Expression and hormonal regulation of Muc-1 at the apical surface of mouse uterine epithelial cells and its role in modulating embryo implantation

Previous studies from our lab have established that large molecular weight mucin glycoproteins are major apically-disposed components of mouse uterine epithelial cells in vitro (Valdizan et al., (1992) J. Cell. Physiol. 151:451-465). The present studies demonstrate that Muc-1 represents one of the apically-disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and mRNA expression are regulated in the peri-implantation stage mouse uterus by ovarian steroids. Muc-1 expression is high in the proestrous and estrous stages, and decreases during diestrous. Both Muc-1 protein and mRNA levels decline to barely detectable levels by day 4 of pregnancy, i.e., prior to the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by administration of exogenous progesterone. Initiation of implantation was triggered by coinjection of progesterone maintained mice with a nidatory dose of 17$\beta$-estradiol. Muc-1 levels in the uterine epithelia of progesterone maintained mice declined to similar low levels as observed on day 4 of normal pregnancy. Coinjection of estradiol did not alter Muc-1 expression suggesting that down-regulation of Muc-1 is a progesterone dominated event. This was confirmed in ovariectomized, non-pregnant mice which displayed stimulation of Muc-1 expression following 6 hr of estradiol injection. Estradiol stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. While progesterone alone had no effect on Muc-1 expression, it antagonized estradiol action in this regard. Injection of pregnant mice with the antiprogestin, RU 486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is progesterone receptor-mediated. Muc-1 appears to function as an anti-adhesive molecule at the apical cell surface of mouse uterine epithelial cells. Treatment of polarized cultures of mouse uterine epithelial cells with O-sialoglycoprotein endopeptidase reduced mucin expression in vitro, by about 50%, and converted polarized uterine epithelia to a functionally receptive state. Similarly, ablation of Muc-1 in Muc-1 null mice resulted in polarized uterine epithelia that were functionally receptive as compared to their wild-type counterparts in vitro. Collectively, these data indicate that Muc-1 and other mucins function as anti-adhesive molecules and that reduction or removal of these molecules is a prerequisite for the generation of a receptive uterine state. ^

Mechanical constraints imposed by 3D cellular geometry and arrangement modulate growth patterns in the Arabidopsis embryo

Morphogenesis occurs in 3D space over time and is guided by coordinated gene expression programs. Here we use postembryonic development in Arabidopsis plants to investigate the genetic control of growth. We demonstrate that gene expression driving the production of the growth-stimulating hormone gibberellic acid and downstream growth factors is first induced within the radicle tip of the embryo. The center of cell expansion is, however, spatially displaced from the center of gene expression. Because the rapidly growing cells have very different geometry from that of those at the tip, we hypothesized that mechanical factors may contribute to this growth displacement. To this end we developed 3D finite-element method models of growing custom-designed digital embryos at cellular resolution. We used this framework to conceptualize how cell size, shape, and topology influence tissue growth and to explore the interplay of geometrical and genetic inputs into growth distribution. Our simulations showed that mechanical constraints are sufficient to explain the disconnect between the experimentally observed spatiotemporal patterns of gene expression and early postembryonic growth. The center of cell expansion is the position where genetic and mechanical facilitators of growth converge. We have thus uncovered a mechanism whereby 3D cellular geometry helps direct where genetically specified growth takes place.

Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count

Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n=16), whereas that of Staph. aureus non-GTB herds was 38% (n=38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9-56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds without purchase of heifers. Furthermore, herds that did not use udder ointment as supportive therapy for acute mastitis had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 8.5, 95% CI: 1.6-58.4) or Staph. aureus non-GTB (odds ratio: 6.1, 95% CI: 1.3-27.8) than herds that used udder ointment occasionally or regularly. Herds in which the milker performed unrelated activities during milking had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds in which the milker did not perform unrelated activities at milking. Awareness of 4 potential risk factors identified in this study guides implementation of intervention strategies to improve udder health in both Staph. aureus GTB and Staph. aureus non-GTB herds.

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Towards a new model of care: integrating mental health, substance use, and somatic care

Although research and clinical interventions for patients with dual disorders have been described since as early as the 1980s, the day-to-day treatment of these patients remains problematic and challenging in many countries. Throughout this book, many approaches and possible pathways have been outlined. Based upon these experiences, some key points can be extracted in order to guide to future developments. (1) New diagnostic approaches are warranted when dealing with patients who have multiple problems, given the limitations of the current categorical systems. (2) Greater emphasis should be placed on secondary prevention and early intervention for children and adolescents at an increased risk of later-life dual disorders. (3) Mental, addiction, and somatic care systems can be integrated, adopting a patient-focused approach to care delivery. (4) Recovery should be taken into consideration when defining treatment intervention and outcome goals. (5) It is important to reduce societal risk factors, such as poverty and early childhood adversity. (6) More resources are needed to provide adequate mental health care in the various countries. The development of European guidance initiatives would provide benefits in many of these areas, making it possible to ensure a more harmonized standard of care for patients with dual disorders.

Psychological distress in medical seeking ED care for somatic reasons: results of a systematic review

Objectives: The aim of this systematic literature review is to investigate (A) currently used instruments for assessing psychological distress, (B) the prevalence of psychological distress in medical emergency department (ED) patients with acute somatic conditions and (C) empirical evidence on how predictors are associated with psychological distress. Methods: We conducted an electronic literature search using three databases to identify studies that used validated instruments for detection of psychological distress in adult patients presented to the ED with somatic (non-psychiatric) complaints. From a total of 1688 potential articles, 18 studies were selected for in-depth review. Results: A total of 13 instruments have been applied for assessment of distress including screening questionnaires and briefly structured clinical interviews. Using these instruments, the prevalence of psychological distress detected in medical ED patients was between 4% and 47%. Psychological distress in general and particularly depression and anxiety have been found to be associated with demographic factors (eg, female gender, middle age) and illness-related variables (eg, urgency of triage category). Some studies reported that coexisting psychological distress of medical patients identified in the ED was associated with physical and psychological health status after ED discharge. Importantly, during routine clinical care, only few patients with psychological distress were diagnosed by their treating physicians. Conclusions: There is strong evidence that psychological distress is an important and prevalent cofactor in medically ill patients presenting to the ED with harmful associations with (subjective) health outcomes. To prove causality, future research should investigate whether screening and lowering psychological distress with specific interventions would result in better patient outcomes.

STUDIES ON THE BIOLOGICAL ACTIVITY OF MACROMOLECULES MICROINJECTED INTO MAMMALIAN SOMATIC CELLS

Red Blood cell mediated and glass needle mediated microinjection technology was used to introduce macromolecules into mammalian somatic cells. The biological activities of DNA synthesis inducing factor(s) (Chapter 1), mitotic factor(s) (Chapter 2), and DNA coding for ovalbumin and thymidine kinase (Chapter 3) were studied following injection into mammalian somatic cells.^ Chapter 1. A cell undergoing DNA replication (S phase) contains a factor(s) that induces DNA synthesis prematurely in a G(,1) nucleus when an S phase cell is fused to a G(,1) cell. An assay for the active factor(s) was developed in which a mixture of s phase extract loaded red blood cells (RBC) and synchronous G(,1) HeLa cells was centrifuged onto Concanavalin A (Con A) treated coverslips and fused by PEG. This technique is called "Centrifusion". The synchronous G(,1) HeLa cells injected with S phase extract initiated DNA synthesis earlier than the control G(,1) cells mock injected with RBC loaded with buffer.^ Chapter 2. It has been demonstrated that fusion between a mitotic and an interphase cell usually leads to breakdown of the interphase nucleus, followed by condensation of the interphase chromatin into discrete chromosomes, a process termed premature chromosome condensation. I wanted to develop an assay for the mitotic factor(s) that induces premature chromosome condensation. Experiments were performed utilizing glass needle mediated microinjection of HeLa cell mitotic extract into interphase somatic mammalian cells in an attempt to induce premature chromosome condensation. However, I was not able to induce premature chromosome condensation in the interphase cells, probably because of an inability to introduce sufficient mitotic factor(s) into the cells.^ Chapter 3. A recombinant plasmid containing the chicken ovalbumin gene and three copies of the Herpes thymidine Kinase gene (pOV12-TK) was introduced into mouse LMTK('-) cell nuclei using glass needle mediated gene transfer resulting in LMTK('+) clones that were selected for in HAT medium. Restriction enzyme analysis of the high molecular weight DNA from 6 HAT medium survivor cell clones revealed the presence of one or at best only a few copies of the 12kb ovalbumin gene per mouse genome. Further analysis showed the ovalbumin DNA was not rearranged and was associated with high molecular weight mouse cell DNA. Each of the analyzed cell clones produced ovalbumin demonstrating that the biological activity of the microinjected ovalbumin was retained. ^

The Differential Requirement of Albumin and Sodium Citrate on the Development of In Vitro Produced Bovine Embryos

In vitro culture for bovine embryos is largely not optimal. Our study was to determine the components necessary for early embryo development. In experiment 1, IVF embryos were cultured for two days in CR1aa medium containing sodium citrate and BSA from two sources (Sigma vs. ICPbio), subsequently for additional five days with cumulus monolayer in 10% FBS CR1aa. We found that supplementation with both Sigma-BSA and sodium citrate significantly increased total blastocyst (BL) development compared with the ICPbio-BSA groups (37% vs. 19-21%), and enhanced the total number of high quality (C1 BL, IETS standard) blastocysts (26% vs. 11-17%) (P < 0.05). In experiment 2 with serum free and/or somatic free culture, we found that CR1aa culture can support a comparable embryo development with a supplement of Sigma BSA. The addition of sodium citrate did not increase blastocyst development in either the Sigma-BSA or the ICPbio-BSA groups. An inferior blastocyst development occurring in ICPbio-BSA culture (1-3%) could be rescued by culture in CRlaa supplemented with 10% FBS (29%), more importantly, by culture in CR1aa with a replacement of Sigma BSA (24%) (P <0.05). C1 blastocysts rescued by FBS and Sigma BSA in ICPbio-BSA culture possessed indistinguishable morphology to embryos developed in a Sigma-BSA, FBS and somatic co-culture system, showing similar cell number/blastocyst (129-180, P > 0.05). Our study found a beneficial effect of sodium citrate and BSA on the in vitro development of bovine IVF embryos during co-culture. We also determined that differential embryotrophic factor(s) contained in BSA and serum, probably not sodium citrate, is necessary for promoting competent morula and blastocyst development in cattle.

Differential Cytoplast Requirement for Embryonic and Somatic Cell Nuclear Transfer in Cattle

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 microM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 micro g/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1-4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P < 0.05). In contrast, when skin fibroblasts were used as donor cells, the use of an MII cytoplast (vs. G1/S phase) was imperative for blastocyst development (30 vs. 6%, P < 0.05). Differential staining showed that parthenogenetic, embryonic, and somatic cloned BL contained 26, 29, and 33% presumptive inner cell mass (ICM) cells, respectively, which is similar to that of frozen-thawed in vivo embryos at a comparable developmental stage (23%). These data indicate that embryonic and somatic nuclei require different recipient cytoplast environment for remodeling/ reprogramming, and this is likely due to the different cell cycle stage and profiles of molecular differentiation of the transferred donor nuclei.

Cloning Animals by Somatic Cell Nuclear Transfer--Biological Factors

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.

Maternal ingestion of radon-222 in drinking water and the absorbed radiation dose to the embryo

Maternal ingestion of high concentrations of radon-222 (Rn-222) in drinking during pregnancy may pose a significant radiation hazard to the developing embryo. The effects of ionizing radiation to the embryo and fetus have been the subject of research, analyses, and the development of a number of radiation dosimetric models for a variety of radionuclides. Currently, essentially all of the biokinetic and dosimetric models that have been developed by national and international radiation protection agencies and organizations recommend calculating the dose to the mother's uterus as a surrogate for estimating the dose to the embryo. Heretofore, the traditional radiation dosimetry models have neither considered the embryo a distinct and rapidly developing entity, the fact that it is implanted in the endometrial layer of the uterus, nor the physiological interchanges that take place between maternal and embryonic cells following the implantation of the blastocyst in the endometrium. The purpose of this research was to propose a new approach and mathematical model for calculating the absorbed radiation dose to the embryo by utilizing a semiclassical treatment of alpha particle decay and subsequent scattering of energy deposition in uterine and embryonic tissue. The new approach and model were compared and contrasted with the currently recommended biokinetic and dosimetric models for estimating the radiation dose to the embryo. The results obtained in this research demonstrate that the estimated absorbed dose for an embryo implanted in the endometrial layer of the uterus during the fifth week of embryonic development is greater than the estimated absorbed dose for an embryo implanted in the uterine muscle on the last day of the eighth week of gestation. This research provides compelling evidence that the recommended methodologies and dosimetric models of the Nuclear Regulatory Commission and International Commission on Radiological Protection employed for calculating the radiation dose to the embryo from maternal intakes of radionuclides, including maternal ingestion of Rn-222 in drinking water would result in an underestimation of dose. ^

Seawater carbonate chemistry, the abiotic conditions in the fluid surrounding the embryo, growth, calcification of the cuttlefish Sepia officinalis in a laboratory experiment

This study investigated the effects of seawater pH (i.e., 8.10, 7.85 and 7.60) and temperature (16 and 19 °C) on (a) the abiotic conditions in the fluid surrounding the embryo (viz. the perivitelline fluid), (b) growth, development and (c) cuttlebone calcification of embryonic and juvenile stages of the cephalopod Sepia officinalis. Egg swelling increased in response to acidification or warming, leading to an increase in egg surface while the interactive effects suggested a limited plasticity of the swelling modulation. Embryos experienced elevated pCO2 conditions in the perivitelline fluid (>3-fold higher pCO2 than that of ambient seawater), rendering the medium under-saturated even under ambient conditions. The growth of both embryos and juveniles was unaffected by pH, whereas 45Ca incorporation in cuttlebone increased significantly with decreasing pH at both temperatures. This phenomenon of hypercalcification is limited to only a number of animals but does not guarantee functional performance and calls for better mechanistic understanding of calcification processes.

Computational methods to create and analyze a digital gene expression atlas of embryo development from microscopy images

Abstract The creation of atlases, or digital models where information from different subjects can be combined, is a field of increasing interest in biomedical imaging. When a single image does not contain enough information to appropriately describe the organism under study, it is then necessary to acquire images of several individuals, each of them containing complementary data with respect to the rest of the components in the cohort. This approach allows creating digital prototypes, ranging from anatomical atlases of human patients and organs, obtained for instance from Magnetic Resonance Imaging, to gene expression cartographies of embryo development, typically achieved from Light Microscopy. Within such context, in this PhD Thesis we propose, develop and validate new dedicated image processing methodologies that, based on image registration techniques, bring information from multiple individuals into alignment within a single digital atlas model. We also elaborate a dedicated software visualization platform to explore the resulting wealth of multi-dimensional data and novel analysis algo-rithms to automatically mine the generated resource in search of bio¬logical insights. In particular, this work focuses on gene expression data from developing zebrafish embryos imaged at the cellular resolution level with Two-Photon Laser Scanning Microscopy. Disposing of quantitative measurements relating multiple gene expressions to cell position and their evolution in time is a fundamental prerequisite to understand embryogenesis multi-scale processes. However, the number of gene expressions that can be simultaneously stained in one acquisition is limited due to optical and labeling constraints. These limitations motivate the implementation of atlasing strategies that can recreate a virtual gene expression multiplex. The developed computational tools have been tested in two different scenarios. The first one is the early zebrafish embryogenesis where the resulting atlas constitutes a link between the phenotype and the genotype at the cellular level. The second one is the late zebrafish brain where the resulting atlas allows studies relating gene expression to brain regionalization and neurogenesis. The proposed computational frameworks have been adapted to the requirements of both scenarios, such as the integration of partial views of the embryo into a whole embryo model with cellular resolution or the registration of anatom¬ical traits with deformable transformation models non-dependent on any specific labeling. The software implementation of the atlas generation tool (Match-IT) and the visualization platform (Atlas-IT) together with the gene expression atlas resources developed in this Thesis are to be made freely available to the scientific community. Lastly, a novel proof-of-concept experiment integrates for the first time 3D gene expression atlas resources with cell lineages extracted from live embryos, opening up the door to correlate genetic and cellular spatio-temporal dynamics. La creación de atlas, o modelos digitales, donde la información de distintos sujetos puede ser combinada, es un campo de creciente interés en imagen biomédica. Cuando una sola imagen no contiene suficientes datos como para describir apropiadamente el organismo objeto de estudio, se hace necesario adquirir imágenes de varios individuos, cada una de las cuales contiene información complementaria respecto al resto de componentes del grupo. De este modo, es posible crear prototipos digitales, que pueden ir desde atlas anatómicos de órganos y pacientes humanos, adquiridos por ejemplo mediante Resonancia Magnética, hasta cartografías de la expresión genética del desarrollo de embrionario, típicamente adquiridas mediante Microscopía Optica. Dentro de este contexto, en esta Tesis Doctoral se introducen, desarrollan y validan nuevos métodos de procesado de imagen que, basándose en técnicas de registro de imagen, son capaces de alinear imágenes y datos provenientes de múltiples individuos en un solo atlas digital. Además, se ha elaborado una plataforma de visualization específicamente diseñada para explorar la gran cantidad de datos, caracterizados por su multi-dimensionalidad, que resulta de estos métodos. Asimismo, se han propuesto novedosos algoritmos de análisis y minería de datos que permiten inspeccionar automáticamente los atlas generados en busca de conclusiones biológicas significativas. En particular, este trabajo se centra en datos de expresión genética del desarrollo embrionario del pez cebra, adquiridos mediante Microscopía dos fotones con resolución celular. Disponer de medidas cuantitativas que relacionen estas expresiones genéticas con las posiciones celulares y su evolución en el tiempo es un prerrequisito fundamental para comprender los procesos multi-escala característicos de la morfogénesis. Sin embargo, el número de expresiones genéticos que pueden ser simultáneamente etiquetados en una sola adquisición es reducido debido a limitaciones tanto ópticas como del etiquetado. Estas limitaciones requieren la implementación de estrategias de creación de atlas que puedan recrear un multiplexado virtual de expresiones genéticas. Las herramientas computacionales desarrolladas han sido validadas en dos escenarios distintos. El primer escenario es el desarrollo embrionario temprano del pez cebra, donde el atlas resultante permite constituir un vínculo, a nivel celular, entre el fenotipo y el genotipo de este organismo modelo. El segundo escenario corresponde a estadios tardíos del desarrollo del cerebro del pez cebra, donde el atlas resultante permite relacionar expresiones genéticas con la regionalización del cerebro y la formación de neuronas. La plataforma computacional desarrollada ha sido adaptada a los requisitos y retos planteados en ambos escenarios, como la integración, a resolución celular, de vistas parciales dentro de un modelo consistente en un embrión completo, o el alineamiento entre estructuras de referencia anatómica equivalentes, logrado mediante el uso de modelos de transformación deformables que no requieren ningún marcador específico. Está previsto poner a disposición de la comunidad científica tanto la herramienta de generación de atlas (Match-IT), como su plataforma de visualización (Atlas-IT), así como las bases de datos de expresión genética creadas a partir de estas herramientas. Por último, dentro de la presente Tesis Doctoral, se ha incluido una prueba conceptual innovadora que permite integrar los mencionados atlas de expresión genética tridimensionales dentro del linaje celular extraído de una adquisición in vivo de un embrión. Esta prueba conceptual abre la puerta a la posibilidad de correlar, por primera vez, las dinámicas espacio-temporales de genes y células.