the method establishing embryonic stem cell lines from five different mouse strains using the medium containing 70% rat heart cell-conditioned medium (RH-CM) as ES cell culture medium, using the primary murine embryo fibroblast as feeder cells, and using the digestive enzyme buffer containing 1% chicken serum and "the series digestive method". We first reported new ES cell lines established from the outbred strain mice KM and ICR using the improved method in our lab and the ratio of establishment of ES cell lines from KM and ICR strain mice is up to 12% and 42.1% respectively. Compared with routine method of establishing ES cell lines, the improved method made distinct differences, increasing the ratio of ES cell line's establishment of 129/ter mouse from 11.8% to 33.3%, that of C57BL/6J mouse from 3.7% to 13.3%, that of BALB/c mouse from 2.9% to 19.4%. We tested the appropriate dispersing occasion, that is proliferating period of the ICM, affected the formation of ES clones and the ratios of ES cell lines established. It was shown that the most appropriate dispersed occasion for the ICM of 129/ter, C57BL/6J, BALB/c, KM and ICR mice was 4-6 d, 3-3.5 d, 4 d, 4-5 d, 4-5 d after ICM proliferation respectively. At the same time, the effects of the concentration of digestive enzyme buffer were discussed. It was found that the ES cells from BALB/c mice were sensitive to the high concentration of digestive enzyme buffer and the 0.05% Trypsin-0.008% EDTA is an ideal concentration for their establishment and maintenance. It was shown that 'the series dispersed method' was much better than 'the once dispersed method' on the aspect of dispersing the proliferating ICM and formation of ES clones. Compared with the routine ES cell culture medium containing mLIF, the RH-CM not only remarkably inhibited the differentiation of murine ES cells and maintained their diploid karyotype, but also promoted the attachment and growth of ES cells. This improved method of establishment and culture of ES cell lines effectively maintained a series of their characteristics of pluripotent embryonic stem cells.

[RNA interference directed by small hairpin RNA expressed in COS-7 cells].

RNA interference is a phenomenon of gene silencing directed by double-stranded RNA. It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in Caenorhabditis elegans, Drosophila melanogaster, etc. For mammalian cells, dsRNA directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma (EC) cells. Our previous work proved the existence of RNAi effect for reporter gene GFP and endogenous gene Oct4 in undifferentiated murine ES cells. Yet in other kinds of mammalian cells, because of the existence of interferon pathway, long dsRNA will induce the cells to shutdown global protein translation and go to apoptosis. Therefore, dsRNA longer than 30 bp cannot be used to induce specific gene knockdown effect in these cells. Elbashir et al found that in vitro synthesized small interfering RNA (siRNA) (19-23 nt) could induce potent RNAi as effective as long dsRNA without showing unspecific effect, so that the interferon pathway could be bypassed. It was shown that during RNAi process, long dsRNA was first degraded into 19-23 nt siRNA and then recruited into RISC (RNA induced silencing complex) to degrade corresponding mRNA. However, the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about a week. Recently, it has been shown that U6 promoter directed small hairpin RNA (shRNA) can induce potent gene knockdown effect in murine P19 Embryonic Carcinoma cell. The RNAi effect of U6 promoter-driven shRNA corresponding to Green Fluorescence Protein (GFP) in COS-7 cells was checked. And it was found that the U6 promoter-driven shRNA for GFP can specifically and potently knockdown the GFP's expression in COS-7 cells. The result established the feasibility of using RNAi technique directed by U6 promoter-driven shRNA to study genes' function in COS-7 cell line.

[RNA interference in three ES cell lines from different mouse strains].

RNA interference phenomenon in three different murine ES cell lines (MESPU13, B3, and R1) is reported. A vector(pdsGFP) was used that transcribed hairpin double-stranded RNA of GFP gene to transfect ES cells by using lipofectin. The transient transcription of dsRNA induced RNAi (RNA interference) in the ES cells. That is, the double-stranded RNA of GFP gene potently turned down the expression of the GFP gene. On the hand, the linearized plasmid pdsGFP-puro was electroporated into MESPU13 ES cells, and the expression level of GFP after puromycin screening was turned down obviously in about 30% ES cell clones; and in a few clones, the expression level of GFP was not observed under the fluorescence microscope and GFP mRNA was not detectable by RT-PCR. Further more, another vector (pdsOCT4) was constructed that transcribed double-stranded RNA of OCT-4 gene which is specifically expressed in ES cells. ES cell clones that stably integrated the vector were screened after the electrotransfection of the cells with the above construct. 51 random-selected clones were amplified and 48 of them were checked by semi-quantitative RT-PCR. In 11 of them the mRNA of OCT-4 was undetectable by RT-PCR. This means that RNAi can be used to study mammal and human gene's function in ES cell lines from different strain mice.

[Discussion of the methods for establishing embryonic stem cell lines from 129/ter. C57BL/6J mice with high efficiency].

A new method for establishing ES cell lines from 129/ter. C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM) for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.