A time- and single-cell-resolved model of murine bone marrow hematopoiesis.

The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution.

The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence.

YTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development.

mRNA 3' uridylation and poly(A) tail length sculpt the mammalian maternal transcriptome.

A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that 3' terminal uridylation of mRNA mediated by TUT4 and TUT7 sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. Uridylation mediated by TUT4 and TUT7 is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome has a shorter poly(A) tail and a higher relative proportion of terminal oligo-uridylation. Deletion of TUT4 and TUT7 leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails, and a loss of 3' oligo-uridylation. By contrast, deficiency of TUT4 and TUT7 does not alter gene expression in a variety of somatic cells. In summary, we show that poly(A) tail length and 3' terminal uridylation have essential and specific functions in shaping a functional maternal transcriptome.

Intracytoplasmic sperm injection (ICSI)-mediated transgenesis in mice.

Over the years many well-described techniques for the introduction of transgene DNA into host organisms have been used, including pronuclear injection, in vitro fertilization-mediated transgenesis, transfection of ES and spermatogenic cells, nuclear transfer of somatic cell nuclei, and lentiviral vectors. The application of these techniques has been limited however either by the time and effort to be executed or by their narrow efficiency with large transgenes. The greatest advantage of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is precisely its ability to stably introduce large DNA molecules into the genome of host organisms with relatively high efficiency, as compared to alternative procedures. In mice, this procedure has been shown to be a reproducible method to generate transgenic offspring with a high efficiency. Recently, it proved also to be a viable method to generate transgenic rats and pigs, and as such, it is foreseen with great interest for the production of transgenic farm animals, where it would constitute an important tool for the production of recombinant proteins and livestock improvement.

The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements.

Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.

Improving the generation of genomic-type transgenic mice by ICSI.

Transgenes included in genomic-type constructs, such as yeast artificial chromosomes (YAC), P1-derived artificial chromosomes, or bacterial artificial chromosomes (BAC), are normally correctly expressed, according to the endogenous expression pattern of the homologous locus, because their large size usually ensures the inclusion of all regulatory elements required for proper gene expression. The use of these large genomic-type transgenes is therefore the method of choice to overcome most position effects, commonly associated with standard-type transgenes, and to guarantee the faithful transgene expression. However, in spite of the different methods available, including pronuclear microinjection and the use of embryonic stem cells as vehicles for genomic transgenes, the generation of transgenic animals with BACs and, particularly, with YACs can be demanding, because of the low efficiencies requiring extensive microinjection sessions and/or higher number of oocytes. Recently, we have explored the use of intracytoplasmic sperm injection (ICSI) into metaphase II oocytes as an alternative method for the generation of YAC transgenic mice. Our results suggest that the use of transgenic strategies based on ICSI significantly enhances the efficiency of YAC transgenesis by at least one order of magnitude.

Generation of yeast artificial chromosome transgenic mice by intracytoplasmic sperm injection.

Genomic-type transgenes are usually expressed in appropriate spatial- and temporal-specific manners. The largest genomic transgenes can be prepared using yeast artificial chromosomes (YACs). Normally, YAC transgenic mice are produced by standard pro-nuclear microinjection, although other methods, involving the use of embryonic stem (ES) cells, have been also devised. To overcome the difficulty and time extension of ES cell-type approaches and to improve the rather usual low efficiency of YAC DNA transgenesis by pronuclear microinjection, that is mostly dependent on the YAC DNA quality of samples, we have devised an updated intracytoplasmic sperm injection (ICSI) method for the stable incorporation of YACs into the germ line of mice. DNA transgenesis efficiencies achieved are often 10 times greater than those usually obtained by standard microinjection, thus enabling the identification of either more transgenic founder animals and the use of reduced numbers of individuals in animal experimentation.

Influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection in pigs.

The purpose of this study was to determine the influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection (ICSI) in pigs. This was done by examining the effect of 1) the conservation method (fresh vs frozen); 2) the sperm treatment preinjection (resuspension in Dulbecco phosphate-buffered saline (DPBS) vs selection by a Percoll gradient); and 3) the acrosomal and live or dead status of the spermatozoa (by incubation with or without calcium ionophore, 1 muM and 5 muM). In vitro matured porcine oocytes were injected with treated spermatozoa according to each experiment. All the experiments were done with non-artificially activated oocytes. The percentages of activation and cleavage were higher (68% vs 43% and 63% vs 43%, respectively, P < .05) in oocytes injected with fresh vs frozen spermatozoa. The DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P < .05). Moreover, a boar effect was observed in the percentage of developing blastocysts. None of the studied parameters was affected by the acrosomal or the live or dead status of the spermatozoa injected. In conclusion, the use of fresh semen is recommended for porcine ICSI, as well as careful selection of the boar; Percoll treatment is only recommended for poor-quality samples or for removing toxic agents, and no exogenous form of activation or induction of the acrosome reaction is necessary for porcine oocytes to develop a male pronucleus and cleave up to the 2-cell stage after ICSI, although experimental conditions to reach the blastocyst stage need to be investigated further.

Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection.

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.

Retinoid-dependent mRNA expression and poly-(A) contents in bovine oocytes meiotically arrested and/or matured in vitro.

The presence of retinoic acid (RA) during in vitro maturation (IVM) improves bovine oocyte quality and developmental potential. In this work, we investigated the underlying molecular mechanisms. Cumulus-oocyte complexes were meiotically arrested by roscovitine and/or matured in defined medium containing RA, 1% ethanol (vehicle), or no additives. Cumulus-free oocytes were analyzed for poly-(A) mRNA contents and relative mRNA expression of genes involved in cell cycle regulation (cyclin B1 and H1) and antioxidative defence (Mn-superoxide dismutase and glucose-6-phosphate dehydrogenase). Poly-(A) mRNA increased after meiotic inhibition and decreased with IVM completion, both in meiotically arrested and permissively matured oocytes, i.e., matured without previous meiotic arrest. RA dramatically increased poly-(A) mRNA in meiotically arrested oocytes, but more than half of the poly-(A) mRNA disappeared during maturation. Irrespective of oocyte origin, transcripts were detected for all the genes analyzed. IVM, with or without previous meiotic inhibition, increased expression of cyclin B1 and glucose-6-phosphate dehydrogenase, and decreased cyclin H1 and Mn-superoxide dismutase. Except for a decreasing of Mn-superoxide dismutase in meiotically arrested and matured oocytes, RA did not affect mRNA expression. Ethanol led to an abnormal poly-(A) mRNA profile and expression of all the genes analyzed. RA does not modify expression of cyclin B1 and HI genes in the bovine oocyte, and probably does not generate oxidative stress. In addition, RA enhanced mRNA amount as measured by poly-(A) mRNA contents.

Architectural defects in pronuclei of mouse nuclear transplant embryos.

Reprogramming somatic nuclear function by transplantation of nuclei into recipient oocytes is associated with a morphological remodeling of the somatic nucleus. Successful cloning of animals by nuclear transplantation (NT) demonstrates that reprogramming somatic cell function is possible. However, low pregnancy rates and high frequencies of lethal abnormalities in animals born suggest that reprogramming is rarely complete. To address this issue, we tested the hypothesis that nuclear transplantation leads to nuclear remodeling deficiencies. We report the identification of several markers of morphological remodeling, or lack thereof, of mouse cumulus cell nuclei after transplantation into oocytes. Notably, nuclear transplant mouse embryos exhibit nuclear assembly of the differentiated cell-specific A-type lamins at the one-cell stage, as a result of misregulation of lamin A gene expression. The transplanted nuclei also display enhanced concentration of the nuclear matrix-associated protein NuMA as a result of translation from maternal mRNA and de novo transcription. The A-kinase anchoring protein 95 (AKAP95), a marker of the nuclear envelope-chromatin interface, is of somatic origin. Furthermore, greater resistance of AKAP95 and DNA to in situ extractions of one-cell stage NT embryos with non-ionic detergent, DNase, RNase and NaCl reflects an enhanced proportion of heterochromatin in these embryos. Passage through first embryonic mitosis does not rescue the defects detected in one-cell stage embryos. We propose that somatic nuclear reprogramming deficiencies by NT might emanate from, at least in part, failure to remodel the somatic nucleus morphologically into a functional embryonic nucleus.

Mouse ICSI with frozen-thawed sperm: the impact of sperm freezing procedure and sperm donor strain.

Normal mouse offspring can be obtained from oocytes injected with frozen-thawed spermatozoa without cryoprotection, however, embryo development can be affected by sperm freezing procedure and sperm donor strain. In this study we observed that direct contact of mouse spermatozoa with liquid nitrogen did not affect their ability to activate injected oocytes but severely restricted subsequent in vitro embryo development to blastocyst stage. Tris-EDTA buffer and M2 were also shown to be better sperm freezing extenders than DPBS, allowing higher developmental potential. In addition, differences in embryo development obtained by intracytoplasmic sperm injection (ICSI) with frozen-thawed spermatozoa were observed between hybrid sperm donor strains. Frozen-thawed B6D2F1 spermatozoa provided higher embryo development than sperm cells from C57CBAF1.