Literature context: X2 R and D Systems Cat# AF1979, RRID:AB_2157172 1:500
During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.
Literature context: polyclonal R&D Systems AF1979; RRID:AB_2157172 1:200
Regulation of rod gene expression has emerged as a potential therapeutic strategy to treat retinal degenerative diseases like retinitis pigmentosa (RP). We previously reported on a small molecule modulator of the rod transcription factor Nr2e3, Photoregulin1 (PR1), that regulates the expression of photoreceptor-specific genes. Although PR1 slows the progression of retinal degeneration in models of RP in vitro, in vivo analyses were not possible with PR1. We now report a structurally unrelated compound, Photoregulin3 (PR3) that also inhibits rod photoreceptor gene expression, potentially though Nr2e3 modulation. To determine the effectiveness of PR3 as a potential therapy for RP, we treated RhoP23H mice with PR3 and assessed retinal structure and function. PR3-treated RhoP23H mice showed significant structural and functional photoreceptor rescue compared with vehicle-treated littermate control mice. These results provide further support that pharmacological modulation of rod gene expression provides a potential strategy for the treatment of RP.
Literature context: Otx2 (R and D, AF1979, 1:1000, RRID:AB_2157172), Oct6 (sc-376143, 1:100, RRID:
Pluripotency is defined by a cell's potential to differentiate into any somatic cell type. How pluripotency is transited during embryo implantation, followed by cell lineage specification and establishment of the basic body plan, is poorly understood. Here we report the transcription factor Zfp281 functions in the exit from naive pluripotency occurring coincident with pre-to-post-implantation mouse embryonic development. By characterizing Zfp281 mutant phenotypes and identifying Zfp281 gene targets and protein partners in developing embryos and cultured pluripotent stem cells, we establish critical roles for Zfp281 in activating components of the Nodal signaling pathway and lineage-specific genes. Mechanistically, Zfp281 cooperates with histone acetylation and methylation complexes at target gene enhancers and promoters to exert transcriptional activation and repression, as well as epigenetic control of epiblast maturation leading up to anterior-posterior axis specification. Our study provides a comprehensive molecular model for understanding pluripotent state progressions in vivo during mammalian embryonic development.
Literature context: # AF1979, RRID:AB_2157172 1:200 (IHC
In the developing murine eye, melanin synthesis in the retinal pigment epithelium (RPE) coincides with neurogenesis of retinal ganglion cells (RGCs). Disruption of pigmentation in the albino RPE is associated with delayed neurogenesis in the ventrotemporal retina, the source of ipsilateral RGCs, and a reduced ipsilateral RGC projection. To begin to unravel how melanogenesis and the RPE regulate RGC neurogenesis and cell subpopulation specification, we compared the features of albino and pigmented mouse RPE cells during the period of RGC neurogenesis (embryonic day, E, 12.5 to 18.5) when the RPE is closely apposed to developing RGC precursors. At E12.5 and E15.5, although albino and pigmented RPE cells express RPE markers Otx2 and Mitf similarly, albino RPE cells are irregularly shaped and have fewer melanosomes compared with pigmented RPE cells. The adherens junction protein P-cadherin appears loosely distributed within the albino RPE cells rather than tightly localized on the cell membrane, as in pigmented RPE. Connexin 43 (gap junction protein) is expressed in pigmented and albino RPE cells at E13.5 but at E15.5 albino RPE cells have fewer small connexin 43 puncta, and a larger fraction of phosphorylated connexin 43 at serine 368. These results suggest that the lack of pigment in the RPE results in impaired RPE cell integrity and communication via gap junctions between RPE and neural retina during RGC neurogenesis. Our findings should pave the way for further investigation of the role of RPE in regulating RGC development toward achieving proper RGC axon decussation. J. Comp. Neurol. 524:3696-3716, 2016. © 2016 Wiley Periodicals, Inc.
The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. We tested the hypothesis that genetic history and timing of gene expression within mDA neuron progenitors impart spatial diversity. Using genetic inducible fate mapping to mark the Sonic hedgehog (Shh) and Gli1 lineages at varying embryonic stages, we performed a quantitative and qualitative comparison of the two lineages' contribution to the mDA neuron domains. Dynamic changes in Shh and Gli1 expression in the vMb primordia delineated their spatial contribution to the embryonic day 12.5 vMb: Both lineages first contributed to the medial domain, but subsequently the Gli1 lineage exclusively contributed to the lateral vMb while the Shh lineage expanded more broadly across the vMb. The contribution of both lineages to the differentiated mDA neuron domain was initially biased anteriorly and became more uniform across the anterior/posterior vMb throughout development. Our findings demonstrate that the early Shh and Gli1 lineages specify mDA neurons of the substantia nigra pars compacta while the late Shh and Gli1 lineages maintain their progenitor state longer in the posterior vMb to extend the production of mDA neurons in the ventral tegmental area. Together, our study demonstrates that the timing of gene expression along with the genetic lineage (Shh or Gli1) within the neural progenitors segregate mDA neurons into distinct spatial domains.