Prevention of development of autoimmune disease in BXSB mice by mixed bone marrow transplantation

Transplantations of fully allogeneic, autoimmune-resistant T-cell-depleted marrow (TCDM) plus syngeneic, autoimmune-prone TCDM into lethally irradiated BXSB mice were carried out to investigate the ability of the mixed bone marrow transplantation (BMT) to prevent development of autoimmune disease and, at the same time, to reconstitute fully the immunity functions of heavily irradiated BXSB recipients. Male BXSB mice were engrafted with mixed TCDM from both allogeneic, autoimmune-resistant BALB/c mice and syngeneic, autoimmune-prone BXSB mice. BMT with mixed TCDM from both resistant and susceptible strains of mice (mixed BMT) prolonged the median life span and inhibited development of glomerulonephritis in BXSB mice. BMT with mixed TCDM also prevented the formation of anti-DNA antibodies that is typically observed in male mice of this strain. Moreover, mixed BMT reconstituted primary antibody production in BXSB recipients, so that no annoying immunodeficiencies that are regularly observed in fully allogeneic chimeras were present in the recipient of the mixed TCDM. These findings indicate that transplanting allogeneic, autoimmune-resistant TCDM plus syngeneic, autoimmune-prone TCDM into lethally irradiated BXSB mice prevents development of autoimmune disease in this strain of mice. In addition, this dual BMT reconstitutes the immunity functions and avoids the immunodeficiencies that occur regularly in fully allogeneic chimeras after total-body irradiation.

Spatial and Temporal Analyses of Expansion and Cell Cycle in Sunflower Leaves1 : A Common Pattern of Development for All Zones of a Leaf and Different Leaves of a Plant

We have investigated the spatial distributions of expansion and cell cycle in sunflower (Helianthus annuus L.) leaves located at two positions on the stem, from leaf initiation to the end of expansion. Relative expansion rate (RER) was analyzed by following the deformation of a grid drawn on the lamina; relative division rate (RDR) and flow-cytometry data were obtained in four zones perpendicular to the midrib. Calculations for determining in situ durations of the cell cycle and of S-G2-M in the epidermis are proposed. Area and cell number of a given leaf zone increased exponentially during the first two-thirds of the development duration. RER and RDR were constant and similar in all zones of a leaf and in all studied leaves during this period. Reduction in RER occurred afterward with a tip-to-base gradient and lagged behind that of RDR by 4 to 5 d in all zones. After a long period of constancy, cell-cycle duration increased rapidly and simultaneously within a leaf zone, with cells blocked in the G0-G1 phase of the cycle. Cells that began their cycle after the end of the period with exponential increase in cell number could not finish it, suggesting that they abruptly lost their competence to cross a critical step of the cycle. Differences in area and in cell number among zones of a leaf and among leaves of a plant essentially depended on the timing of two events, cessation of exponential expansion and of exponential division.

Layer-specific programs of development in neocortical projection neurons.

How are long-range axonal projections from the cerebral cortex orchestrated during development? By using both passively and actively transported axonal tracers in fetal and postnatal ferrets, we have analyzed the development of projections from the cortex to a number of thalamic nuclei. We report that the projections of a cortical area to its corresponding thalamic nuclei follow highly cell-specific programs of development. Axons from cells in the deepest layers of the cerebral cortex (layer 6 and superficial subplate neurons) appear to grow very slowly and be delayed for several weeks in the cerebral white matter, reaching the thalamus over a protracted period. Neurons of layer 5, on the other hand, develop their projections much faster; despite being born after the neurons of deeper layers, layer 5 neurons are the first to extend their axons out of the cortical hemisphere and innervate the thalamus. Layer 5 projections are massive in the first postnatal weeks but may become partly eliminated later in development, being overtaken in number by layer 6 cells that constitute the major corticothalamic projection by adulthood. Layer 5 projections are area-specific from the outset and arise as collateral branches of axons directed to the brainstem and spinal cord. Our findings show that the early development of corticofugal connections is determined not by the sequence of cortical neurogenesis but by developmental programs specific for each type of projection neuron. In addition, they demonstrate that in most thalamic nuclei, layer 5 neurons (and not subplate or layer 6 neurons) establish the first descending projections from the cerebral cortex.

A modern human pattern of dental development in Lower Pleistocene hominids from Atapuerca-TD6 (Spain)

The study of life history evolution in hominids is crucial for the discernment of when and why humans have acquired our unique maturational pattern. Because the development of dentition is critically integrated into the life cycle in mammals, the determination of the time and pattern of dental development represents an appropriate method to infer changes in life history variables that occurred during hominid evolution. Here we present evidence derived from Lower Pleistocene human fossil remains recovered from the TD6 level (Aurora stratum) of the Gran Dolina site in the Sierra de Atapuerca, northern Spain. These hominids present a pattern of development similar to that of Homo sapiens, although some aspects (e.g., delayed M3 calcification) are not as derived as that of European populations and people of European origin. This evidence, taken together with the present knowledge of cranial capacity of these and other late Early Pleistocene hominids, supports the view that as early as 0.8 Ma at least one Homo species shared with modern humans a prolonged pattern of maturation.

Microtubule-based Endoplasmic Reticulum Motility in Xenopus laevis: Activation of Membrane-associated Kinesin during Development

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts of Xenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopus egg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development in Xenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute ∼19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower (∼0.4 μm/s) than minus end-directed motility (∼1.3 μm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other.

Molecular heterochrony in the early development of Drosophila

Heterochrony, the relative change of developmental timing, is one of the major modes of macroevolutionary change; it identifies temporally disassociated units of developmental evolution. Here, we report the results of a fine-scale temporal study for the expression of the developmental gene hairy and morphological development in three species of Drosophila, D. melanogaster, D. simulans, and D. pseudoobscura. The results suggest that between and among closely related species, temporal displacement of ontogenetic trajectory is detected even at the earliest stage of development. Overall, D. simulans shows the earliest expression, followed by D. melanogaster, and then by D. pseudoobscura. Setting D. melanogaster as the standard, we find the approximate time to full expression is accelerated by 13 min, 48 s in D. simulans and retarded by 24 min in D. pseudoobscura. Morphologically, again with D. melanogaster setting the standard, initiation of cellularization is faster in D. simulans by 15 min, 42 s; and initiation of morphogenesis is faster in D. simulans by 18 min, 7 s. These results seem to be consistent with the finding that the approximate time to full expression of hairy is accelerated by 13 min, 48 s in D. simulans. On the other hand, the same morphological events are delayed by 5 min, 32 s, and by 11 min, 32 s, respectively, in D. pseudoobscura. These delays are small, compared with the 24-min delay in full expression. The timing changes, in total, seem consistent with continuous phyletic evolution of temporal trajectories. Finally, we speculate that epigenetic interactions of hairy expression timing and cell-cycle timing may have led to morphological differences in the terminal system of the larvae.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2Ek/Ek) was lethal in mid-gestation (the equivalent of mouse E9.5–E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long–Evans strain Tsc2Ek/Ek embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants.

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta- and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development.

Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.

We have recently cloned the human fms-like tyrosine kinase 4 gene FLT4, whose protein product is related to two vascular endothelial growth factor receptors FLT1 and KDR/FLK1. Here the expression of FLT4 has been analyzed by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA in adult human tissues. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. Our results suggest that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also support the theory on the venous origin of lymphatic vessels.