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Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal–ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal. This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

The pairing of homologous chromosomes (homologs) in meiosis is essential for distributing the correct numbers of chromosomes into haploid gametes. In budding yeast, pairing depends on the formation of 150-200 Spo11-mediated double-strand breaks (DSBs) that are distributed among 16 homolog pairs, but it is not known if all, or only a subset of these DSBs, contribute to the close juxtaposition of homologs. Having established a system to measure the position of fluorescently tagged chromosomal loci in 3D space over time, we analyzed locus trajectories to determine how frequently, and how long, loci spend colocalized or apart. Continuous imaging revealed highly heterogeneous cell-to-cell behavior of foci, with the majority of cells exhibiting a “mixed” phenotype where foci move into and out of proximity, even at late stages of prophase, suggesting that the axial structures of the synaptonemal complex may be more dynamic than anticipated. The observed plateaus of the mean-squared change in distance (MSCD) between foci informed the development of a biophysical model of two diffusing polymers that captures the loss of centromere linkages as cells enter meiosis, nuclear confinement, and the formation of Spo11-dependent linkages. The predicted number of linkages per chromosome in our theoretical model closely approximates the small number (~2-4) of estimated synapsis-initiation sites, suggesting that excess DSBs have negligible effects on the overall juxtaposition of homologs. These insights into the dynamic in-terchromosomal behavior displayed during homolog pairing demonstrate the power of combining time-resolvedin vivoanalysis with modeling at the granular level.Significance StatementEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process is the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent “pairing” of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study is the first to provide observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

InDrosophilaembryos, genes expressed along the dorsal-ventral axis are responsive to concentration of the Dorsal (Dl) transcription factor, which varies in space; however, levels of this morphogen also build over time. Since expression of high-threshold Dl target genes such assnail(sna) is supported before Dl levels peak, it is unclear what role increasing levels have if any. Here we investigated action of two enhancers that controlsnaexpression in embryos, demonstrating using genome editing that Dl binding sites within one enhancer located promoter proximally,sna.prox, can limit the ability of the other distally-located enhancer,sna.dis, to increasesnalevels. In addition, MS2-MCP live imaging was used to studysnatranscription rate in wildtype,dlheterozygote, and a background in which a photo-sensitive degron is fused to Dl (dl-BLID). The results demonstrate that, when Dl levels are high, Dl acts throughsna.proxto limit the activity ofsna.disand thereby influencesnatranscription rate. In contrast, when Dl levels are kept low usingdl-BLID, sna.proxpositively influencessnatranscription rate. Collectively, our data support the view that Dl’s effect on gene expression changes over time, switching from promotingsnaexpression at low concentration to dampeningsnaexpression at high concentration by regulating enhancer interactions. We propose this differential action of the Dl morphogen is likely supported by occupancy of this factor first to high and then low affinity binding sites over time as Dl levels rise to coordinate action of these two co-acting enhancers.Significance statementA gradient of the maternal transcription factor Dorsal is important for establishing spatial expression of target genes along the dorsal-ventral axis ofDrosophilaembryos. Dorsal levels are also dynamic as nuclear concentration builds in time. Surprisingly, expression of high-threshold target genes such assnailis supported before levels peak, raising the question why levels continue to build. Our data support the view that peak Dorsal levels act to preferentially support activity of one enhancer over another to effectively decreasesnailexpression. In addition, while the morphogen Dorsal acts early to support gene expression, later it effectively acts as a damper to limit gene expression. Our results suggest other morphogens also have effects on gene expression that change over time.

Homologous recombination drives structural reorganization of the nucleus in early meiosis. In order to investigate the connection between homolog pairing, meiotic progression, and the dynamics of the underlying chromatin, we tracked flourescently labeled homolog pairs in synchronized S. cerevisae. Various previously unreported statistics of the anomalous inter-loci motion correlate with meiotic progression and can be quantitatively reproduced by a simple polymer model of the sister chromatids. The first of these is the distribution of waiting times for the homologous loci to come into and out of contact with each other (loosely, inter-locus “looping” and “unlooping” times). The full shape of the looping time distribution can be quantitatively reproduced by a simple model of two polymers diffusing independently in a spherical confinement. This finding suggests a dominant role for diffusion-limited, undirected search in homolog pairing in early meiosis. This is in sharp contrast with the intuition that a heavy-tailed search process could never drive such a critical cell-cycle stage. We further show that the inter-locus velocity-velocity correlation (VVC) quantitatively matches analytical results for the inter-locus VVC of our polymer model, allowing us to leverage our analytical theory to extract the time scale of stress communication between the labeled loci along the chromosome. We show that stresses can take tens of minutes to propagate between loci on paired chromosomes, and that the increasing connectivity between the chromosomes as the cell progresses through meiosis can be quantified by the shortening of this communication time. Our study highlights the power of coarse-grained polymer models to analyze dynamic structural properties of the nucleus in vivo and the importance of analytical theory for uncovering intracellular connections that might be obscured by lag times of many minutes.

Interstitial fluid pressures in the lung were estimated in 39 dogs from pressure measurements made in implanted perforated capsules. Special operative procedures were used to minimize operative trauma of the lungs and infection and inflammation in and around the implanted capsules. In 86% of the preparations, the x-ray findings indicated little remaining inflammation and edema around the capsules, though autopsy findings indicated that a few of these did still have some edema not detectable by x-rays. In 29 of 34 animals in which x-ray findings indicated no edema, the intracapsular pressure was negative. The average of the measurements in all 34 animals was -5.8 mm Hg (± 0.8 SEM), and in the 29 animals with negative pressure values the average was -7.3 mm Hg (± 0.6 SEM). In eight capsules the pressures were below -10 mm Hg, ranging to -16 mm Hg. Because the tissues around some of the capsules undoubtedly still had varying degrees of inflammation and edema, despite the failure of roentgenograms to show these, it is suggested that the normal interstitial fluid pressure of pulmonary tissues in dogs is somewhat more negative than the capsule pressure measurements indicate, perhaps as low as -10 mm Hg.