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NASA NTRS1999

Avian Blood-Vessel Formation in Space

Based on previous studies, we hypothesized that the developmental anomalies observed in the past might be related to or caused by delayed or improper vascular development. The objective of our research is to test the hypothesis that exposure to microgravity during space flight cause delayed or improper vascular development during embryogenesis. The effects of microgravity on the time course and extent of avian blood-vessel formation are assessed using two models, one for angiogenesis and one for vasculogenesis. The methodological approach is dictated by the constraints of the tissue preservation method used in space. Thus, both in the chorioallantoic membrane (CAM) and in the adrenal, we will evaluate microscopically the vascular architecture and immunostain endothelial cells with specific antibodies (anti- vWF and QH1). The extent of ECM protein deposition will be assessed by immunohistochemistry and correlated with the degree of vascularization, using computer-based image analysis. Also, the cellular source for ECM proteins will be assessed by in situ hybridization.

Lelkes, Peter I.↗

NASA NTRS2002

XCTK1: A Xenopus C-terminal Kinesin-like Protein

XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

Winfree, Seth↗

NASA NTRS2003

Mammalian development in space

Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young mammals develop, comprised of its mother and siblings, is of paramount importance in interpreting spaceflight effects.

Review↗

NASA NTRS2002

Testing putative hemichordate homologues of the chordate dorsal nervous system and endostyle: expression of NK2.1 (TTF-1) in the acorn worm Ptychodera flava (Hemichordata, Ptychoderidae)

Recent phylogenetic investigations have confirmed that hemichordates and echinoderms are sister taxa. However, hemichordates share several cardinal characterstics with chordates and are thus an important taxon for testing hypotheses of homology between key chordate characters and their putative hemichordate antecedents. The chordate dorsal nervous system (DNS) and endostyle are intriguing characters because both hemichordate larval and adult structures have been hypothesized as homologues. This study attempts to test these purported homologies through examination of the expression pattem of a Ptychodera flava NK2 gene, PfNK2.1, because this gene is expressed both in the DNS and endostyle/thyroid in a wide range of chordate taxa. We found that PfNK2.1 is expressed in both neuronal and pharyngeal structures, but its expression pattem is broken up into distinct embryonic and juvenile phases. During embryogenesis, PfNK2.1 is expressed in the apical ectoderm, with transcripts later detected in presumable neuronal structures, including the apical organ and ciliated feeding band. In the developing juvenile we detected PfNK2.1 signal throughout the pharynx, including the stomochord, and later in the hindgut. We conclude that the similar utilization of NK2.1 in apical organ development and chordate DNS is probably due to a more general role for NK2.1 in neurogenesis and that hemichordates do not possess a homologue of the chordate DNS. In addition, we conclude that P. flava most likely does not possess a true endostyle; rather during the evolution of the endostyle NK2.1 was recruited from its more general role in pharynx development.

NASA Discipline Evolutionary Biology↗

NASA NTRS2002

The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest

Amphioxus, the closest living invertebrate relative of the vertebrates, has a notochord, segmental axial musculature, pharyngeal gill slits and dorsal hollow nerve cord, but lacks neural crest. In amphioxus, as in vertebrates, exogenous retinoic acid (RA) posteriorizes the embryo. The mouth and gill slits never form, AmphiPax1, which is normally downregulated where gill slits form, remains upregulated and AmphiHox1 expression shifts anteriorly in the nerve cord. To dissect the role of RA signaling in patterning chordate embryos, we have cloned the single retinoic acid receptor (AmphiRAR), retinoid X receptor (AmphiRXR) and an orphan receptor (AmphiTR2/4) from amphioxus. AmphiTR2/4 inhibits AmphiRAR-AmphiRXR-mediated transactivation in the presence of RA by competing for DR5 or IR7 retinoic acid response elements (RAREs). The 5' untranslated region of AmphiTR2/4 contains an IR7 element, suggesting possible auto- and RA-regulation. The patterns of AmphiTR2/4 and AmphiRAR expression during embryogenesis are largely complementary: AmphiTR2/4 is strongly expressed in the cerebral vesicle (homologous to the diencephalon plus anterior midbrain), while AmphiRAR expression is high in the equivalent of the hindbrain and spinal cord. Similarly, while AmphiTR2/4 is expressed most strongly in the anterior and posterior thirds of the endoderm, the highest AmphiRAR expression is in the middle third. Expression of AmphiRAR is upregulated by exogenous RA and completely downregulated by the RA antagonist BMS009. Moreover, BMS009 expands the pharynx posteriorly; the first three gill slit primordia are elongated and shifted posteriorly, but do not penetrate, and additional, non-penetrating gill slit primordia are induced. Thus, in an organism without neural crest, initiation and penetration of gill slits appear to be separate events mediated by distinct levels of RA signaling in the pharyngeal endoderm. Although these compounds have little effect on levels of AmphiTR2/4 expression, RA shifts pharyngeal expression of AmphiTR2/4 anteriorly, while BMS009 extends it posteriorly. Collectively, our results suggest a model for anteroposterior patterning of the amphioxus nerve cord and pharynx, which is probably applicable to vertebrates as well, in which a low anterior level of AmphiRAR (caused, at least in part, by competitive inhibition by AmphiTR2/4) is necessary for patterning the forebrain and formation of gill slits, the posterior extent of both being set by a sharp increase in the level of AmphiRAR. Supplemental data available on-line.

NASA Discipline Evolutionary Biology↗

NASA NTRS2001

Multiple inductive signals are involved in the development of the ctenophore Mnemiopsis leidyi

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e(1) micromeres (e(11) and e(12)) and a single daughter of the four m(1) micromeres (the m(12) micromeres). Although isolated e(1) micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e(1) descendents. Thus, the m(1) lineage requires the inductive interaction of the e(1) lineage to contribute to comb plate formation. Here we show that, although m(12) cells are normally the only m(1) derivatives to contribute to comb plate formation, m(11) cells are capable of generating comb plates in the absence m(12) cells. The reason that m(11) cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e(1) descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m(12). In addition, we show that the signals provided by the e(1) lineage are not sufficient for m(1)-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required. Copyright 2001 Academic Press.

NASA Discipline Evolutionary Biology↗

NASA NTRS1998

Control of seed development in Arabidopsis thaliana by atmospheric oxygen

Seed development is known to be inhibited completely when plants are grown in oxygen concentrations below 5.1 kPa, but apart from reports of decreased seed weight little is known about embryogenesis at subambient oxygen concentrations above this critical level. Arabidopsis thaliana (L.) Heynh. plants were grown full term under continuous light in premixed atmospheres with oxygen partial pressures of 2.5, 5.1, 10.1, 16.2 and 21.3 kPa O2, 0.035 kPa CO2 and the balance nitrogen. Seeds were harvested for germination tests and microscopy when siliques had yellowed. Seed germination was depressed in O2 treatments below 16.2 kPa, and seeds from plants grown in 2.5 kPa O2 did not germinate at all. Fewer than 25% of the seeds from plants grown in 5.1 kPa oxygen germinated and most of the seedlings appeared abnormal. Light and scanning electron microscopic observation of non-germinated seeds showed that these embryos had stopped growing at different developmental stages depending upon the prevailing oxygen level. Embryos stopped growing at the heart-shaped to linear cotyledon stage in 5.1 kPa O2, at around the curled cotyledon stage in 10.1 kPa O2, and at the premature stage in 16.2 kPa O2. Globular and heart-shaped embryos were observed in sectioned seeds from plants grown in 2.5 kPa O2. Tissue degeneration caused by cell autolysis and changes in cell structure were observed in cotyledons and radicles. Transmission electron microscopy of mature seeds showed that storage substances, such as protein bodies, were reduced in subambient oxygen treatments. The results demonstrate control of embryo development by oxygen in Arabidopsis.

NASA Discipline Plant Biology↗

NASA NTRS1997

Vertebrate development in the environment of space: models, mechanisms, and use of the medaka

With the advent of space travel, it is of immediate interest and importance to study the effects of exposure to various aspects of the altered environment of space, including microgravity, on Earth-based life forms. Initial studies of space travel have focused primarily on the short-term effects of radiation and microgravity on adult organisms. However, with the potential for increased lengths of time in space, it is critical to now address the effects of space on all phases of an organism's life cycle, from embryogenesis to post-natal development to reproduction. It is already possible for certain species to undergo multiple generations within the confines of the Mir Space Station. The possibility now exists for scientists to consider the consequences of even potentially subtle defects in development through multiple phases of an organism's life cycle, or even through multiple generations. In this discussion, we highlight a few of the salient observations on the effects of the space environment on vertebrate development and reproductive function. We discuss some of the many unanswered questions, in particular, in the context of the choice of appropriate models in which to address these questions, as well as an assessment of the availability of hardware already existing or under development which would be useful in addressing these questions.

Flight Experiment↗

NASA NTRS1995

Models and molecular approaches to assessing the effects of the microgravity environment on vertebrate development

The extent to which gravity, and especially the lack thereof, can affect normal development in higher organisms is poorly understood. Underlying this question is the assumption that normal development depends on the embryo's ability to maintain a programmed temporal and spatial coordination of morphogenetic events. There are several reports documenting the apparently normal development of several vertebrate species, including mammals, under conditions of exposure to space flight during various periods of the development process. Evidence to the contrary also exists and it is therefore likely that some alterations in morphology do occur in a microgravity environment. Although subsequent development may appear overtly normal, more subtle abnormalities result. In all studies, the evaluation is restricted by the few numbers of specimens that can be examined and the relatively insensitive techniques for assessing potentially subtle effects. In the present discussion, we summarize some observations of mammalian development made in microgravity and consider which stages might be expected to be differentially sensitive to altered gravity conditions. While we emphasize mammalian development, we discuss the suitability of another model system for examining such effects in a cross-species context. Furthermore, we consider recent developments in our understanding of the molecular genetic program regulating embryogenesis that could serve as markers for assessing perturbations of development.

Cosmos 1129 Project↗

NASA NTRS1990

Revival of Dobell's "chromidia" hypothesis: chromatin bodies in the amoebomastigote Paratetramitus jugosus

Multiple fission of a mature Paratetramitus jugosus (approx. 10 micrometers long) resulted in the production of many small, roughly spherical (2-7 micrometers in diameter) amoebae. Our observation of live material and examination of over two hundred micrographs lead us to suggest that DNA-containing membrane-bounded chromatin bodies bud amitotically from the nucleus. DAPI-stained bodies of these were observed in the cytoplasm of amoebae, mastigotes, and cysts, and at least some of these chromatin bodies seemed to be released into the medium. This interpretation revives for P. jugosus the "chromatin hypothesis" of Dobell. Our data, consistent with the descriptions of Dobell, Hogue, and Wherry, indicate that encysting amoebae may reproduce by chromidia. Dobell's original chromidia concept was limited to amoebae. Others claimed for it far-reaching consequences: "chromidia" were touted as an explanation for embryogenesis and histogenesis of metazoa. Although there is no evidence for chromidia in animals, outright rejection of Dobell's chromidia hypothesis sensu stricto as an amitotic multiple fission process in amoebae is unjustified.

Non-NASA Center↗

NASA NTRS1992

Drosophila melanogaster as a model system for assessing development under conditions of microgravity

More is known about the regulation of early developmental events in Drosophila than any other animal. In addition, its size and short life cycle make it a facile experimental system. Since developmental perturbations have been demonstrated when both oogenesis and embryogenesis occur in the space environment, there is a strong rationale for using this organism for the elucidation of specific gravity-sensitive developmental events.

NASA Discipline Developmental Biology↗

NASA NTRS2003

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Pluripotent Stem Cells/cytology/physiology↗

NASA NTRS1994

Insect gravitational biology: ground-based and shuttle flight experiments using the beetle Tribolium castaneum

Many of the traditional experimental advantages of insects recommend their use in studies of gravitational and space biology. The fruit fly, Drosophila melanogaster, is an obvious choice for studies of the developmental significance of gravity vectors because of the unparalleled description of regulatory mechanisms controlling oogenesis and embryogenesis. However, we demonstrate that Drosophila could not survive the conditions mandated for particular flight opportunities on the Space Shuttle. With the exception of Drosophila, the red flour beetle, Tribolium castaneum, is the insect best characterized with respect to molecular embryology and most frequently utilized for past space flights. We show that Tribolium is dramatically more resistant to confinement in small sealed volumes. In preparation for flight experiments we characterize the course and timing of the onset of oogenesis in newly eclosed adult females. Finally, we present results from two shuttle flights which indicate that a number of aspects of the development and function of the female reproductive system are not demonstrably sensitive to microgravity. Available information supports the utility of this insect for future studies of gravitational biology.

NASA Discipline Developmental Biology↗

NASA NTRS2001

Complex expression patterns of lymphocyte-specific genes during the development of cartilaginous fish implicate unique lymphoid tissues in generating an immune repertoire

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish-the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult.

Non-NASA Center↗

NASA NTRS2000

Spatial expression of Hox cluster genes in the ontogeny of a sea urchin

The Hox cluster of the sea urchin Strongylocentrous purpuratus contains ten genes in a 500 kb span of the genome. Only two of these genes are expressed during embryogenesis, while all of eight genes tested are expressed during development of the adult body plan in the larval stage. We report the spatial expression during larval development of the five 'posterior' genes of the cluster: SpHox7, SpHox8, SpHox9/10, SpHox11/13a and SpHox11/13b. The five genes exhibit a dynamic, largely mesodermal program of expression. Only SpHox7 displays extensive expression within the pentameral rudiment itself. A spatially sequential and colinear arrangement of expression domains is found in the somatocoels, the paired posterior mesodermal structures that will become the adult perivisceral coeloms. No such sequential expression pattern is observed in endodermal, epidermal or neural tissues of either the larva or the presumptive juvenile sea urchin. The spatial expression patterns of the Hox genes illuminate the evolutionary process by which the pentameral echinoderm body plan emerged from a bilateral ancestor.

Non-NASA Center↗

NASA NTRS2000

Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

Non-NASA Center↗

NASA NTRS1999

Laser ablation of persistent twist cells in Drosophila: muscle precursor fate is not segmentally restricted

In Drosophila the precursors of the adult musculature arise during embryogenesis. These precursor cells have been termed Persistent Twist Cells (PTCs), as they continue to express the transcription factor Twist after that gene ceases expression elsewhere in the mesoderm. In the larval abdomen, the PTCs are associated with peripheral nerves in stereotypic ventral, dorsal, and lateral clusters, which give rise, respectively, to the ventral, dorsal, and lateral muscle fiber groups of the adult. We tested the developmental potential of the PTCs by using a microbeam laser to ablate specific clusters in larvae. We found that the ablation of a single segmental PTC cluster does not usually result in the deletion of the corresponding adult fibers of that segment. Instead, normal or near normal numbers of adult fibers can form after the ablation. Examination of pupae following ablation showed that migrating PTCs from adjacent segments are able to invade the affected segment, replenishing the ablated cells. However, the ablation of homologous PTCs in multiple segments does result in the deletion of the corresponding adult muscle fibers. These data indicate that the PTCs in an abdominal segment can contribute to the formation of muscle fibers in adjacent abdominal segments, and thus are not inherently restricted to the formation of muscle fibers within their segment of origin.

NASA Discipline Neuroscience↗

NASA NTRS1998

[Characteristics of morphogenesis of the Japanese quail embryos during microgravity]

Experiments performed in the period of 1995-1996 cooperatively with US investigators within the MIR/SHUTTLE and MIR/NASA space science projects continued exploration of avian embryogenesis in microgravity. Evaluation of Japanese quail embryos incubated in spaceflight microgravity showed that for the most part they were normally developed and compliant with duration of incubation. One of the major morphometric characteristics of embryo are its mass and size. Comparative analysis of body mass values in the space and laboratory and synchronous control groups pointed to a slight retardation. Body length of space embryos mimicked their mass curve. Data on the dynamics of mass and length of Japanese quail embryos support the well-known theory according to which growth and formation are distinguished by equifinality. No differences were revealed by the investigations of individual parts of embryonic bodies in the space and control groups. However, this finding was true only with regard to the embryos that had no developmental abnormalities. A part of embryos had defective eyes (microphtalmia), limbs (twisted fingers), and beaks.

manned↗