Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Optical and electron microscopy of HIPK4-null male germ cells reveals defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts and that HIPK4 deficiency alters the subcellular distribution of an F-actin capping protein in the testis, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.
Doppelfunktion: Die Titelverbindung, ein bereits identifizierter wirksamer Hemmstoff des Hedgehog‐Signalwegs, der embryonischen Zellen notwendige Informationen für die Entwicklung liefert, moduliert die Genexpression von Hedgehog‐Targets, indem sie Mikrotubuli depolymerisiert. Auf diesem Weg wurde eine doppelte Steuerungsfunktion des Zytoskeletts nachgewiesen (siehe Bild).
Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Glidriven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore conducted a screen of 325,120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.
33Mammalian spermiogenesis is a remarkable cellular transformation, during which round 34 spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that 35 coordinate this process are not well understood, and we demonstrate here that homeodomain-36 interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. 37
The application of primary organoid cultures to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. We have previously described a single air-liquid interface culture method that can be used to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach, and pancreas (Ootani et al., Nature Medicine 15(6):701-6 (2009); Li et al., in press). We have recently modified this methodology to allow the robust long-term 3-dimensional culture of a wide variety of primary neoplasms from human biopsy samples. These primary tumor organoids accurately recapitulate the architecture and histopathology of the tumors from which they were derived, including epithelial and mesenchymal compartments. These tumor organoids can be further passaged into 96-well format to enable a variety of genetic and pharmacologic manipulations with the overall goal of enabling mechanistic studies of tumorigenesis and obtaining clinically actionable information regarding chemosensitivity and resistance to therapy. Citation Format: James T. Neal, Michael Cantrell, Paul Rack, Calvin J. Kuo. 3-Dimensional air-liquid interface organoid culture of primary human tumor biopsies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-34. doi:10.1158/1538-7445.AM2014-LB-34
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