Restricted Developmental Potential Research Articles

P-155 Reproductive Potential of Metaphase-I (M-I) Intra Cytoplasmic Sperm Injection (ICSI)

Abstract Study question To evaluate the reproductive potential of performing ICSI with metaphase-I (M-I) oocytes derived from In Vitro Fertilization (IVF)-ICSI cycles Summary answer M-I ICSI oocytes can fertilize and develop into blastocysts, but with low rates and limited efficiency in producing more embryos, highlighting challenges in reproductive outcomes. What is known already ICSI for initially immature oocytes undergoing in vitro maturation in IVF cycles is a topic of controversy with varying practices. While it has the potential to increase usable embryos, it may be time-intensive and yield uncertain results. Assessing the maturation status of M-I oocytes multiple times on collection day is challenging. If viable embryos can be obtained after ICSI with M-I oocytes, it simplifies workload management in busy embryology labs for patients with a limited number of mature oocytes. Despite a few reported cases of healthy live births resulting from ICSI with M-I oocytes, their reproductive potential remains inadequately explored. Study design, size, duration The study included IVF cycles (n = 929) with both immature and mature oocytes from Jan, 2022-Dec, 2023. Fertilization and blastocyst were assessed based on oocyte maturity. Participants/materials, setting, methods The study included IVF-ICSI cycles obtaining at least one immature (Germinal vesicle (GV), M-I) and one mature (M-II) oocyte at oocyte denudation. Analyzing 9,793 inseminated oocytes from 929 women (mean age: 34.0), we divided them into groups: M-II (Group 1: n = 7678), M-I at ICSI (Group 2; n = 835), M-IàM-II at ICSI (Group 3; n = 643), M-IàM-II on day 1 (Group 4; n = 129), and GVàM-II on day 1 (Group 5: n = 508). Fertilization and blastocyst development were then examined. Main results and the role of chance Overall, embryos originating from initially immature oocytes exhibited lower cleavage and blastulation rates compared to those from initially mature oocytes (P < 0.05). In Group 2, where ICSI was performed with M-I oocytes, fertilization and blastocyst rates were 23.6% and 16.1%, respectively. However, these rates were significantly lower than in other groups (P < 0.05). In Group 3, where ICSI was performed with M-II oocytes matured from M-I within 2 hours after oocyte denudation, the fertilization rate per oocyte (55.2%) was lower than in Group 4 (75.7%) (P < 0.05). However, the blastocyst rate per 2PN embryo was higher (30.7%) in Group 3 than that of Group 4 (18.0%) (P < 0.05). The blastocyst rate from the inseminated oocytes was directly associated with oocyte maturation: 29.3% (2250/7678) for in vivo M-II, 14.9% (96/643) for Group 3, 11.6% (15/129) for Group 4, 8.9% (45/508) for Group 5, and 2.9% (24/835) for M-I (Group 2). There were few pregnancies resulting from fresh transfers with only embryos produced from day 1 M-II oocytes (3/18=16.7%). While there were pregnancies after transferring only embryos generated from M-II matured from M-I on day 0 (4/10=40%), no patients transferred only embryos produced from M-I oocytes. Limitations, reasons for caution Despite the study possessing a substantial sample size, limited availability of embryos for fresh transfers arises from the deprioritization of those originating from immature oocytes. Consequently, it is crucial to conduct further research involving frozen embryo transfers and chromosome analysis to determine the efficacy of M-I ICSI. Wider implications of the findings Immature M-I oocytes exhibit a blastocyst formation rate of less than 3%. Consequently, these immature M-I oocytes demonstrate very limited developmental potential in progressing to the blastocyst stage, and ICSI for the M-I stage should be re-evaluated. Trial registration number Bnoon20240101

P–204 Dynamics of cavitation as a potential non-invasive predictor of mammalian blastocyst quality

Abstract Study question We wished to investigate whether dynamics of cavity formation can be used in embryo quality assessment. Summary answer Dynamics of mouse embryo cavitation reflects to certain extent blastocysts’ developmental capabilities. It can be potentially used as a biomarker of mammalian embryo quality. What is known already During cavity expansion blastocyst pulsates, i.e. changes its volume in an oscillatory way. Recent studies performed on a mouse model have shown, that dynamics of cavitation, biomechanical properties of the trophectoderm (TE) and embryo size are intertwined. Presence or absence of blastocyst contractions has been linked to particular parameters related to positive outcome of the in vitro fertilization procedures, but the data on influence of contractions on human embryos’ developmental capabilities is often contradictory. Moreover, mostly in those studies only strong contractions (leading to a high volume loss) have been taken into consideration. Study design, size, duration We tested how postovulatory (in vitro or in vivo) or maternal aging of mouse oocytes affects dynamics of cavity formation and expansion in the resulting embryos (n = 27, n = 26 and n = 30, respectively). Furthermore, we also analyzed almost 100 mouse blastocysts in order to correlate dynamics of their cavitation with their ability to form correct outgrowths (in vitro model of implantation). Participants/materials, setting, methods Mouse oocytes subjected to postovulatory (either in vivo or in vitro) or maternal aging were fertilized in vitro. Dynamics of cavity formation and expansion was assessed by time-lapse imaging; equatorial images were taken every 10 minutes. Blastocyst area was measured over time and compared to the outcome from control embryos. In another set of experiments, after the filming mouse blastocysts were cultured for additional 4 days to test their ability to form outgrowths. Main results and the role of chance We noticed, that mouse embryos which represent limited developmental potential (obtained from either postovulatory or maternally aged oocytes) and blastocysts developed from freshly fertilized young females’ oocytes differ in terms of some parameters related to dynamics of cavitation, e.g. time of the initiation of cavity formation, frequency of contractions or mean loss of blastocyst’s area during contraction. We observed that embryos obtained from oocytes subjected to maternal or postovulatory aging have distinct dynamics of cavitation. Moreover, we noticed slightly different effect on particular parameters related to cavitation between in vivo and in vitro version of postovulatory ageing. We also showed that blastocysts, which are unable to create proper outgrowths (i.e. too small or without epiblast cells), differ from embryos that differentiate into correct outgrowths in terms of certain parameters of cavitation dynamics. Our data indicates, that dynamics of cavity formation and expansion might be related to developmental potential of mouse embryo. Limitations, reasons for caution Further studies with extended group size and testing embryos’ ability to implant in vivo are required to confirm our results. Moreover, we examined dynamics of cavitation only in a mouse model, so additional studies performed on other mammalian species are needed. Wider implications of the findings: Our data proves, that dynamics of embryo cavitation reflects, to certain extent, developmental capabilities of mouse blastocysts. Therefore, it is possible that it can be a biomarker of embryo quality (in combination with parameters provided by other methods or solely) of other mammalian species, including humans. Trial registration number Not applicable

From head to tail: regionalization of the neural crest.

The neural crest is regionalized along the anteroposterior axis, as demonstrated by foundational lineage-tracing experiments that showed the restricted developmental potential of neural crest cells originating in the head. Here, we explore how recent studies of experimental embryology, genetic circuits and stem cell differentiation have shaped our understanding of the mechanisms that establish axial-specific populations of neural crest cells. Additionally, we evaluate how comparative, anatomical and genomic approaches have informed our current understanding of the evolution of the neural crest and its contribution to the vertebrate body.

Identification of a Siglec-F+ granulocyte-macrophage progenitor.

In recent years multi-parameter flow cytometry has enabled identification of cells at major stages in myeloid development; from pluripotent hematopoietic stem cells, through populations with increasingly limited developmental potential (common myeloid progenitors and granulocyte-macrophage progenitors), to terminally differentiated mature cells. Myeloid progenitors are heterogeneous, and the surface markers that define transition states from progenitors to mature cells are poorly characterized. Siglec-F is a surface glycoprotein frequently used in combination with IL-5 receptor alpha (IL5Rα) for the identification of murine eosinophils. Here, we describe a CD11b+ Siglec-F+ IL5Rα− myeloid population in the bone marrow of C57BL/6 mice. The CD11b+ Siglec-F+ IL5Rα− cells are retained in eosinophil deficient PHIL mice, and are not expanded upon overexpression of IL-5, indicating that they are upstream or independent of the eosinophil lineage. We show these cells to have GMP-like developmental potential in vitro and in vivo, and to be transcriptionally distinct from the classically described GMP population. The CD11b+ Siglec-F+ IL5Rα− population expands in the bone marrow of Myb mutant mice, which is potentially due to negative transcriptional regulation of Siglec-F by Myb. Lastly, we show that the role of Siglec-F may be, at least in part, to regulate GMP viability.

Stem Cell Therapies for Neurodegenerative Diseases.

Stem cell therapies have been proposed as a treatment option for neurodegenerative diseases, but the best stem cell source and therapeutic efficacy for neuroregeneration remain uncertain. Embryonic stem cells (ESCs) and neural stem cells (NSCs), which can efficiently generate neural cells, could be good candidates but they pose ethical and practical issues. Not only difficult to find the good source of those cells but also they alway pose immunorejection problem since they may not be an autologous cells. Even if we overcome the immunorejection problem, it has also been reported that transplantation of ESCs develop teratoma. Although adult stem cells are more accessible, they have a limited developmental potential. We developed technologies to increase potency of mesenchymal stem cells, which allow them to develop into neural cells, by over expression of the ESC gene, nanog. We also developed a small molecule compound, which significantly increases endogenous NSCs by peripheral administration, eliminating even the necessity of stem cell injection to the brain. These novel technologies may offer neuroregenerative therapies for Alzheimers disease (AD). However, we found that AD pathological condition prevent neurogenesis from NSCs. This chapter discusses how to overcome the problem associated stem cell therapy under AD pathology and introduces exosome as a tool to improve the modification of adult stem cells. These new technologies may open a door for the new era for AD therapy.

Capturing and Interconverting Embryonic Cell Fates in a Dish.

Cells of the early embryo are totipotent because they will differentiate to produce the fetus and its surrounding extraembryonic tissues. By contrast, embryonic stem (ES) cells are considered to be merely pluripotent because they lack the ability to efficiently produce extraembryonic cell types. The relatively limited developmental potential of ES cells can be explained by the observation that ES cells are derived from the embryo after its cells have already begun to specialize and lose totipotency. Meanwhile, at the time that pluripotent ES cell progenitors are specified, so are the multipotent progenitors of two extraembryonic stem cell types: trophoblast stem (TS) cells and extraembryonic endoderm stem (XEN) cells. Notably, all three embryo-derived stem cell types are capable of either self-renewing or differentiating in a lineage-appropriate manner. These three types of embryo-derived stem cell serve as paradigms for defining the genes and pathways that define and maintain unique stem cell identities. Remarkably, some of the mechanisms that maintain the specific developmental potential of each stem cell line do so by preventing conversion to another stem cell fate. This chapter highlights noteworthy studies that have identified the genes and pathways that normally limit the interconversion of stem cell identities.

Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors.

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

High Resolution Mapping of Human Lymphopoiesis Reveals a Common Lymphoid Progenitor (CLP) Population

In vivo reconstitution studies have established the phenotype of murine CLP, a progenitor population with restricted developmental potential for T, NK, B lymphoid and plasmacytoid dendritic cell (pDC) lineages. Co-transplantation of murine CLP with purified HSC more rapidly restores lymphopoiesis, immune function and resistance to viral challenge in experimental models of HSC transplantation. Thus, the identification of human CLP would have translational impact in clinical HSCT. Based on the expression of IL-7Rα (CD127) as a key distinguishing marker of murine CLP, we studied the CD127+ subset of immature CD34+ Lin- CD38+ cells for lymphoid committed progenitors, comprising approximately 0.1-0.2% of total marrow. Like murine CLP, the CD34+ Lin- CD38+ CD127+ cells also expressed Kit and Flt3. OP9 stromal cells were transduced with a vector expressing a doxycycline inducible delta-like ligand 1 (dll1) gene to test the differentiation potential in a single-well switch culture. Progenitors co-cultured with uninduced OP9Δdll1 cells can differentiate into B and myeloid cells; after induction of dll1, the culture is permissive for T and NK differentiation in the same well. In the presence of Kit and Flt3 ligands, erythromyeloid growth factors (IL-6, IL-3, GM-CSF, EPO, TPO) and lymphoid factors (IL-7, IL-15), CD34+ Lin- CD38- CD127- HSC generated erythroid (E), myelomonocytic (MM), myeloid DC (mDC), T, NK, and B lymphocytes and pDC. In contrast, CD34+ Lin- CD38+ CD127+ cells generated T/NK/B/pDC progeny, distributed in varying proportions, but did not generate E/MM/mDC. Singly plated CD34+ Lin- CD38+ CD127+ cells generate T/NK/B cells in the same well, consistent with these cells being human CLP. CD34+ Lin- CD38+ CD127- "non-CLP" generated E/MM/mDC, but not T/NK/B/pDC progeny. Limiting dilution analyses under lymphoid conditions demonstrated that the CD34+ Lin- CD38+ CD127+ CLP had approximately 2-3-fold greater efficiency of lymphocyte generation than HSC under the same conditions. Transcriptional profiling of the human CLP showed patterns of gene expression distinct from that of HSC, CD34+ Lin- CD38+ CD127- progenitors and immature CD3-CD4-CD8- thymocytes. Approximately 50% of the CD34+ Lin- CD38+ CD127+ CLP also express CD10, a subpopulation which CyTOF analyses revealed to have other markers of B-cell commitment and/or differentiation, e.g., CD179a. Human CD34+ Lin- CD38+ CD127+ marrow cells are a unique multipotent, lymphoid-restricted progenitor population for T/NK/B/pDC cell development, and within which subsets of cells biased for individual lymphoid lineages may exist. Identification of human CLP will permit further analyses of lymphoid specification, commitment to lymphoid lineages, and potential adoptive transfer as a short-term source of polyclonal lymphocytes. DisclosuresNolan:Fluidigm: Consultancy.

Embryonic expression of endothelins and their receptors in lamprey and frog reveals stem vertebrate origins of complex Endothelin signaling.

Neural crest cells (NCCs) are highly patterned embryonic cells that migrate along stereotyped routes to give rise to a diverse array of adult tissues and cell types. Modern NCCs are thought to have evolved from migratory neural precursors with limited developmental potential and patterning. How this occurred is poorly understood. Endothelin signaling regulates several aspects of NCC development, including their migration, differentiation, and patterning. In jawed vertebrates, Endothelin signaling involves multiple functionally distinct ligands (Edns) and receptors (Ednrs) expressed in various NCC subpopulations. To test the potential role of endothelin signaling diversification in the evolution of modern, highly patterned NCC, we analyzed the expression of the complete set of endothelin ligands and receptors in the jawless vertebrate, the sea lamprey (Petromyzon marinus). To better understand ancestral features of gnathostome edn and ednr expression, we also analyzed all known Endothelin signaling components in the African clawed frog (Xenopus laevis). We found that the sea lamprey has a gnathsotome-like complement of edn and ednr duplicates, and these genes are expressed in patterns highly reminiscent of their gnathostome counterparts. Our results suggest that the duplication and specialization of vertebrate Endothelin signaling coincided with the appearance of highly patterned and multipotent NCCs in stem vertebrates.

Functional constraints on SoxE proteins in neural crest development: The importance of differential expression for evolution of protein activity

Vertebrate SoxE genes (Sox8, 9, and 10) are key regulators of neural crest cell (NCC) development. These genes arose by duplication from a single SoxE gene in the vertebrate ancestor. Although SoxE paralogs are coexpressed early in NCC development, later, Sox9 is restricted to skeletogenic lineages in the head, and Sox10 to non-skeletogenic NCC in the trunk and head. When this subfunctionalization evolved and its possible role in the evolution of the neural crest are unknown. Sea lampreys are basal vertebrates that also possess three SoxE genes, while only a single SoxE is present in the cephalochordate amphioxus. In order to address the functional divergence of SoxE genes, and to determine if differences in their biochemical functions may be linked to changes in neural crest developmental potential, we examined the ability of lamprey and amphioxus SoxE genes to regulate differentiation of NCC derivatives in zebrafish colourless (cls) mutants lacking expression of sox10. Our findings suggest that the proto-vertebrate SoxE gene possessed both melanogenic and neurogenic capabilities prior to SoxE gene duplication. Following the agnathan-gnathostome split, lamprey SoxE1 and SoxE3 largely lost their melanogenic and/or enteric neurogenic properties, while gnathostome SoxE paralogs have retained functional conservation. We posit that this difference in protein subfunctionalization is a direct consequence of the independent regulation of SoxE paralog expression between the two lineages. Specifically, we propose that the overlapping expression of gnathostome SoxE paralogs in early neural crest largely constrained the function of gnathostome SoxE proteins. In contrast, the largely non-overlapping expression of lamprey SoxE paralogs allowed them to specialize with regard to their DNA-binding and/or protein interaction properties. Restriction of developmental potential among cranial and trunk neural crest in lampreys may be related to constraints on SoxE activity among duplicates, but such specialization does not appear to have occurred in gnathostomes. This highlights an important difference in the evolution of SoxE activity between these two divergent vertebrate lineages and provides insights for understanding how cell fate restriction in different NCC populations may be dependent on subfunctionalization among SoxE duplicates.

Molecular markers for oocyte competence in bovine cumulus cells

This study aimed to quantify the expression of candidate genes in cumulus cells (CCs) from cumulus-oocyte complexes (COCs) with high and low potential for in vitro development up to the blastocyst stage. First, the effects of individual culture and biopsy on embryo development were evaluated. Individuals cultured using the well of the well system were compared with individuals cultured in 20 μL droplets (microdroplets) and those cultured in groups (control). Blastocyst rates were lower for the individual culture systems (P < 0.05; well of the well = 17.9%, n = 95; microdrop = 26.3%, n = 95) than for the control group (45.0%, n = 209). Second, the effects of biopsy on embryo production were compared between the control and microdroplet cultures, and no effects (P > 0.05) were observed for either group. Finally, the expression profiles of glypican 4 (GPC4), IGF4-binding protein, follicle-stimulating hormonereceptor, growth hormone receptor, epidermal growth factor receptor, fibroblast growth factor 11, solute carrier family 2 member 1, solute carrier family 2 member 3,sprouty homolog 1, versican, and keratin protein 8 in CCs obtained by biopsy were quantified by real-time polymerase chain reaction. Cumulus cells were categorized on the basis of the fates of the COCs: expanded blastocyst, cleaved and arrested, and uncleaved. The GPC4 gene was overexpressed (P = 0.007) in CCs from oocytes that formed embryos compared with those that produced cleaved and arrested embryos. We concluded that individual culture reduced blastocyst production; however, biopsy did not affect embryo development. The profile of GPC4 expression can be used as a marker to distinguish COCs with potential for embryo development from those with limited developmental potential.

The Drosophila Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors

Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. In this study, we report that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs.

Individual bovine in vitro embryo production and cumulus cell transcriptomic analysis to distinguish cumulus-oocyte complexes with high or low developmental potential

Studying cumulus cell (CC) transcriptome is of great interest as it could provide a noninvasive method to assess oocyte quality. In cattle, the search for quality markers has not been done with cumulus-oocyte complexes (COCs) cultured individually from maturation to blastocyst stage. Here, differences between high- and low-potential COCs were examined by transcriptomic analysis of CC biopsies obtained from COCs of 2 to 6 mm follicles (n = 249; eight replicates) before individual in vitro maturation, fertilization, and culture until Day 8 after fertilization. Each COC was individually tracked and categorized based on his fate: embryo at blastocyst stage (CC-Blast) or embryo arrested at 2- to 8-cell stage (CC-2-8-cells). Average blastocyst rates were 27.7% for individual culture and 31.2% for group control (not significantly different). For transcriptomic analysis, five cumulus biopsies per replicate were pooled for each fate. Three CC replicates underwent transcriptomic analysis using RNA microarray assay. Some clear differences in gene expression between the CC-Blast and the CC-2-8-cell groups were identified. Considering a 1.5-fold change (P < 0.05), 68 genes were differentially expressed between the CC-Blast and CC-2-8-cells. Quantitative reverse transcription–polymerase chain reaction validations were performed for 12 selected genes: six upregulated genes for each COC fate. Higher expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) (lipid metabolism), Chloride intracellular channel 3 (CLIC3), Keratin 8 (KRT8), and Lumican (LUM) (molecular transport) was observed in CC-2-8-cells (P < 0.05). The CC-Blast fate analysis revealed a significantly higher expression of Glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM) (posttranslational modification, amino acid metabolism, and free radical scavenging). This newly identified set of genes could provide new markers to distinguish COCs associated with good quality embryos from COCs with limited developmental potential.

Earmuff restricts progenitor cell potential by attenuating the competence to respond to self-renewal factors

Despite expressing stem cell self-renewal factors, intermediate progenitor cells possess restricted developmental potential, which allows them to give rise exclusively to differentiated progeny rather than stem cell progeny. Failure to restrict the developmental potential can allow intermediate progenitor cells to revert into aberrant stem cells that might contribute to tumorigenesis. Insight into stable restriction of the developmental potential in intermediate progenitor cells could improve our understanding of the development and growth of tumors, but the mechanisms involved remain largely unknown. Intermediate neural progenitors (INPs), generated by type II neural stem cells (neuroblasts) in fly larval brains, provide an in vivo model for investigating the mechanisms that stably restrict the developmental potential of intermediate progenitor cells. Here, we report that the transcriptional repressor protein Earmuff (Erm) functions temporally after Brain tumor (Brat) and Numb to restrict the developmental potential of uncommitted (immature) INPs. Consistently, endogenous Erm is detected in immature INPs but undetectable in INPs. Erm-dependent restriction of the developmental potential in immature INPs leads to attenuated competence to respond to all known neuroblast self-renewal factors in INPs. We also identified that the BAP chromatin-remodeling complex probably functions cooperatively with Erm to restrict the developmental potential of immature INPs. Together, these data led us to conclude that the Erm-BAP-dependent mechanism stably restricts the developmental potential of immature INPs by attenuating their genomic responses to stem cell self-renewal factors. We propose that restriction of developmental potential by the Erm-BAP-dependent mechanism functionally distinguishes intermediate progenitor cells from stem cells, ensuring the generation of differentiated cells and preventing the formation of progenitor cell-derived tumor-initiating stem cells.

Evidence for dynamic rearrangements but lack of fate or position

Neural crest cells emerge from the dorsal trunk neural tube and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the neural tube have similar or differential ability to contribute to neural crest cells and their derivatives. To this end, we precisely labeled neural tube precursors at specific dorsoventral levels of the chick neural tube using fluorescent dyes and a photoconvertible fluorescent protein. Neural tube and neural crest cell dynamics were then examined in vivo and in slice culture using 2‐photon and confocal time‐lapse imaging. The results show that neural crest precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the neural tube, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the neural tube to contribute to neural crest derivatives, with the exception of sympathetic ganglia, which appeared to be “filled” by the first population to emigrate. Rather than restricted developmental potential, however, this is likely due to a matter of timing.Grant Funding Source: NIH grant 1RO1HD057922

Interchangeable potential varies among mesothelial cell populations in the embryo

Mesothelium is an epithelial sheet that covers coelomic organs and is involved in development of the vascular system. In the developing heart, the proepicardium (PE), migrates over the heart forming the epicardium, and undergoes epithelial‐mesenchymal transition (EMT) to contribute cells to the coronary blood vessels. The gut mesothelium also serves as a major source of vascular smooth muscle cells for the gut tube in development. We hypothesize that heart and gut mesothelial cell populations possess an interchangeable potential in coelomic organs. First, we characterized avian mesothelial cells using histological and molecular techniques to determine the cell types present at time of transplant. In addition, mesothelial cells were cultured to investigate the differentiative properties the mesothelial isolates. To test the interchangeable potential of mesothelial cells, we used the chick‐quail chimera system, transplanting quail PE and epicardial cells into the peritoneal cavity of a chick embryo and quail gut mesothelial cells into chick pericardial cavities. Our findings have revealed that all mesothelia have the potential to migrate into organs in the analogous coelomic cavity. However, PE and epicardial cells did not incorporate into the endogenous gut mesothelium and retained epithelial characteristics. Alternatively, gut mesothelial cells incorporated into the endogenous epicardium and contributed vascular smooth muscle cells, fibroblasts, and myofibroblasts to the heart. Taken together, our current data suggest a limited developmental potential between heart and gut mesothelial cell populations.

The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar toESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon invitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

Evidence for dynamic rearrangements but lack of fate or position restrictions in premigratory avian trunk neural crest

Neural crest (NC) cells emerge from the dorsal trunk neural tube (NT) and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the NT have similar or differential ability to contribute to NC cells and their derivatives. To this end, we precisely labeled NT precursors at specific dorsoventral levels of the chick NT using fluorescent dyes and a photoconvertible fluorescent protein. NT and NC cell dynamics were then examined in vivo and in slice culture using two-photon and confocal time-lapse imaging. The results show that NC precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the NT, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the NT to contribute to NC derivatives, with the exception of sympathetic ganglia, which appeared to be 'filled' by the first population to emigrate. Rather than restricted developmental potential, however, this is probably due to a matter of timing.

Early cell fate decisions in the mouse embryo

During mammalian preimplantation development, the fertilised egg gives rise to a group of pluripotent embryonic cells, the epiblast, and to the extraembryonic lineages that support the development of the foetus during subsequent phases of development. This preimplantation period not only accommodates the first cell fate decisions in a mammal's life but also the transition from a totipotent cell, the zygote, capable of producing any cell type in the animal, to cells with a restricted developmental potential. The cellular and molecular mechanisms governing the balance between developmental potential and lineage specification have intrigued developmental biologists for decades. The preimplantation mouse embryo offers an invaluable system to study cell differentiation as well as the emergence and maintenance of pluripotency in the embryo. Here we review the most recent findings on the mechanisms controlling these early cell fate decisions. The model that emerges from the current evidence indicates that cell differentiation in the preimplantation embryo depends on cellular interaction and intercellular communication. This strategy underlies the plasticity of the early mouse embryo and ensures the correct specification of the first mammalian cell lineages.

Klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division

Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription factor Klumpfuss (Klu) as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led us to propose that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division.