A cellular lineage analysis of the chick limb bud

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.     

Position specific growth regulation of 3T3 cells in vivo.

In this study we have investigated the mechanism by which spatial growth is regulated by monitoring 3T3 cells, introduced into the developing mouse limb using exo utero surgery. The 3T3 cells were labeled with a human cell surface glycoprotein, CD8, and injected into stage 7-9 mouse limbs. At 24 and 48 hr after injection embryos were labeled with [3H]thymidine and processed for immunohistochemistry and autoradiography. The labeling index of CD8 positive cells was compared to that of neighboring limb bud cells and also to the position of the injection site within the limb. We find that the labeling index of 3T3 cells is in accord with that of the limb cells that immediately surround them; 3T3 cells display a high labeling index in limb regions of high growth and a low labeling index in limb regions of low growth. In addition, we find that both limb bud cells and injected 3T3 cells display a general proximal (low) to distal (high) gradient of growth at the stages analyzed. We conclude from these results that position-specific regulation of growth occurs in a non-cell autonomous manner and is likely to be mediated by mitogenic signals that are localized within the limb environment. In addition, our results demonstrate the usefulness of utilizing established cell lines as in vivo probes to monitor developmental mechanisms.     

Conditional inactivation of Fgfr1 in mouse defines its role in limb bud establishment, outgrowth and digit patterning

Previous studies have implicated fibroblast growth factor receptor 1 (FGFR1) in limb development. However, the precise nature and complexity of its role have not been defined. Here, we dissect Fgfr1 function in mouse limb by conditional inactivation of Fgfr1 using two different Cre recombinase-expressing lines. Use of the T (brachyury)-cre line led to Fgfr1 inactivation in all limb bud mesenchyme (LBM) cells during limb initiation. This mutant reveals FGFR1 function in two phases of limb development. In a nascent limb bud, FGFR1 promotes the length of the proximodistal (PD) axis while restricting the dimensions of the other two axes. It also serves an unexpected role in limiting LBM cell number in this early phase. Later on during limb outgrowth, FGFR1 is essential for the expansion of skeletal precursor population by maintaining cell survival. Use of mice carrying the sonic hedgehog(cre) (Shh(cre)) allele led to Fgfr1 inactivation in posterior LBM cells. This mutant allows us to test the role of Fgfr1 in gene expression regulation without disturbing limb bud growth. Our data show that during autopod patterning, FGFR1 influences digit number and identity, probably through cell-autonomous regulation of Shh expression. Our study of these two Fgfr1 conditional mutants has elucidated the multiple roles of FGFR1 in limb bud establishment, growth and patterning.     

A quantitative study of growth variability of tumour cell clones in vitro

Abstract.   Objectives : In this study, we quantify growth variability of tumour cell clones from a human leukaemia cell line. Materials and methods : We have used microplate spectrophotometry to measure growth kinetics of hundreds of individual cell clones from the Molt3 cell line. Growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results : Growth rates were observed to vary between different clones. Up to six clones with growth rates above or below mean growth rate of the parent population were further cloned and growth rates of their offspring were measured. Distribution of growth rates of the subclones did not significantly differ from that of the parent population, thus suggesting that growth variability has an epigenetic origin. To explain observed distributions of clonal growth rates, we have developed a probabilistic model, assuming that fluctuation in the number of mitochondria through successive cell cycles is the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the average maximum number of mitochondria in Molt3 cells. The model fits nicely observed distributions in growth rates; however, cells in which mitochondria were rendered non-functional (ρ⁰ cells) showed only 30% reduction in clonal growth variability with respect to normal cells. Conclusions : A tumour cell population is a dynamic ensemble of clones with highly variable growth rates. At least part of this variability is due to fluctuations in the initial number of mitochondria in daughter cells.     

Specification of cell fate along the proximal-distal axis in the developing chick limb bud

Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. In the present study, detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. Our fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. Our findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development.     

Development of the embryonic chick wing bud from stage 24 to stage 32

If a graft is placed in an early chick wing bud, the location of the graft after several days of further development cannot be predicted solely from the rate of proximal-distal outgrowth. The movement of the graft depends on the rate of outgrowth of the wing but also on morphogenetic tissue movements intrinsic to the wing and on accommodation to the growth and morphogenetic movements of the body of the embryo. Numerous experiments have been reported in which tissue grafted into ectopic sites in the wing causes abnormal wing development. These experiments have been discussed in terms of pattern formation or positional information. However, until the movement of wing tissue during normal development is understood, it cannot be known in what way the development of grafts placed in ectopic sites is abnormal. Previous experiments have demonstrated that carbon particles placed in the wing move in the same manner as grafts of wing mesenchyme, but the carbon particles do not affect normal wing development. Carbon particles were placed in the wing, dorsal to the base of the wing, and cranial and caudal to the wing, to plot the expected movement of a graft and to discover how this movement can be predicted from the tissue movements at the base of the wing. It is concluded that three tissue movements are responsible for the movement of a graft. These are outgrowth at a rate determined by the rate of cell division, formation of the shoulder through caudal movement of the tissues cranial to the wing, and ventral movement of prospective flank ventral to somite 19. These three tissue movements and their influence on normal wing development are discussed.     

Music Attenuates Excessive Visual Guidance of Skilled Reaching in Advanced but Not Mild Parkinson's Disease

Parkinson''s disease (PD) results in movement and sensory impairments that can be reduced by familiar music. At present, it is unclear whether the beneficial effects of music are limited to lessening the bradykinesia of whole body movement or whether beneficial effects also extend to skilled movements of PD subjects. This question was addressed in the present study in which control and PD subjects were given a skilled reaching task that was performed with and without accompanying preferred musical pieces. Eye movements and limb use were monitored with biomechanical measures and limb movements were additionally assessed using a previously described movement element scoring system. Preferred musical pieces did not lessen limb and hand movement impairments as assessed with either the biomechanical measures or movement element scoring. Nevertheless, the PD patients with more severe motor symptoms as assessed by Hoehn and Yahr (HY) scores displayed enhanced visual engagement of the target and this impairment was reduced during trials performed in association with accompanying preferred musical pieces. The results are discussed in relation to the idea that preferred musical pieces, although not generally beneficial in lessening skilled reaching impairments, may normalize the balance between visual and proprioceptive guidance of skilled reaching.     

The analysis of clonal expansions in normal and autoimmune B cell repertoires

Clones are the fundamental building blocks of immune repertoires. The number of different clones relates to the diversity of the repertoire, whereas their size and sequence diversity are linked to selective pressures. Selective pressures act both between clones and within different sequence variants of a clone. Understanding how clonal selection shapes the immune repertoire is one of the most basic questions in all of immunology. But how are individual clones defined? Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development. Next, we discuss how clones are defined using different experimental methods. We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape. We discuss methods that visualize sequence variants within the same clone and allow us to consider collections of shared mutations to determine which sequences share a common ancestry. Finally, we comment on features of frequently encountered expanded B cell clones that may be of particular interest in the setting of autoimmunity and other chronic conditions.     

Phytochrome control of short-day-induced bud set in black cottonwood

In trees and other woody perennial plants, short days (SDs) typically induce growth cessation, the initiation of cold acclimation, the formation of a terminal bud and bud dormancy. Phytochrome control of SD-induced bud set was investigated in two northern clones of black cottonwood (Populus trichocarpa Torr. & Gray) by using night breaks with red light (R) and far-red light (FR). For both clones (BC-1 and BC-2), SD-induced bud set was prevented when R night breaks as short as 2 min were given in the middle of the night. When night breaks with 2 min of R were immediately followed by 2 min of FR, substantial reversibility of bud set was observed for BC-1 but not for BC-2. By comparing the effects of the R night breaks on bud set and the length of specific internodes, we determined that the R night breaks influenced internode elongation in two opposing ways. First, the addition of a R night break to the SD treatment prevented the cessation of internode elongation that is associated with bud set. Those internodes that would not have elongated under SDs (and would have been found within the terminal bud) elongated in the R treatment. Second, the R night breaks decreased internode length relative to the long-day (LD) control. In contrast to the clonal differences in reversibility that we observed for bud set, the decrease in internode length (i.e. the second effect of R) was R/FR reversible in both clones. Based on these results, we conclude that internode elongation is influenced by two distinct types of phytochrome-mediated response. The first response is a typical response to photpperiod, whereas the second response is a typical “end-of-day” response to light quality. Our results demonstrate that SD-induced bud set in black cottonwood is controlled by phytochrome but that clonal differences have an important influence on the R/FR reversibility of this response. The availability of an experimental system in which SD-induced bud set is R/FR reversible will be valuable for studying the physiological genetics of photoperiodism in trees.     

Dynamical mechanisms for skeletal pattern formation in the vertebrate limb

We describe a 'reactor-diffusion' mechanism for precartilage condensation based on recent experiments on chondrogenesis in the early vertebrate limb and additional hypotheses. Cellular differentiation of mesenchymal cells into subtypes with different fibroblast growth factor (FGF) receptors occurs in the presence of spatio-temporal variations of FGFs and transforming growth factor-betas (TGF-betas). One class of differentiated cells produces elevated quantities of the extracellular matrix protein fibronectin, which initiates adhesion-mediated preskeletal mesenchymal condensation. The same class of cells also produces an FGF-dependent laterally acting inhibitor that keeps condensations from expanding beyond a critical size. We show that this 'reactor-diffusion' mechanism leads naturally to patterning consistent with skeletal form, and describe simulations of spatio-temporal distribution of these differentiated cell types and the TGF-beta and inhibitor concentrations in the developing limb bud.     

Role of chondrogenic tissue in programmed cell death and BMP expression in chick limb buds

In the developing chick leg bud, massive programmed cell death occurs in the interdigital region. Previously, we reported the inhibition of cell death by separation of the interdigital region from neighboring digit cartilage. In this study, we examined the relationship between cell death and cartilaginous tissue in vitro. First, cell fate was observed with DiI that was used to examine cell movement in the distal tip of leg bud. Labeled cells in the prospective digital region were distributed only in the distal region as a narrow band, while cells in the prospective interdigital region expanded widely in the interdigit. In coculture of monolayer cells and a cell pellet tending to differentiate into cartilage, monolayer cells migrated into the cell pellet. These results suggested that digit cartilage tends to recruit neighboring cells into the cartilage during limb development. Next, we observed the relationship between cell death and chondrogenesis in monolayer culture. Apoptotic cell death that could be detected by TUNEL occurred in regions between cartilaginous nodules in mesenchymal cell culture. More apoptotic cell death was detected in the cell culture of leg bud mesenchyme of stage 25/26 than that of leg bud mesenchyme of stage 22 or that of stage 28. The most developed cartilaginous nodules were observed in the cell culture of stage 25/26. Finally, we observed Bmp expression in vitro and in vivo. Bmp-2, Bmp-4 and Bmp-7 were detected around the cartilage nodules. When the interdigit was separated from neighboring digit cartilage, Bmp-4 expression disappeared near the cut region but remained near the digit cartilage. This correlation between cell death and cartilaginous region suggests that cartilage tissue can induce apoptotic cell death in the developing chick limb bud due to cell migration accompanying chondrogenesis and Bmp expression.     

Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.     

Extensive Hidden Genomic Mosaicism Revealed in Normal Tissue

Genomic mosaicism arising from post-zygotic mutation has recently been demonstrated to occur in normal tissue of individuals ascertained with varied phenotypes, indicating that detectable mosaicism may be less an exception than a rule in the general population. A challenge to comprehensive cataloging of mosaic mutations and their consequences is the presence of heterogeneous mixtures of cells, rendering low-frequency clones difficult to discern. Here we applied a computational method using estimated haplotypes to characterize mosaic megabase-scale structural mutations in 31,100 GWA study subjects. We provide in silico validation of 293 previously identified somatic mutations and identify an additional 794 novel mutations, most of which exist at lower aberrant cell fractions than have been demonstrated in previous surveys. These mutations occurred across the genome but in a nonrandom manner, and several chromosomes and loci showed unusual levels of mutation. Our analysis supports recent findings about the relationship between clonal mosaicism and old age. Finally, our results, in which we demonstrate a nearly 3-fold higher rate of clonal mosaicism, suggest that SNP-based population surveys of mosaic structural mutations should be conducted with haplotypes for optimal discovery.