Production and characterization of monoclonal antibodies to <i>Xenopus</i> proteins

Horr, Brett; Kurtz, Ryan; Pandey, Ankit; Hoffstrom, Benjamin G.; Schock, Elizabeth N.; LaBonne, Carole; Alfandari, Dominique

doi:10.1242/dev.201309

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…Fig 1B). A Xenopus -specific Sox3 antibody (Horr et al, 2023) was used in combination with a hybridization chain reaction (HCR) probe for pax3 to mark the forming neural plate border (Choi et al, 2018). We asked if pax3 -positive neural plate border cells were also Sox3-postive during gastrulation and neurulation.…”

Section: Resultsmentioning

confidence: 99%

SoxB1 transcription factors are essential for initiating and maintaining the neural plate border gene expression

Schock,

York,

et al. 2023

Preprint

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SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). UsingXenopus laevis, we set out to determine if SoxB1 transcription factors have a regulatory function in NPB formation. Herein, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and, surprisingly, in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as a potential SoxB1 partners in regulating the formation of the NPB and show their combined activity is needed to maintain NPB gene expression. Together, these data identify a novel role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

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Section: Resultsmentioning

confidence: 99%

SoxB1 transcription factors are essential for initiating and maintaining the neural plate border gene expression

Schock,

York,

et al. 2023

Preprint

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“…The rat monoclonal to flag tag (Agilent Cat# 200474, RRID: AB_10597743 ). The monoclonal to X. laevis Sox3 (DSHB Cat# DA5H6, RRID: AB_2876376 ) was developed in the laboratory ( Horr et al, 2023 ) against the full length X. laevis protein produced in Hek293T cells. It has been characterized by Western blot and immunofluorescence (R24OD21485).…”

Section: Methodsmentioning

confidence: 99%

“…Rat monoclonal against N-cadherin (DSHB Cat# MNCD2, RRID: AB_528119 ). Mouse monoclonal to X. laevis Adam11.L (3C5) was developed in the lab by injecting a bacterial fusion protein containing the DC (disintegrin and cystein rich) domain and isolating the clone by the methods described in ( Horr et al, 2023 ).…”

Section: Methodsmentioning

confidence: 99%

ADAM11 a novel regulator of Wnt and BMP4 signaling in neural crest and cancer

Pandey,

Cousin,

Horr

et al. 2023

Front. Cell Dev. Biol.

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Introduction: Cranial neural crest (CNC) cells are induced at the border of the neural plate by a combination of FGF, Wnt, and BMP4 signaling. CNC then migrate ventrally and invade ventral structures where they contribute to craniofacial development.Methods: We used loss and gain of function experiments to determine phenotypes associated with the perturbation of Adam11 expression in Xenopus Laevis. Mass spectrometry to identify partners of Adam11 and changes in protein expression in CNC lacking Adam11. We used mouse B16 melanoma to test the function of Adam11 in cancer cells, and published database analysis to study the expression of ADAM11 in human tumors.Results: Here we show that a non-proteolytic ADAM, Adam11, originally identified as a putative tumor suppressor binds to proteins of the Wnt and BMP4 signaling pathway. Mechanistic studies concerning these non-proteolytic ADAM lack almost entirely. We show that Adam11 positively regulates BMP4 signaling while negatively regulating β-catenin activity. In vivo, we show that Adam11 influences the timing of neural tube closure and the proliferation and migration of CNC. Using both human tumor data and mouse B16 melanoma cells, we further show that ADAM11 levels similarly correlate with Wnt or BMP4 activation levels.Discussion: We propose that ADAM11 preserves naïve cells by maintaining low Sox3 and Snail/Slug levels through stimulation of BMP4 and repression of Wnt signaling, while loss of ADAM11 results in increased Wnt signaling, increased proliferation and early epithelium to mesenchyme transition.

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“…A monoclonal antibody, DA5C7-TRPM7, was produced in-house and generated against a bacterial fusion protein corresponding to the C-term portion of the cytoplasmic domain of mTRPM7 (amino acids 1159-1413). We followed standard hybridoma procedures and techniques described in the noted reference 81 . Briefly, BalbC mice (Jackson Laboratories) were immunized with the TRPM7 protein.…”

Section: Methodsmentioning

confidence: 99%

Production and characterization of monoclonal antibodies to Xenopus proteins

Cited by 7 publications

References 27 publications

SoxB1 transcription factors are essential for initiating and maintaining the neural plate border gene expression

SoxB1 transcription factors are essential for initiating and maintaining the neural plate border gene expression

ADAM11 a novel regulator of Wnt and BMP4 signaling in neural crest and cancer

Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes

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