RESUMO
Three central cell populations play roles in morphogen action, the cells that produce, the cells that distribute, and the cells that respond to the morphogen. Taking advantage of the properties of embryonic stem cell to aggregate and readily differentiate into neural progenitor tissue, we describe an approach using genetically modified murine stem cell lines to individually address the contribution of these cells in the establishment and response to a morphogenetic gradient in mosaic spinal cord organoids.
Assuntos
Células-Tronco Embrionárias Murinas , Organoides , Animais , Camundongos , Transdução de Sinais , Medula EspinalRESUMO
BACKGROUND: Sonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination motif, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and are involved in the extracellular distribution of Shh. RESULTS: We found that the zinc-coordination center of Shh is required for its association to the ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 µM zinc and is prevented by mutations affecting critical conserved catalytical residues. Consistent with the presence of a conserved calcium binding domain, we find that extracellular calcium inhibits ECM association of Shh. CONCLUSIONS: Our results indicate that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling, possibly by enzymatically altering ECM characteristics.
Assuntos
Matriz Extracelular/metabolismo , Proteínas Hedgehog/metabolismo , Zinco/química , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cálcio , Células Cultivadas , Colesterol , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Camundongos , Mutação , Peptídeo Hidrolases/química , Domínios Proteicos , Transdução de SinaisRESUMO
Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor.
Assuntos
Receptor Patched-1/metabolismo , Receptor Patched-2/metabolismo , Receptor Smoothened/antagonistas & inibidores , Animais , Técnicas de Inativação de Genes , Proteínas Hedgehog/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Receptor Patched-1/genética , Receptor Patched-2/genética , Transdução de Sinais , Receptor Smoothened/genéticaRESUMO
A case of an unusual unilocular cystic lesion of diameter 7 cm located retroperitoneally in the pelvis in close connection to the right adnexa of a 61 year-old woman is presented. Macroscopically, the lesion had a smooth outer and inner surface and was filled with translucent fluid. Histological examination revealed a fibrous and hyalinized wall which lacked a specific lining. Numerous nerve bundles in the cyst wall constituted the most conspicuous element of its histology possibly with some contribution of perineurial and/or mesothelial components. The morphology and immunohistochemistry speak for an intraneural pseudocyst sometimes called intraneural ganglion cyst which is rare in this location. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1357862917132314.