Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor selfrenewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNAseq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.

Highlights d The expression domain of the Dorsal target gene sog narrows in the absence of Zelda d Using MS2 reporter transgenes, this can be accurately recapitulated in living embryos d Without Zelda, the onset and degree of reporter activation becomes graded like Dorsal d Zelda promotes accumulation of Dorsal protein at the site of the enhancer

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program.

Highlights d RNA polymerase II (RNA Pol II) accumulates into nuclear speckles at genome activation d Without the pioneer factor Zelda, speckle number and size are greatly reduced d RNA Pol II moves instead to the histone locus bodies and histone RNA synthesis increases d Genes in close 3D proximity do not share a RNA Pol II speckle

1 SummaryConnecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal/ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (DL), a homologue of the NF-κB family of transcriptional activators found in humans [1,2]. A second maternally deposited factor, Zelda (ZLD), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors such as DL [3][4][5][6][7][8][9]. Here we utilized the MS2 live imaging system to evaluate the expression of the DL target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that ZLD modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of DL at the site of transcription, suggesting that transcription factor "hubs" induced by ZLD [10] functionally regulate transcription. ResultsOur study focused on the DL target gene sog as its expression domain spans a large dynamic range of the DL gradient, allowing us to examine how ZLD potentiates DL activity across the dorsal/ventral axis. Previous experiments have demonstrated that the lateral stripe of sog expression narrows dramatically in zelda null embryos [5,11] (Figure 1A,B), and that progressively removing ZLD DNA binding sites from the sog shadow enhancer shrinks the domain of activation of reporter genes in a linear manner [7]. In order to understand how ZLD alters transcription at different points along the DL gradient, we revisited these constructs with the aim of visualizing transcription in real time by adding 24 MS2 loops to the 5' end of the lacZ reporter. Since previously utilized MS2 loops [12][13][14][15] contained potential ZLD binding sites [16], we revised the MS2v5 [17] sequence to make a ZLD binding site-free non-repetitive version, referred to as MS2v5(-TAG) (see Methods and Table S1).Constructs also contained either the sog distal (shadow) enhancer [18,19] with its three native canonical ZLD binding sites, CAGGTAG (hereafter referred to as "3TAG"), or without these sites (hereafter referred to as 2 "0TAG") ( Figure 1C; see Table S2 for enhancer sequences; [7]). The narrowing effect of removing ZLD binding sites was confirmed by in situ hybridization ( Figure 1D,E).By crossing these transgenic reporter lines to females with a nanos promoter-driven MCP-GFP fusion transgene [14], we visualized the transcriptional activation of each reporter as fluorescent foci. These embryos also carry an H2Av(histone 2A variant)-RFP transgene [20], allowing us to track nuclear cycles and record transcriptional ac...

The extended persistence of filarial nematodes within a host suggests immunomodulatory mechanisms that allow the parasites to resist or evade the host immune response. There is increasing evidence for immunomodulatory glycans expressed by a diversity of parasitic worms. In this study, we integrate multiple layers of the host-parasite interface to investigate the glycome of a model filarial parasite, Brugia malayi. We report a significant overrepresentation of terminal GalNAc moieties in adult female worms coupled with an overall upregulation in O-glycosylation, T-antigen expression, and a bias for galactose containing glycans. Adult males preferentially displayed a bias for terminal GlcNAc containing glycans, and fucosylated epitopes. Subsequent proteomic analysis confirmed sex-biases in protein glycosylation and highlighted the sex-specific glycosylation of well characterized immunomodulators expressed and secreted by B. malayi. We identify sex-specific effectors at that interface and suggest approaches to selectively interfere with the parasitic life cycle and potentially control transmission.

Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called “shadow enhancers,” which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, however the biological significance of this remains unclear. In this study we characterize the shadow enhancer pair controlling the gene short gastrulation (sog). We removed either the intronic proximal enhancer or the upstream distal enhancer, and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation, and RNA Polymerase II loading rates. Additionally, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. While activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that can be explained by the differences in transcriptional kinetics measured.

SummaryThe early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the whole genome. Using Drosophila embryos undergoing the first wave of genome activation, we detected discrete “speckles” of RNA Polymerase II (Pol II), and showed that they overlap with transcribing loci. We characterized the spatial distribution of Pol II speckles and quantified how this distribution changes in the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda. Although the number and size of Pol II speckles were reduced, indicating that Zelda promotes Pol II speckle formation, we observed a uniform distribution of distances between active genes in the nuclei of both wildtype and zelda mutant embryos. This suggests that the topologically associated domains identified by Hi-C studies do little to spatially constrain groups of transcribed genes at this time. We provide evidence that linear genomic distance between transcribed genes is the primary determinant of measured physical distance between the active loci. Furthermore, we show active genes can have distinct Pol II pools even if the active loci are in close proximity. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an “individualist” model of gene control at early genome activation in Drosophila. This model is in contrast to a “collectivist” model where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.