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

BACKGROUND: Little 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. RESULTS: We 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. CONCLUSIONS: Using 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.

Derivation of the human embryonic stem cell line RCM1.

The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Truncation of the Mll gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryos.

The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.

Differentiation potential of intestinal mesenchyme and its interaction with epithelial cells: a study using beta-galactosidase-expressing fibroblast lines.

Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either beta-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene was more efficient than electroporation. The expression of beta-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of beta-galactosidase were isolated. These clones expressed beta-galactosidase even after prolonged passage in the absence of selection. The beta-galactosidase tagged lines (F1:G9gal and A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9gal and A1:F1gal cells differed in their morphological organisation but this did not change their expression of smooth muscle alpha-actin.

Stability of a model beta-sheet in water.

We have used molecular dynamics simulations to determine the stability in water of a model beta-sheet formed by two alanine dipeptide molecules with two intermolecular hydrogen bonds in the closely spaced antiparallel arrangement. In this paper we describe our computations of the binding free energy of the model sheet and a portion of the free energy surface as a function of a reaction co-ordinate for sheet formation. We used the free energy surface to identify stable conformations along the reaction co-ordinate. To determine whether or not the model sheet with two hydrogen bonds is more stable than a single amide hydrogen bond in water, we compared the results of the present calculations to results from our earlier study of linear hydrogen bond formation between two formamide molecules (the formamide "dimer"). The free energy surfaces for the sheet and formamide dimer each have two minima corresponding to locally stable hydrogen-bonded and solvent-separated configurations. The binding free energies of the model sheet and the formamide dimer are -5.5 and -0.34 kcal/mol, respectively. Thus, the model sheet with two hydrogen bonds is quite stable while the simple amide hydrogen bond is only marginally stable. To understand the relative stabilities of the model sheet and formamide dimer in terms of solute-solute and solute-water interactions, we decomposed the free energy differences between hydrogen-bonded and solvent-separated conformations into energetic and entropic contributions. The changes in the peptide-peptide energy and the entropy are roughly twice as large for the sheet as they are for the formamide dimer. The magnitude of the peptide-water energy difference for the sheet is less than twice (by about 3.5 kcal/mol) that for the formamide dimer, and this accounts for the stability of the sheet. The presence of the side-chains and/or blocking groups apparently prevents the amide groups in the sheet from being solvated as favorably in the separated arrangement as in the formamide dimer, where the amide groups are completely exposed to the solvent.

The role of packing interactions in stabilizing folded proteins.

In order to investigate the role of nonpolar side chains in determining protein stability, we have carried out a molecular dynamics simulation study of the thermodynamics of interconverting isoleucine and valine side chains in the core of ribonuclease T1. The free energy change in the unfolded state, which we take to be fully solvated, was small and agrees qualitatively with experimental studies of alkane solvation. In the two Ile----Val mutations studied, the protein was able to relax around the smaller side chains, while in the case of the two Val----Ile mutations, the ability of the core to accommodate the extra methylene group depended on where the mutation took place. We argue that the experimentally observed decrease in stability for mutating isoleucine into valine results from a loss of favorable packing interactions of the side chain in the folded form of the protein. This supports the view that packing interactions in the folded state are an important contributor to the overall stability of the folded protein and that the core of the native protein is packed efficiently and almost completely.

Efficient method for the generation and display of electrostatic potential surfaces from ab-initio wavefunctions.

A cost effective color graphics representation of molecular electrostatic potential surfaces employing the cumulative atomic or bond multipole moments has been described. A general description of the method used to obtain cumulative multipole moments directly from ab-initio wavefunctions is given, along with an outline of the algorithm for generating electrostatic potential surfaces in the molecular graphics programs MOL17 (FORTRAN 77, Silicon Graphics 3130 and 4D series workstations) and PCMCAMM (Turbo Pascal, IBM PC and PS/2 computers). Examples are given that illustrate the convergence of the multiple expansion, the degree of basis-set dependence compensated by the use of higher atomic moments, and the effect of placing additional expansion centers along the bonds.

Reverse turns in blocked dipeptides are intrinsically unstable in water.

We have carried out molecular dynamics simulations to study the conformational equilibria of two blocked dipeptides, Ac-Ala-Ala-NHMe and trans-Ac-Pro-Ala-NHMe, in water (Ac, amino-terminal blocking group COCH3; NHMe, carboxy-terminal blocking group NHCH3). Using specialized sampling techniques we computed free-energy surfaces as functions of a conformation co-ordinate that corresponds to hydrogen-bonded reverse turns at small values and to extended conformations at large values. The free-energy difference between hydrogen-bonded reverse turn conformations and extended conformations, determined from the equilibrium constants for reverse turn unfolding, is approximately -5 kcal/mole for Ac-Ala-Ala-NHMe, and -10 kcal/mole for Ac-Pro-Ala-NHMe. These results demonstrate that reverse turns in blocked dipeptides are intrinsically unstable in water. That is, in the absence of strongly stabilizing sequence-specific inter-residue interactions involving side-chains and/or charged terminal groups, the extended conformations of small peptides are highly favored in solution. By thermodynamically decomposing the free-energy differences, we found that the peptide-water entropy is the primary reason for the exceptional stability of the extended conformations of both peptides, and that the differences between the two peptides are primarily due to differences in the peptide-water interactions. In addition, we assessed the "proline effect" on the conformational equilibria by comparing the differences in configurational entropies between the reverse turn and extended conformations of the two peptides. As expected, the extended conformation of the Pro-Ala peptide is destabilized by reduced configurational entropy, but the effect is negligible in the blocked dipeptides. Finally, we compared our results with the results of several other experimental studies to identify some of the specific interactions that may be responsible for stabilizing reverse turns in small peptides in solution.

Thermodynamics of amide hydrogen bond formation in polar and apolar solvents.

We present the initial findings of a theoretical study of hydrogen bond formation between two formamide molecules in water and in carbon tetrachloride. These systems were chosen as the simplest models for secondary structure formation in the polar environment near the protein surface and the apolar environment of the protein interior. We have employed thermodynamic simulation methods to obtain absolute binding free energies and free energy profiles for the formation of peptide hydrogen bonds in the two solvents. We find that the amide hydrogen bond is stable by 8.4 kcal/mol in CCl4, and by 0.3 kcal/mol in water. Our results indicate also that the hydrogen-bonded dimer is 2.2 kcal/mol more stable in water than it is in CCl4. We compare our results with those from experiment, and discuss their use in interpreting mechanisms of protein folding.

Microcomputer-based three-dimensional stereoscopic macromolecular graphics display.

Software which permits an IBM AT and two IBM Professional Graphics Displays to be used to display high-quality three-dimensional space-filling stereoscopic images of macromolecules is described. Stereo image pairs generated on two screens are visually fused using a simple mirror system to provide binocular depth perception. Images are colored to identify atomic type, residue type, charge or hydrophobicity according to user-specified codes and can be rotated and rescaled. Macromolecules containing over 16,000 atoms can be rapidly drawn using Brookhaven Protein Data Bank or user-supplied coordinates.

A new classification of the amino acid side chains based on doublet acceptor energy levels.

We describe a new classification of the amino acid side chains based on the potential energy level at which each will accept an extra (doublet) electron. The doublet acceptor energy level, and the doublet acceptor orbital were calculated using semiempirical INDO/2-UHF molecular orbital theory. The results of these calculations show that the side chains fall into four groups. We have termed these groups repulsive, insulating, semiconducting, and attractive in accordance with where each lies on the relative energy scale. We use this classification to examine the role of residues between the donor and acceptor in modulating the rate and mechanism of electron transfer in proteins. With the calculated acceptor levels, we construct a potential barrier for those residues between the donor and acceptor. It is the area beneath this barrier that determines the decay of electronic coupling between donor and acceptor, and thus the transfer rate. We have used this schematic approach to characterize the four electron transfer pathways in myoglobin recently studied by Mayo et al. (Mayo, S.L., W.R. Ellis, R.J. Crutchley, and H.B. Gray. 1986. Science [Wash. DC]. 233:948-952).

Accuracy of spirometric and flow-volume indices obtained by digitizing volume-time tracings.

We tested the accuracy of a new system for deriving standard spirometric indices, maximal expiratory flow rates, and slope ratios from volume-time tracings. A computer-based technique employing a hand-operated cursor was used to put discrete values of volume and time into a memory array. Spirometric values obtained on 102 subjects using the computer system were compared with the corresponding "hand-read" values. The difference between the 2 measuring techniques were not significant for the FVC, the FEV75, and the FEF25-75; however, the average FEV1 differed by 6.7 ml (SD, 20.3 ml), which was significant. In addition, 10 subjects performed FVC maneuvers through a spirometer and flowmeter connected in series. Flows and slope ratios obtained from the volume-time tracings were compared with those obtained directly from the flowmeter. There was a high degree of correlation between the 2 types of flow measurements (r = 0.989), whereas slope ratios were less well correlated (r = 0.589). Configurational detail such as the presence of "bumps" on slope ratio versus volume plots were recovered with the computer technique. Using this new system, it is possible for 1 operator to process 10 to 12 sets of spirometry tracings per hour.

Breathing patterns influence aerosol deposition sites in excised dog lungs.

Particles' suspended in inhaled air can deposit on lung surfaces. The amount deposited in different lung regions is determined by distribution of ventilation and local efficiency of particle collection. We examined how the pattern of ventilation influences sites of aerosol deposition in excised dog lungs. The lungs breathed a 99mTc-labeled aerosol according to one of three patterns: slow-deep (f less than 25/min, VT greater than 0.35 TLC); rapid-shallow (f greater than 65/min, VT less than 0.26 TLC); and slow-shallow (f = 8.4/min, VT = 0.1 TLC). After exposure, lungs were inflated, dried, and sliced transversely at 1-cm intervals. Distribution of deposited aerosol in each slice was measured by 1) gamma camera, 2) autoradiography, and 3) dissection of each slice into pieces whose activity, weight, and airway content were recorded. Our results indicate that a) total deposition decreases as f increases, b) slow-deep breathing produces uniform deposition throughout the lung, but with little aerosol collection in large airways, c) rapid-shallow ventilation results in enhanced large-airway deposition and marked heterogeneity in deposition distribution, and d) slow-shallow breathing enhances small-airway deposition. The deposition distributions characteristic of different breathing patterns were apparent on the visual displays from the gamma camera and autoradiographs. These techniques showed particle retention changing across lobar divisions, as a function of distance from the hilum, and across the lungs in both the caudal-to-cranial and dorsal-to-ventral directions.

Steady expiratory flow in dog lungs: an isovolume preparation.

By supplying air and other gases through discs glued to the pleural surface, we studied steady expiratory flow at constant volume. Dog lungs were studied at constant PA - Ppl (alveolar minus pleural pressure) of 7 to 10 cmH2O, as increasing flow was achieved by increasing driving pressure [Ppl - Pao (airway opening pressure)]. Flow became limited (independent of further increases in Ppl - Pao) at between 3.5 and 5.5 l/s. Isovolume-pressure-flow (IVPF) curves constructed from forced expirations at graded efforts yielded similar maximal flows. When the airways were made rigid by drying, flow limitation was abolished. When various gases were passed through the dried lung Moody plots of normalized pressure drop (CD) vs. Reynolds number (Re) showed that all of the data could be plotted on a single curve. Although variable among animals, all Moody plots showed a laminar flow region at Re below 100 and an inertial region at Re above 10,000, with a distinct transition.