Numerous well known master regulators were recovered alongside previously unappreciated factors for either broad tissue groups e. Remarkably, in several instances approaching half of the genes with the most extreme PC loadings imputed co-regulation by a single transcription factor, such as HNF4A in the liver or SRF in the heart.
At the lowest extreme of PC5 liver the twenty-two transcription factors contained all three of those required for reprogramming fibroblasts directly to hepatocytes Huang et al.
This suggests novel fate programming roles for transcription factors at the extremes of other PCs including new potential regulators of pluripotency amongst the sixteen factors containing zinc fingers in PC1. In keeping with these regulatory roles, the extreme PC loadings in the LgPCA data also prioritized those transcription factors responsible for major congenital disorders Supplementary file 1G.
In several examples, individual transcription factors e. Mutations in genes encoding transcription factors are over-represented causes of congenital disorders, most likely due to their critical function during organogenesis and inadequacy when haploinsufficient.
The enrichment of transcription factors with specific disease-associations at the extremes of the LgPCA implicates the co-enriched genes as leading candidates for unanswered clinical syndromes. To test this model we identified some of the earliest chromosomal mapping or patient deletion data for the known disease-associated transcription factors from Supplementary file 1G. Non-coding transcription has emerged as a critical regulator of cell and developmental biology Goff and Rinn, A dedicated programme operating during human organogenesis seemed likely as 81 out of the genes enriched in embryogenesis compared to the fetal datasets were annotated long intergenic non-coding LINC transcripts Supplementary file 1B.
These lncRNAs were classified as either bidirectional, antisense or overlapping, or by exclusion intergenic, according to orientation and position in relation to the annotated genome Mattick and Rinn, Transcripts were most commonly —1, bp but could extend to over Kb Figure 4c and showed high tissue-specificity with the median Tau value Yanai et al. We investigated the association between this novel human embryonic transcriptome and the annotated genome. Reduced physical distance to expressed annotated genes markedly increased the likelihood of novel transcript co-expression Figure 4e , although the best correlations were by no means always with the closest gene Figure 4f—g.
The median distance to the closest annotated gene was 7. While LINC RNAs can harbour important regulatory function, how to forecast their relationship s with the protein-coding genome and prioritize the investigation of thousands of new transcripts is immensely challenging Goff and Rinn, As a first step, the multi-tissue nature of our dataset allowed intricate correlative patterns to be deciphered implying putative relationships; for instance over a 2 Mb window and across numerous genes on chromosome 7 between HE-LINC-C7T and TBX20 , which encodes a developmental cardiac transcription factor mutated in a wide range of congenital heart disease Figure 4h.
Details on exon and read counts, and proximity to surrounding genes are shown in Figure 4—figure supplement 1. Mean correlation was near zero beyond 1 Mb. There was no strong anticorrelation. Taken together, this study reports the first comprehensive transcriptomic atlas during human organogenesis to complement parallel initiatives from later development and adulthood Jaffe et al. Subjecting transcription from many sites to a method of analysis that incorporated developmental lineage deciphered novel genetic signatures, predicted causality in many human developmental disorders and associated novel non-coding transcription with expression from the surrounding protein-coding genome.
At present, the data arise from a relatively narrow window of embryonic development but set the stage for future longitudinal studies for individual organs over time. The tiny amounts and scarcity of human embryonic tissue also necessitated aspects of pooling across different Carnegie stages for some sites but it is striking that this had no impact on ascertaining organ and tissue-specific transcriptomic signatures by LgPCA.
The integrated data are expected to be particularly valuable to stem cell researchers examining the fidelity of PSC differentiation in vitro or searching for transcription factors for direct reprogramming of chosen cell lineages. Finally, the discovery of a major new programme of non-coding transcription adds a fresh layer of detail on the spatiotemporal regulation of the human genome.
Human embryonic material was collected under ethical approval, informed consent and according to the Codes of Practice of the Human Tissue Authority and staged by the Carnegie classification as described previously Jennings et al. This clinical material was collected on site overseen by our research team with immediate transfer to the laboratory. Individually identified tissues and organs details in Supplementary file 1A were immediately dissected. The adrenal gland, whole brain, heart, kidney, liver, entire limb buds, lung, stomach, testis, thyroid and anterior two-thirds of the tongue were readily identifiable as discrete organs and tissues.
All visible adherent mesenchyme was removed from organs and tissues under a dissecting microscope. For the adrenal gland, this includes the capsule which allowed separation from the kidney. The ureter, emerging from the renal pelvis, was removed separately from the kidney. For the heart, a window of tissue was removed from the lateral wall of the left ventricle. A segment of the liver was dissected from each embryo that avoided the developing gall bladder. The trachea was removed from the lung at its entry point into the lung parenchyma.
The stomach was identified between the gastro-oesophageal and pyloric junctions. The testis was dissected free from the attached mesonephros.
The palatal shelves were dissected on either side of the midline. The eye was dissected and the RPE peeled off mechanically from its posterior surface with validation possible under the dissecting microscope because the RPE is very darkly pigmented compared to the other ocular structures. Once the quality of each RNA sample had been confirmed, samples were pooled in order to obtain sufficient RNA for each biological replicate Supplementary file 1A.
The pancreas dataset derived from the same tissue collection was re-used from a previous study Cebola et al. Briefly, total RNA 0. The mRNA was then fragmented using divalent cations under elevated temperature and then reverse transcribed into first strand cDNA using random primers. Adapter indices were used to multiplex libraries, which were pooled prior to cluster generation using a cBot instrument. The RNA-seq was conducted in three batches at different times as a necessity of how human embryonic tissue was collected over time Supplementary file 1A.
RNA-seq reads from the Illumina platform were mapped to the human genome hg19 strand-specifically using TopHat 2. We also remapped the published pancreas RNA-seq dataset Cebola et al. Additionally, a dataset of hepatocyte differentiation RNA-seq Du et al. Commonly applied RNA-seq normalisation methods such as TMM assume a small proportion of differentially expressed genes in any one dataset Dillies et al.
Because the highly distinct tissues surveyed here differed strongly on the scale of thousands of genes for instance liver versus brain we used quantile normalisation which gave a lower median coefficient of variation than either no or TMM normalization.
Read counts from the different datasets were quantile normalized using the R package preprocessCore Bolstad, Tissue-specificity was scored per gene using Tau Yanai et al. Initial genome-wide relationships were assessed using PCA Figure 1—figure supplement 3 and hierarchical clustering heatmap, Figure 1—figure supplement 4.
Exon level counts were then summed into a single total per gene per sample. Counts were quantile normalized across samples. For the analysis of human embryonic RNA-seq with comparable Roadmap fetal data adrenal gland, heart, kidney, lung, limbs, stomach and testis a single pairwise differential expression test was undertaken using the R package edgeR Robinson et al.
Non-normalised read counts were filtered to remove all Y-linked genes, the X-inactivation gene XIST and genes with fewer than reads across all samples.
The maximal cophenetic distance was used to select the value of r. Subsequently, runs using the optimal rank were performed to assess consistency of sample groupings between runs. Non-overlapping i. A broad user-defined guide tree Figure 2b based on well-established knowledge of mammalian gastrulation and downstream lineage relationships was imposed on the different organ and tissue types following which the adephylo R package weighted the principal components by the lineage auto-correlation between samples; increased if related samples were similar and lessened if related samples were more different.
These patterns were not apparent if lineage relationships were not included nor were they altered if any one tissue, such as palate, was altered within the broad lineage structure data not shown. The global patterns in PCs 1—15 infer co-regulatory patterns of gene expression across human organogenesis. We used the Abouheif distance as implemented in adephylo Jombart et al. Sample-specific transcriptomes were assembled with Cufflinks version 2.
Names were assigned to these novel transcripts following suggested criteria Mattick and Rinn, Figure 4a with the sole adaptation that bidirectional BI transcripts were defined as having their TSS within 1 Kb of the TSS of the associated annotated gene. No transcripts mapped to the same strand within the introns of any annotated gene excluding the possibility of unspliced transcripts from annotated genes being erroneously defined as novel transcripts.
Where there were multiple transcripts from a single locus, the longest transcript was retained in assembling the final dataset of novel transcripts. Transcript level read counts for the embryonic samples and NIH Roadmap samples Supplementary file 1J were generated for the merged transcriptome using bedtools multicov vers 2. The correlations between each of the transcripts and all annotated genes within 1 Mb were calculated using only the human embryonic data from this study.
Mapping coordinates against multiple genome versions using a range of common pipelines and summary count data are hosted at www. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Therefore, damage to any of the organ systems of the body which may ultimately result in some type of birth defect usually strikes during this time frame.
By week five, the buds of tissue which will become the limbs are in place. The structures which will become the skeleton, nervous system , and circulatory system of the face, neck, and jaws are in place. A five-week-old embryo has the early developmental structures of the esophagus, stomach, intestine, liver, and pancreas. The heart is already functioning, and continues to develop and change over this period of time.
The respiratory system begins developing, as do blood vessels, blood cells, nervous and endocrine organs. Clearly, the most crucial organs of the human form are developing during organogenesis. Normal development of the conceptus—the zygote, blastocyst, embryo, and fetus, depending on its stage of development, plus its supporting membranes and placenta—can be adversely affected by poor maternal health and nutrition, genetic mutation, exposures to exogenous agents or a combination of these factors.
The focus of this chapter is on the timing of important events during pregnancy that may be disrupted by a teratogenic exposure Fig. The conceptus is susceptible to such exposures throughout in utero development and even postnatally, although there are critical periods in which the conceptus is highly susceptible that are dependent on the endpoint that is affected by the exposure.
Moreover, the conceptus is conceived during fertilization as the male and female gametes—the sperm and egg—join together in the upper end of the oviduct Fallopian tube to form a single-cell zygote. Consequently, environmental exposures over the life span of the prospective mother or father both prenatally and postnatally; see discussion of gametogenesis below can impact the ability of their gametes to produce a successful pregnancy and the birth of a healthy child.
Thus, susceptibility to teratogen exposure for a given conceptus actually begins even prior to when it is conceived. Figure 1. Timeline of important events during pregnancy that may be disrupted by a teratogenic exposure. Shown are the lengths of pregnancy in days , from conception or fertilization to birth , the span of the three trimesters 3 months each , the three periods of prenatal development egg, embryo, and fetus , key developmental events fertilization, cleavage, implantation, gastrulation, primary morphogenesis, organogenesis, and birth , and some outcomes of teratogenic exposure.
The period of the egg is generally defined as the time during pregnancy that precedes implantation—that is, the time from formation of the zygote until the blastocyst burrows into the wall of the receptive, that is, hormonally primed, uterus initiated by the end of the first week postfertilization. Although the conceptus at this stage is not truly an egg or oocyte, as traditionally defined, for all intents and purposes it looks like an egg, being grossly spherical, throughout this first period.
The second period, the period of the embryo, is roughly defined as the time from implantation through the 8th week of development. During the period of the embryo, part of the conceptus takes on the shape of what can be readily recognized as an embryo Fig. The remainder of the conceptus contributes to the so-called extraembryonic membranes, including the amnion, which enclose and protect the embryo during its development, and the fetal component of the placenta.
The final period, the period of the fetus, extends from the beginning of the 9th week of gestation until birth. This period is characterized by rapid growth of the fetus and the differentiation of cells, resulting in the formation of distinct tissue types that become assembled into functional organ systems.
Figure 2. Photographs of human embryos at five stages of gestation reproduced from the collection of the Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, courtesy of Dr. Kohei Shiota, Ms. Chigako Uwabe, and Dr. Shigehito Yamada.
Shown from left to right are Carnegie Stages 9, 10, 13, 17, and 23 during the period of the embryo. The Stage 9 embryo has initiated neurulation, which is largely completed by Stage 10 with the exception that the cranial and caudal ends of the neural tube remain open as the neuropores. By Stage 13, body folding has established the tube-within-a-tube body plan of the early embryo, with a distinct head, trunk, and tail, and paddle-like limb buds.
By Stage 17, the developing eyes are readily identifiable, and the limb buds now have bulbous distal plate-like structures that will form the hands and the feet. By Stage 23—the last stage in the period of the embryo—all external structures have taken on morphologies similar to those of the adult. Periods of development broadly define the structure of the developing organism at three different times during pregnancy. The embryo contains the rudiments of the organs of the fetus; the link between the structure of the embryo and fetus is more intuitive, but, for example, the paddle-like limb buds present in the early embryo are non-functional and have a very different structure than that of the upper and lower limbs of the fetus, which at birth are fully functional.
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