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Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8–12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain–hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal–ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development. Improved integration of spatial and single-cell transcriptomic data provides insights into mouse development.

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods-in germ cells and in preimplantation embryos-in which methylation patterns are reprogrammed genome wide, generating cells with a broad developmental potential. Epigenetic reprogramming in germ cells is critical for imprinting; reprogramming in early embryos also affects imprinting. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. A role of reprogramming in stem cell differentiation is also envisaged.

Imprinted genes tend to be clustered in the genome. Most of these clusters have been found to be under the control of discrete DNA elements called imprinting centres (ICs) which are normally differentially methylated in the germline. ICs can regulate imprinted expression and epigenetic marks at many genes in the region, even those which lie several megabases away. Some of the molecular and cellular mechanisms by which ICs control other genes and regulatory regions in the cluster are becoming clear. One involves the insulation of genes on one side of the IC from enhancers on the other, mediated by the insulator protein CTCF and higher-order chromatin interactions. Another mechanism may involve non-coding RNAs that originate from the IC, targeting histone modifications to the surrounding genes. Given that several imprinting clusters contain CTCF dependent insulators and/or non-coding RNAs, it is likely that one or both of these two mechanisms regulate imprinting at many loci. Both mechanisms involve a variety of epigenetic marks including DNA methylation and histone modifications but the hierarchy of and interactions between these modifications are not yet understood. The challenge now is to establish a chain of developmental events beginning with differential methylation of an IC in the germline and ending with imprinting of many genes, often in a lineage dependent manner.

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

In mammals, imprinted genes have an important role in feto-placental development. They affect the growth, morphology and nutrient transfer capacity of the placenta and, thereby, control the nutrient supply for fetal growth. In particular, the reciprocally imprinted Igf2-H19 gene complex has a central role in these processes and matches the placental nutrient supply to the fetal nutrient demands for growth. Comparison of Igf2P0 and complete Igf2 null mice has shown that interplay between placental and fetal Igf2 regulates both placental growth and nutrient transporter abundance. In turn, epigenetic modification of imprinted genes via changes in DNA methylation may provide a mechanism linking environmental cues to placental phenotype, with consequences for development both before and after birth. Changes in expression of imprinted genes, therefore, have major implications for developmental programming and may explain the poor prognosis of the infant born small for gestational age and the wide spectrum of adult-onset diseases that originate in utero.

Restricted fetal growth is associated with postnatal mortality and morbidity and may be directly related to alterations in the capacity of the placenta to supply nutrients. We proposed previously that imprinted genes can regulate nutrient supply by the placenta. Here, we tested the hypothesis that the insulin-like growth factor 2 gene (Igf2) transcribed from the placental-specific promoter (P0) regulates the development of the diffusional permeability properties of the mouse placenta. Using mice in which placental-specific Igf2 had been deleted (P0), we measured the transfer in vivo of three inert hydrophilic solutes of increasing size (14C-mannitol,51CrEDTA, and14C-inulin). At embryonic day 19, placental and fetal weights in P0 conceptuses were reduced to 66% and 76%, respectively, of wild type. In P0 mutants, the permeability·surface area product for the tracers at this stage of development was 68% of that of controls; this effect was independent of tracer size. Stereological analysis of histological sections revealed the surface area of the exchange barrier in the labyrinth of the mouse placenta to be reduced and thickness increased in P0 fetuses compared to wild type. As a result, the average theoretical diffusing capacity in P0 knockout placentas was dramatically reduced to 40% of that of wild-type placentas. These data show that placental Igf2 regulates the development of the diffusional exchange characteristics of the mouse placenta. This provides a mechanism for the role of imprinted genes in controlling placental nutrient supply and fetal growth. Altered placental Igf2 could be a cause of idiopathic intrauterine growth restriction in the human.

To test the significance of the observed and expected frequencies we used a Poisson approximation to the binomial distribution and obtained a two tailed p value of 0.018. [...]the observed frequency (n=6) of IVF and ICSI births in the BWS series is significantly greater than the expected (1.7252), with an associated 95% confidence interval on the excess risk of 1.5, 8.8. Had such data been available, then this would have very probably reduced the expected number of BWS births after IVF or ICSI, as the birth rate in the general population has been declining since 1989 and the annual number of ART births before 1995 would have been less than during the five years included in the comparison. [...]our comparison is likely to be conservative in relation to calendar year.

H2020 European Research Council, Grant/Award Number: ERC-2014-AdG/669622; Fundación Científica Asociación Española Contra el Cáncer, Grant/Award Number: PROYE18061FERN; Ministerio de Ciencia e Innovación, Grant/Award Number: SAF2013-48256-R; the Asturias Regionla Government (PCTI) co-funding 2018- 2022/FEDER (IDI/2018/146), the Health Institute Carlos III (Plan Nacional de I+D+I) co-funding FEDER (PI18/01527)...

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications 1 , 2 , 3 , 4 , 5 . The paternally expressed gene insulin-like growth-factor 2 ( Igf2 ) is separated by ∼100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries 6 , 7 , 8 , 9 , silencers 10 , 11 and activators 12 and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers 13 , 14 , 15 . Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains 16 , 17 , 18 , 19 . Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.