Cell Lineage Allocation Research Articles

Taurine, alpha lipoic acid and vitamin B6 ameliorate the reduced developmental competence of immature mouse oocytes exposed to methylglyoxal

Advanced glycation end products (AGEs) are the final products of the Maillard reaction, formed through the interaction of carbohydrates and proteins. Reactive dicarbonyl compounds such as methylglyoxal (MGO) serve as precursors for AGEs formation. Elevated levels of MGO/AGEs are observed in conditions like obesity, polycystic ovarian syndrome (PCOS), and diabetes, negatively impacting oocyte development. Previous studies have shown that hydrogen sulfide, a gasotransmitter with anti-AGEs effects, is produced in a process influenced by vitamin B6. R-α-lipoic acid (ALA) inhibits protein glycation and AGEs formation while stimulating glutathione (GSH) production. Taurine mitigates oxidative stress and acts as an anti-glycation compound, preventing in vitro glycation and AGEs accumulation. This study aimed to explore the ameliorative effects of a micronutrient support (Taurine, ALA and B6: TAB) on mouse oocytes challenged with MGO. Our results indicate that MGO reduces oocyte developmental competence, while TAB supplementation improves maturation, fertilization, and blastocyst formation rates. TAB also restores cell lineage allocation, redox balance and mitigates mitochondrial dysfunction in MGO-challenged oocytes. Furthermore, cumulus cells express key enzymes in the transsulfuration pathway, and TAB enhances their mRNA expression. However, TAB does not rescue MGO-induced damage in denuded oocytes, emphasizing the supportive role of cumulus cells. Overall, these findings suggest that TAB interventions may have significant implications for addressing reproductive dysfunctions associated with elevated MGO/AGEs levels. This study highlights the potential of TAB supplementation in preserving the developmental competence of COCs exposed to MGO stress, providing insights into mitigating the impact of dicarbonyl stress on oocyte quality and reproductive outcomes.

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

18S ribosomal RNA methyltransferase METTL5-mediated CDX2 translation regulates porcine early embryo development1

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

In vitro-produced equine blastocysts exhibit dispersed ICM cell allocation

Although in vitro production (IVP) of horse embryos has increased markedly in clinical practice, pregnancy rates after transfer are lower, and the incidence of pregnancy loss and monozygotic twins, higher than for in vivo-derived (IVD) embryos. Cell lineage specification is critical to normal early embryo development, and starts with differentiation of trophectoderm (TE) from inner cell mass (ICM) cells, followed by division of the ICM into primitive endoderm (PE) and epiblast (EPI). We examined the influence of developmental stage and speed, IVP and culture (in vivo versus in vitro) on expression of markers for the 3 early cell lineages, namely CDX-2 (TE), GATA-6 (PE) and SOX-2 (EPI). Day 7 IVD blastocysts (n=3 early; n=3 expanded), and IVP embryos identified as blastocysts on day 7 (n=5) or day 9 (n=9) were examined using confocal microscopy to compare total cell number, proportions and distribution of cells expressing CDX-2, GATA-6 and SOX-2. Additional day 7 IVP blastocysts were cultured for 2 days, either in vitro (n=5) or in vivo (after transfer into recipient mares; n=3), to examine the impact on subsequent development. Numerical data were analyzed using T- or Mann-Whitney U-tests. In IVD embryos, CDX-2 expression was exclusive to the TE, whereas SOX-2 expression was detected in both ICM and TE cells in early blastocysts but only thepresumed EPI in expanded blastocysts. GATA-6 was co-expressed with SOX-2 in outer ICM cells in early blastocysts but specific to PE cells lining the EPI and TE in expanded blastocysts. In IVP blastocysts, CDX-2 was also specific to the TE. However, SOX-2 and GATA-6 positive cells were intermingled in the ICM, and both SOX-2 and GATA-6 were co-expressed in TE cells. Strikingly, the EPI cells of IVP blastocysts were more dispersed, with larger mean inter-EPI cell distances (day 7 blastocysts, 52±6µm; day 9, 68±9µm), than in IVD embryos (35±3µm). Total cell number was higher in day 7 IVD (486±81) than IVP (day 7, 317±21; day 9, 377±104) blastocysts, but proportions of the different lineages didn't differ. Cell distribution in day 7 IVP embryos didn't change during an extra 2-day in vitro culture whereas, after 2 days in a mare's uterus, the SOX-2 (EPI) cells had aggregated and compacted, and segregated from PE cells. Overall, with respect to cell lineage allocation, IVP embryos resemble early IVD blastocysts in co-expressing SOX-2 and GATA-6 in TE cells, but differ by having a more dispersed ICM/EPI. While the EPI cells do compact following transfer to a recipient mare, the initial dispersal might contribute to both lower developmental competence (inadequate EPI aggregation) and a higher incidence of monozygotic twins (separated EPIs).

DMRT1 regulation of TOX3 modulates expansion of the gonadal steroidogenic cell lineage in the chicken embryo.

During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data indicate that that chicken steroidogenic cells, derive from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and down-regulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We identified the gene TOX3 as a novel transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1 positive Leydig cells. TOX3 over-expression in male and female gonads resulted in a significant decline in CYP17A1 positive steroidogenic cells. In ovo knock-down of the testis determinant, DMRT1, in male gonads resulted in a down-regulation of TOX3 expression. Conversely, DMRT1 over-expression caused an increase in TOX3 expression. Taken together, this data indicates that DMRT1 regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly via signaling from the supporting to steroidogenic cell populations.

Highly efficient generation of blastocyst-like structures from spliceosomes-repressed mouse totipotent blastomere-like cells.

Mammalian embryogenesis begins with a totipotent zygote. Blastocyst-like structures can be captured by aggregated cells with extended pluripotent properties in a three-dimensional (3D) culture system. However, the efficiency of generating blastoids is low, and it remains unclear whether other reported totipotent-like stem cells retain a similar capacity. In this study, we demonstrated that spliceosomal repression-induced totipotent blastomere-like cells (TBLCs) form blastocyst-like structures within around 80% of all microwells. In addition, we generated blastoids initiating from a single TBLC. TBLC-blastoids express specific markers of constituent cell lineages of a blastocyst and resemble blastocyst in cell-lineage allocation. Moreover, single-cell RNA sequencing revealed that TBLC-blastoids share a similar transcriptional profile to natural embryos, albeit composed of fewer primitive endoderm-like cells. Furthermore, TBLC-blastoids can develop beyond the implantation stage in vitro and induce decidualization in vivo. In summary, our findings provided an alternative cell type to efficiently generate blastoids for the study of early mouse embryogenesis.

Short-Term Exposure with Long-Term Effects: The Ketone Body, Beta-Hydroxybutyrate, Alters Mouse Embryo Developmental Programming and Reduces Viability

Background: Diet-induced nutritional changes in the maternal reproductive tract stimulate embryonic adaptations to maintain growth and survival, directly impacting child and adult health via developmental programming. This is plausibly facilitated by an interrelationship between metabolism and epigenetic control of gene expression, termed ‘metaboloepigenetics’, resulting in heritable modifications that affect long-term offspring health and viability. The ketogenic diet (KD) is increasingly popular amongst sub-fertile women trying to conceive, and results in elevated maternal ketone levels, including beta-hydroxybutyrate (bOHB). The impact of high bOHB levels on embryonic development, physiology, and developmental programming remains unknown. Aim: To assess how bOHB affects preimplantation mouse embryo development, metaboloepigenetic regulation, and post-transfer viability. Method: Preimplantation mouse embryos were cultured in vitro with or without 2 mM bOHB, representing serum concentrations with KD consumption. Day 5 blastocyst cell number and lineage allocation was assessed via differential nuclear stain, metabolism of bOHB and carbohydrates was assessed by ultramicrofluorescence, and acetylation of histone 3 lysine 9 (H3K9ac) and lysine 27 (H3K27ac) were assessed by immunofluorescence. Day 4 blastocysts were transferred to pseudo-pregnant females for analysis of embryonic day 14.5 placental and fetal development. Results: A reduction in total and trophectoderm cell number (P<0.05) was observed, indicating reduced viability. Blastocysts were shown to consume bOHB in vitro, with uptake increasing with bOHB concentration. Further, bOHB increased glycolytic rate (P<0.01), while epigenetic analyses revealed H3K9ac and H3K27ac were unaffected by bOHB. Of significance, post-transfer implantation rates were reduced (P<0.05), concurrent with smaller placental diameter (P<0.01) and fetal crown-rump length (P<0.05), indicating developmental programming alterations. Conclusion: These data infer that bOHB-stimulated metabolic alterations are a contributing factor to ketone-induced developmental programming, which may incorporate epigenetic components other than H3K9ac or H3K27ac. Therefore, consumption of a KD during conception and pregnancy may have negative impacts on the long-term viability and health of offspring.

In vitro investigation of mammalian peri-implantation embryogenesis†.

The embryos attach and invade the uterus, establishing the connection with their mother in peri-implantation development. During this period, the pluripotent epiblast cells of the embryo undergo symmetry breaking, cell lineage allocation, and morphogenetic remodeling, accompanied by the dramatic changes of transcriptomic, epigenomic, and signaling pathways, and preparing the stage for their differentiation and gastrulation. The progress in mouse genetics and stem cell biology has advanced the knowledge of these transformations, which are still largely hindered by the hard accessibility of natural embryos. To gain insight into mammalian peri-implantation development, much effort has been made in the field. Recently, advances in the prolonged in vitro culture of blastocysts, the derivation of multiple pluripotent stem cells, and the construction of stem cell-based embryo-like models have opened novel avenues to investigate peri-implantation development in mammals, especially humans. Combining with other emerging new technologies, these new models will substantially promote the comprehension of mammalian peri-implantation development, thus accelerating the progress of reproductive and regenerative medicine.

P–176 Effects of high non-esterified fatty acids exclusively during bovine in vitro fertilization on cell lineage allocation of blastocysts

Abstract Study question Are there effects of high non-esterified fatty acids exclusively during bovine in vitro fertilization on cell lineage allocation of blastocysts? Summary answer Under the conditions of the present study, high exposure to NEFA during bovine IVF significantly decreases embryo production and alters cell allocation of resultant blastocysts. What is known already Cattle models have shown that a high exposure to non-esterified fatty acids (NEFA) such as steric acid (SA), palmitic acid (PA]) and oleic acid (OA) during in vitro oocyte maturation and embryo development can disrupt both embryo formation and quality. However, the fertilization process per se have been less studied, which is needed to identify developmental stages were potential therapies could be develop to ameliorate NEFA toxicity during the periconceptional period Study design, size, duration Day–8 blastocysts were immunostained for CDX2, a transcription factor involved in trophectoderm differentiation, to examine cell allocation of blastocysts derived from oocytes fertilised under high NEFA levels. VF (19 h) was carried with different NEFA levels(4 replicates) representing physiological (Control–1[C1], 28μM SA, 23μM PA, 21μM OA) and pathophysiological (NEFA, 280μM SA, 230μM PA, 210μM OA) relevant concentrations. A second control (C2) group contained solvent. Blastocysts (C1; n = 14, C2; n = 12, NEFA; n = 8) Participants/materials, setting, methods All blastocysts were examined by confocal microscopy and cell counting was done with the Imaris software. Data were analysed by ANOVA (mean±SEM) with percentage data arcsine transformed before analysis. Main results and the role of chance Blastocyst formation was decreased by high NEFA levels (C1=25.6±2.7%, C2=26.0±2.3%, NEFA=9.4±0.4%, P < 0.001) which was associated with a decreased cleavage rate (C1=70.1±6.5%, C2=71.5±3.1%, NEFA=42.5±4.1%, P = 0.006) and an increase in embryo degeneration (C1=47.6±3.5%, C2=47.7±5.8%, NEFA=63.0±4.9%, P = 0.05). A lower total cell (TC) the number was observed in high NEFA-derived blastocyst (C1=125.2±6.6, C2=132.3±8.4, NEFA=67.3±5.6, P < 0.001) associated with a low cell number in both the trophectoderm (CDX2 positive cells, C1=90.2±5.9, C2=96.7±6.4, NEFA=41.3±4.1, P < 0.001) and the inner cell mass (ICM, C1=35.0±2.4, C2=35.7±3.5, NEFA=26.0±2.2, P < 0.001). Furthermore, high NEFA-derived blastocyst showed an increased allocation of cells towards the ICM (ICM/TC proportion, C1=28.2±1.7%, C2=26.9±1.8%, NEFA=39.1±2.2%, P < 0.001) Limitations, reasons for caution It will be better if the number of blastocysts reached increases. Wider implications of the findings: such research can be widely applied to the human model due to the similarities between both specie. Trial registration number Royal Embassy of Saudi Arabia Cultural Bureau

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Gonadal Sex Differentiation: Supporting Versus Steroidogenic Cell Lineage Specification in Mammals and Birds.

The gonads of vertebrate embryos are unique among organs because they have a developmental choice; ovary or testis formation. Given the importance of proper gonad formation for sexual development and reproduction, considerable research has been conducted over the years to elucidate the genetic and cellular mechanisms of gonad formation and sexual differentiation. While the molecular trigger for gonadal sex differentiation into ovary of testis can vary among vertebrates, from egg temperature to sex-chromosome linked master genes, the downstream molecular pathways are largely conserved. The cell biology of gonadal formation and differentiation has long thought to also be conserved. However, recent discoveries point to divergent mechanisms of gonad formation, at least among birds and mammals. In this mini-review, we provide an overview of cell lineage allocation during gonadal sex differentiation in the mouse model, focusing on the key supporting and steroidogenic cells and drawing on recent insights provided by single cell RNA-sequencing. We compare this data with emerging information in the chicken model. We highlight surprising differences in cell lineage specification between species and identify gaps in our current understanding of the cell biology underlying gonadogenesis.

SUPPLEMENTATION OF CULTURE MEDIUM WITH MELATONIN IMPROVES BLASTOCYST DEVELOPMENT AND REVERSES GLUCOSE INTOLERANCE IN IVF MICE

This study aimed to determine if the adverse effects of IVF on blastocyst and offspring could be reversed by supplementation of culture medium with melatonin in mice. A cross-sectional mouse model was utilized, wherein blastocysts were generated by natural mating (control group) or IVF with or without melatonin (10-6M) (mIVF and IVF group respectively) in clinical grade fertilization and culture media. Blastocysts were transferred to pseudo-pregnant ICR females. Cell lineage allocation was assessed for 30-40 blastocysts from each group. Body weight, glucose tolerance, energy expenditure, hepatic gene expression was measured in 6-10 mice from each group. C57BL/6 female mice and DBA2 male mice were used to generate blastocysts by natural mating (control group) or IVF with or without melatonin (10-6M) (mIVF and IVF group respectively) in clinical grade fertilization and culture media. Blastocysts were transferred to pseudo-pregnant recipients. Embryo development was recorded for IVF groups. Cell numbers of inner cell mass (SOX2) or trophectoderm (Cdx2+) were determined by immunohistochemistry. Males were weaned at 3 weeks of age onto a chow diet or a high-fat diet (60% fat) for 8 weeks. Glucose tolerance was assessed by an intraperitoneal glucose tolerance test (2g/Kg). Energy metabolism was examined in metabolic cages. Hepatic gene expression was measured by RNA-Seq and validated by real-time quantitative PCR. Blastocyst rates were similar in the two IVF groups. Reduced inner cell mass, trophectoderm and total cell number were observed in IVF blastocysts, compared with the control group (P<0.05). IVF pups had significantly lower birth weight and body weight before weaning, but exhibited increased body weight and liver weight at 11 weeks of age compared with controls (P<0.05), independently of diet. IVF mice were glucose intolerant shown as an increased glucose area under the curve, and had decreased energy expenditure as well as a large number of differentially expressed genes related to metabolic pathways in the liver tissue compared with the control group (P<0.05), independently of diet. The mIVF group showed intermediate cell numbers in blastocysts, body weight, liver weight and differentially expressed hepatic genes compared with the IVF group and control group, and normalized glucose tolerance and energy expenditure compared with the IVF group (P<0.05), independently of diet. Importantly, there was no significant difference in glucose tolerance and energy expenditure between the mIVF group and the control group. The data suggests that supplementation of culture medium with melatonin improved IVF blastocyst differentiation, reduced excessive weight gain after birth and normalized glucose intolerance in adult male offspring of IVF mice.

Advanced glycation end products present in the obese uterine environment compromise preimplantation embryo development

Research questionProinflammatory advanced glycation end products (AGE), highly elevated within the uterine cavity of obese women, compromise endometrial function. Do AGE also impact preimplantation embryo development and function? DesignMouse embryos were cultured in AGE equimolar to uterine fluid concentrations in lean (1–2 µmol/l) or obese (4–8 µmol/l) women. Differential nuclear staining identified cell allocation to inner cell mass (ICM) and trophectoderm (TE) (day 4 and 5 of culture). Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay (day 5). Day 4 embryos were placed on bovine serum albumin/fibronectin-coated plates and embryo outgrowth assessed 93 h later as a marker of implantation potential. AGE effects on cell lineage allocation were reassessed following pharmacological interventions: either 12.5 nmol/l AGE receptor (RAGE) antagonist; 0.1 nmol/l metformin; or combination of 10 µmol/l acetyl-l-carnitine, 10 µmol/l N-acetyl-l-cysteine, and 5 µmol/l alpha-lipoic acid. Results8 µmol/l AGE reduced: hatching rates (day 5, P < 0.01); total cell number (days 4, 5, P < 0.01); TE cell number (day 5, P < 0.01), and embryo outgrowth (P < 0.01). RAGE antagonism improved day 5 TE cell number. ConclusionsAGE equimolar with the obese uterine environment detrimentally impact preimplantation embryo development. In natural cycles, prolonged exposure to AGE may developmentally compromise embryos, whereas following assisted reproductive technology cycles, placement of a high-quality embryo into an adverse ‘high AGE’ environment may impede implantation success. The modest impact of short-term RAGE antagonism on improving embryo outcomes indicates preconception AGE reduction via pharmacological or dietary intervention may improve reproductive outcomes for overweight/obese women.

Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development invitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

SummaryCellular responses to transforming growth factor β (TGF-β) depend on cell context. Here, we explored how TGF-β/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

Comprehensive single cell mRNA profiling reveals a detailed roadmap for pancreatic endocrinogenesis.

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed Ngn3low endocrine progenitors, Ngn3high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7260 profiled Ngn3-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis in vivo.

Differential impacts of gonadotrophins, IVF and embryo culture on mouse blastocyst development.

Conception via assisted reproductive technology (ART) increases the risk of type 2 diabetes and cardiovascular disease in adulthood. Underlying differences between ART-conceived and in-vivo-conceived embryos that contribute to this increased risk are, however, not known. This study examined the developmental characteristics of mouse blastocysts derived from ART- compared with in-vivo-conceived embryos. To determine the effect of ovarian stimulation versus IVF versus in-vitro embryo culture on phenotype, six distinct groups of blastocysts were generated. Female mice were naturally cycling or treated with high or mild doses of gonadotrophin, followed by natural mating or IVF under clinical conditions. Embryo morphokinetics were assessed by continuous time-lapse monitoring. Cell lineage allocation to the inner cell mass (Oct4+) or trophectoderm (Cdx2+) was determined by immunohistochemistry, and mitochondrial DNA (mtDNA) copy number was measured by quantitative PCR. Ovarian stimulation increased embryo number but reduced the percentage of blastocysts. Morphokinetic analysis showed that gonadotrophin treatment led to advanced development (P<0.05) due to earlier post-pronuclear breakdown. The blastocyst rate was reduced in IVF embryos compared with those fertilized in vivo before culture (P<0.001). Morphokinetics showed that embryo development was slower in all the IVF groups (P<<0.05), due to a delay from the 3-cell stage. A reduced total and trophectoderm cell number was observed in all groups of cultured blastocysts compared with naturally conceived blastocysts (P<0.01). Gonadotrophin treatment did not affect the blastocyst mtDNA copy number; however, IVF embryos exhibited reduced mtDNA copy number compared with naturally conceived embryos. Ovarian stimulation, IVF and in-vitro culture differentially impair blastocyst developmental kinetics, differentiation and mtDNA copy number.

Systems based analysis of human embryos and gene networks involved in cell lineage allocation

BackgroundLittle is understood of the molecular mechanisms involved in the earliest cell fate decision in human development, leading to the establishment of the trophectoderm (TE) and inner cell mass (ICM) stem cell population. Notably, there is a lack of understanding of how transcriptional networks arise during reorganisation of the embryonic genome post-fertilisation.ResultsWe identified a hierarchical structure of preimplantation gene network modules around the time of embryonic genome activation (EGA). Using network models along with eukaryotic initiation factor (EIF) and epigenetic-associated gene expression we defined two sets of blastomeres that exhibited diverging tendencies towards ICM or TE. Analysis of the developmental networks demonstrated stage specific EIF expression and revealed that histone modifications may be an important epigenetic regulatory mechanism in preimplantation human embryos. Comparison to published RNAseq data confirmed that during EGA the individual 8-cell blastomeres are transcriptionally primed for the first lineage decision in development towards ICM or TE.ConclusionsUsing multiple systems biology approaches to compare developmental stages in the early human embryo with single cell transcript data from blastomeres, we have shown that blastomeres considered to be totipotent are not transcriptionally equivalent. Furthermore we have linked the developmental interactome to individual blastomeres and to later cell lineage. This has clinical implications for understanding the impact of fertility treatments and developmental programming of long term health.

Mist1 Expression Is Required for Paneth Cell Maturation.

BackgroundPaneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells.MethodsMist1+/+ and Mist1–/– mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function.ResultsAbsence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1–/– mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo.ConclusionsMIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.

Sox4 Promotes Atoh1-Independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates

The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation. We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation. Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1. We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection.