Genome-wide occupancy links Hoxa2 to Wnt–β-catenin signaling in mouse embryonic development

The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx–Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt–β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Integration of Signals along Orthogonal Axes of the Vertebrate Neural Tube Controls Progenitor Competence and Increases Cell Diversity

<div><p>A relatively small number of signals are responsible for the variety and pattern of cell types generated in developing embryos. In part this is achieved by exploiting differences in the concentration or duration of signaling to increase cellular diversity. In addition, however, changes in cellular competence—temporal shifts in the response of cells to a signal—contribute to the array of cell types generated. Here we investigate how these two mechanisms are combined in the vertebrate neural tube to increase the range of cell types and deliver spatial control over their location. We provide evidence that FGF signaling emanating from the posterior of the embryo controls a change in competence of neural progenitors to Shh and BMP, the two morphogens that are responsible for patterning the ventral and dorsal regions of the neural tube, respectively. Newly generated neural progenitors are exposed to FGF signaling, and this maintains the expression of the Nk1-class transcription factor Nkx1.2. Ventrally, this acts in combination with the Shh-induced transcription factor FoxA2 to specify floor plate cells and dorsally in combination with BMP signaling to induce neural crest cells. As development progresses, the intersection of FGF with BMP and Shh signals is interrupted by axis elongation, resulting in the loss of Nkx1.2 expression and allowing the induction of ventral and dorsal interneuron progenitors by Shh and BMP signaling to supervene. Hence a similar mechanism increases cell type diversity at both dorsal and ventral poles of the neural tube. Together these data reveal that tissue morphogenesis produces changes in the coincidence of signals acting along orthogonal axes of the neural tube and this is used to define spatial and temporal transitions in the competence of cells to interpret morphogen signaling.</p></div

Hoxa2 downregulates Six2 in the neural crest-derived mesenchyme

The Hoxa2 transcription factor acts during development of the second branchial arch. As for most of the developmental processes controlled by Hox proteins, the mechanism by which Hoxa2 regulates the morphology of second branchial arch derivatives is unclear. We show that Six2, another transcription factor, is genetically downstream of Hoxa2. High levels of Six2 are observed in the Hoxa2 loss-of-function mutant. By using a transgenic approach to overexpress Six2 in the embryonic area controlled by Hoxa2, we observed a phenotype that is reminiscent of the Hoxa2 mutant phenotype. Furthermore, we demonstrate that Hoxa2 regulation of Six2 is confined to a 0.9 kb fragment of the Six2 promoter and that Hoxa2 binds to this promoter region. These results strongly suggest that Six2 is a direct target of Hoxa2

A model for the specification of FP and NCC.

<p>Cells in the posterior open neural plate area (pre-neural tube) (time T₁) are exposed to FGF (drawn as a grey band). FGF and Nkx1.2 form a positive feedback loop and RAR/Irx3/Pax6 activity is low. Shh (red) and BMP (light blue) signal to progenitors at the poles of the forming neural plate. As a consequence of axis elongation, progenitors are displaced anteriorly into the neural tube, FGF signal decreases (time T₂), and Nkx1.2 is down-regulated. Cells are no longer competent to induce FP or NCC. The combination of RAR/Irx3/Pax6 inhibits Nkx1.2 expression, either directly or indirectly (as indicated by the dashed line), and provides the competence for Shh and BMP signaling to induce the neuronal progenitors (time T₃).</p

FGF signaling and Nkx1.2 expression provides competence for neural crest induction.

<p>(A–G) Neural crest induction requires early BMP signaling and the timing of competence is regulated by FGF signaling. Explants were cultured in the conditions indicated in (A) and analyzed by immunohistochemistry for Snail2 (B, C, D, E), Olig3 (B′, C′, D′, E′), and HNK1 (B″, C″, D″, E″). Note that the Olig3 staining, which gives a weaker signal, is shown at a higher magnification (B′, C′, D′, E′; 125 µm per side) than the other images (B, B″, C, C″, D, D″, E, E″; 375 µm per side). Quantification is shown in (F). (G) qRT-PCR was used to assay the expression of Snail2, Sox10, Olig3, and Lhx2 in explants cultured as indicated by the schema in (A). The relative expression levels compared to condition (i) are shown. (H–L) Transient expression of Nkx1.2 provides competence for the neural crest induction. Explants electroporated with GR-Nkx1.2 were cultured in the conditions described in (H). Explants indicated (PD) were treated with 500 nM PD184352 and all explants assayed by immunohistochemistry for Snail2 (I, J, red in I″, J″) and GFP (green in I″, J″). Quantification of (J–J″) provided in (K) suggests that the majority of Snail2-expressing cells derive from cells electroporated with GR-Nkx1.2. (L) qRT-PCR for expression of Snail2, Sox10, Olig3, and Lhx2. Relative expression levels compared to levels at time 0 were calculated. (M–R′) Inhibiting Nkx1.2 or enhancing of RAR-VP16 activity blocks neural crest induction. Control GFP (M, M′, P, P′), Nkx1.2^DBD-VP16 (N, N′, Q, Q′), or RAR-VP16 (O, O′, Q, Q′) was electroporated at HH stage 8+ and embryos cultured for 12 h to reach HH stage 12. Assaying Snail2 (M, N, O, red in M′, N′, O′) and Pax6 (P, Q, R, red in P′, Q′, R′) expression indicated an inhibition of Snail2 expression in the dorsal midline of the neural tube and a dorsal expansion of Pax6. Affected cells are labeled with arrowheads. Scale bar in (M) for (M–R′) = 50 µm.</p

FP induction by FoxA2 requires FGF signaling.

<p>(A–F′) The ability of FoxA2 to induce Arx is time-dependent. Following the electroporation of a FoxA2 expression construct at HH stage 11 or at HH stage 14, embryos were cultured for 48 h. The expression of Arx (A, D red in A′, D′), Nkx2.2 (B, E, red in B′, E′), and Shh (C, F) were analyzed with immunohistochemistry (for Arx and Nkx2.2) or in situ hybridization (for Shh). Electroporated cells were identified by GFP (green in A′, B′, D′, E′, white in C′, F′). Scale bar (A for A, A′, B′, B′, C, C′ and D for D, D′, E, E′, F, F′) = 100 µm. (G–K′) Arx induction requires the early expression of FoxA2. ER-FoxA2 (the hormone-binding domain of the estrogen receptor fused to a full-length FoxA2 coding region) was electroporated into embryos. Explants were prepared and cultured as indicated in (G). 4-hydroxy-tamoxifen (4-OHT) was used at 1 µM. Explants were assayed for Arx (H, I, J, K, red in H′, I′, J′, K′) and GFP (green in H′, I′, J′, K′) expression. (L–Q′) The induction of Arx by FoxA2 requires FGF-derived factor(s). Expression plasmids for FoxA2 and MKP3 (L–N′) or Nkx1.2^DBD-VP16 (O–Q′) were coelectroporated into the embryos at HH stage 11. Embryos were harvested at 48 hpt and analyzed for Arx (L, O, red in L′, O′), Nkx2.2 (M, P, red in M′, P′), and Shh (N, Q) expression. Electroporated cells were identified by GFP (green in L′, M′, O′, P′, white in N′, Q′). (R–T′) Sequential induction of Nkx1.2 and FoxA2 allows FP differentiation in vitro. GR-Nkx1.2 and ER-FoxA2 were electroporated and explants were prepared. These were cultured as indicated in (R). Dexthamethazone (DEX) was used at 10 µM. Arx (S, T, red in S′, T′) and GFP (green in S′, T′) expression was analyzed by immunohistochemistry. (U–Z) Comparison of the expression patterns of the indicated genes. HH stage 9+ embryos were analyzed by in situ hybridization with antisense RNA probes for Nkx1.2 (U, u1, u2, u3), FoxA2 (V, v1, v2, v3), FGF8 (W, w1, w2, w3), and Shh (X, x1, x2, x3), Pax6 (Y), and Irx3 (Z). The levels of the sections are indicated by lines in (U–X). Scale bars are in (U) = 200 µm (for U, V, W, X, Y, Z) and in (u3) = 100 µm (for u1–u3, v1–v3, x1–x3).</p

Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs

In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues