Zinc finger gene nolz1 regulates the formation of retinal progenitor cells and suppresses the Lim3/Lhx3 phenotype of retinal bipolar cells in chicken retina.

BACKGROUND: The zinc-finger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the transcription factors Isl1, Lim1, and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal development. We, therefore, studied Nolz1 during retinal development. RESULTS: Nolz1 expression was seen in two waves during development: one early (peak at embryonic day 3-4.5) in retinal progenitors and one late (embryonic day 8) in newly differentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors, and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpression of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells. CONCLUSIONS: We conclude that Nolz1 has multiple functions during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progenitor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differentiation. Developmental Dynamics 247:630-641, 2018. © 2017 Wiley Periodicals, Inc.

A core transcriptional network composed of Pax2/8, Gata3 and Lim1 regulates key players of pro/mesonephros morphogenesis.

Translating the developmental program encoded in the genome into cellular and morphogenetic functions requires the deployment of elaborate gene regulatory networks (GRNs). GRNs are especially crucial at the onset of organ development where a few regulatory signals establish the different programs required for tissue organization. In the renal system primordium (the pro/mesonephros), important regulators have been identified but their hierarchical and regulatory organization is still elusive. Here, we have performed a detailed analysis of the GRN underlying mouse pro/mesonephros development. We find that a core regulatory subcircuit composed of Pax2/8, Gata3 and Lim1 turns on a deeper layer of transcriptional regulators while activating effector genes responsible for cell signaling and tissue organization. Among the genes directly affected by the core components are the key developmental molecules Nephronectin (Npnt) and Plac8. Hence, the pro/mesonephros GRN links together several essential genes regulating tissue morphogenesis. This renal GRN sheds new light on the disease group Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) in that gene mutations are expected to generate different phenotypic outcomes as a consequence of regulatory network deficiencies rather than threshold effects from single genes.

LIM1 contributes to the malignant potential of endometrial cancer.

Introduction: The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor. Methods: Gene expression profile datasets for EC cases registered in The Cancer Genome Atlas (TCGA) was reanalyzed. Highly expressed genes in advanced-stage EC (110 cases) compared with early-stage EC (255 cases) were extracted and Gene Ontology (GO) enrichment analysis was performed. Among the enriched genes, Kaplan-Meier (KM) plotter analysis was performed. Candidate genes expression was analyzed in HEC50B cells and Ishikawa cells by RT-qPCR. In HEC50B cells, LIM homeobox1 (LIM1) was knocked down (KD) and cell proliferation, migration, and invasion ability of the cells were evaluated. Xenografts were generated using LIM1-KD cells and tumor growth was evaluated. Ingenuity Pathway Analysis (IPA) of RNA-seq data using LIM-KD cells was performed. Expression of phospho-CREB and CREB-related proteins were evaluated in LIM1-KD cells by western blotting and in xenograft tissue by immunofluorescent staining. Two different CREB inhibitors were treated in HEC50B and cell proliferation was evaluated by MTT assay. Results: Reanalysis of TCGA followed by GO enrichment analysis revealed that homeobox genes were highly expressed in advanced-stage EC. Among the identified genes, KM plotter analysis showed that high LIM1 expression was associated with a significantly poorer prognosis in EC. Additionally, LIM1 expression was significantly higher in high-grade EC cell lines, HEC50B cells than Ishikawa cells. Knockdown of LIM1 showed reduced cell proliferation, migration and invasion in HEC50B cells. Xenograft experiments revealed that tumor growth was significantly suppressed in LIM1-KD cells. IPA of RNA-seq data using LIM-KD cells predicted that the mRNA expression of CREB signaling-related genes was suppressed. Indeed, phosphorylation of CREB was decreased in LIM1-KD cells and LIM1-KD cells derived tumors. HEC50B cells treated by CREB inhibitors showed suppression of cell proliferation. Conclusion and discussion: Collectively, these results suggested that high LIM1 expression contributed to tumor growth via CREB signaling in EC. Inhibition of LIM1 or its downstream molecules would be new therapeutic strategies for EC.

Biological Evaluation of the Osteoinductive Potential of Dry Teeth after Chemical Demineralization Treatment Using the Tooth Transformer Device.

Several studies have already demonstrated the biocompatibility of a tooth as a grafting material in the regeneration of bone tissue, showing its osteoconductive potential, while no studies have verified whether the osteoinductive potential of a tooth remains constant or is altered after its treatment with the Tooth Transformer (TT) device. The aim of the study was to demonstrate that the treatment with the TT device did not alter the osteoinductivity of an extracted tooth that was stored dry. Twelve extracted human teeth were collected from real patients. Caries, tartar and filling materials were removed from each tooth; each tooth was coarsely cut and stored at room temperature (RT) until use. Each sample was shredded, demineralized and disinfected, using the TT device. Protein extraction was carried out for each sample, and Western Blot analysis was performed to test the presence of mineralization protein LIM-1 and transforming growth factor-ß. The presence of the human Bone Morphogenetic Protein 2 (BMP-2) and human collagen Type I (COL-I) was found in dry tooth samples processed with the TT device and subjected to Enzyme-Linked Immunosorbent Assay (ELISA) testing. The treatment of chemical demineralization using the TT device does not alter the osteoinductive potential of a dry tooth.

LIM homeodomain proteins and associated partners: Then and now.

The field of molecular embryology started around 1990 by identifying new genes and analyzing their functions in early vertebrate embryogenesis. Those genes encode transcription factors, signaling molecules, their regulators, etc. Most of those genes are relatively highly expressed in specific regions or exhibit dramatic phenotypes when ectopically expressed or mutated. This review focuses on one of those genes, Lim1/Lhx1, which encodes a transcription factor. Lim1/Lhx1 is a member of the LIM homeodomain (LIM-HD) protein family, and its intimate partner, Ldb1/NLI, binds to two tandem LIM domains of LIM-HDs. The most ancient LIM-HD protein and its partnership with Ldb1 were innovated in the metazoan ancestor by gene fusion combining LIM domains and a homeodomain and by creating the LIM domain-interacting domain (LID) in ancestral Ldb, respectively. The LIM domain has multiple interacting interphases, and Ldb1 has a dimerization domain (DD), the LID, and other interacting domains that bind to Ssbp2/3/4 and the boundary factor, CTCF. By means of these domains, LIM-HD-Ldb1 functions as a hub protein complex, enabling more intricate and elaborate gene regulation. The common, ancestral role of LIM-HD proteins is neuron cell-type specification. Additionally, Lim1/Lhx1 serves crucial roles in the gastrula organizer and in kidney development. Recent studies using Xenopus embryos have revealed Lim1/Lhx1 functions and regulatory mechanisms during development and regeneration, providing insight into evolutionary developmental biology, functional genomics, gene regulatory networks, and regenerative medicine. In this review, we also discuss recent progress at unraveling participation of Ldb1, Ssbp, and CTCF in enhanceosomes, long-distance enhancer-promoter interactions, and trans-interactions between chromosomes.

Evolution of cis-regulatory modules for the head organizer gene goosecoid in chordates: comparisons between Branchiostoma and Xenopus.

BACKGROUND: In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated. RESULTS: To address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5' upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5' upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5' region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos. CONCLUSIONS: On the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5' upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate.

The cell biology and molecular genetics of Müllerian duct development.

The Müllerian ducts are part of the embryonic urogenital system. They give rise to mature structures that serve a critical function in the transport and development of the oocyte and/or embryo. In most vertebrates, both sexes initially develop Müllerian ducts during embryogenesis, but they regress in males under the influence of testis-derived Anti-Müllerian Hormone (AMH). A number of regulatory factors have been shown to be essential for proper duct development, including Bmp and Wnt signaling molecules, together with homeodomain transcription factors such as PAX2 and LIM1. Later in development, the fate of the ducts diverges between males and females and is regulated by AMH and Wnt signaling molecules (duct regression in males) and Hox genes (duct patterning in females). Most of the genes and molecular pathways known to be involved in Müllerian duct development have been elucidated through animal models, namely, the mouse and chicken. In addition, genetic analysis of humans with reproductive tract disorders has further defined molecular mechanisms of duct formation and differentiation. However, despite our current understanding of Müllerian duct development, some questions remain to be answered at the molecular genetic level. This article is categorized under: Early Embryonic Development > Development to the Basic Body Plan.