Biology subjects

Joachim Wittbrodt

Publications and source records attributed to Joachim Wittbrodt.

4 recordsLinked to original sources

bioRxiv2016

iDamIDseq and iDEAR: An improved method and a computational pipeline to profile chromatin-binding proteins of developing organisms

DNA adenine methyltransferase identification (DamID) has emerged as an alternative for profiling protein-DNA interactions, however critical issues in the method limit its applicability. Here we present iDamlDseq, a protocol that improves specificity and robustness making its use compatible with developing organisms. In addition, we present the analysis tool iDEAR (iDamlDseq Enrichment Analysis with R) to determine protein-DNA interactions genome wide. The combination of both allows establishing highly reliable transcription factor profiles, even in transient assays. For tissue specific expression we improved the Dam coding sequence to overcome predominant aberrant splicing of Dam fusions we discovered with the commonly used sequence.

Genomics↗

bioRxiv2015

Maternal expression of the novel centrosome assembly factor Wdr8 is required for vertebrate embryonic mitosis and development

The assembly of the first centrosome occurs upon fertilisation when the male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. While inaccuracy in the assembly of centrosomes leads to infertility and abnormal embryogenesis, the mechanism that ensures accurate assembly in vertebrate embryos remains obscure. Here we applied a CRISPR-Cas9-mediated gene knockout to show that Wdr8, a novel centrosomal protein, is maternally essential for PCM assembly during embryonic mitoses of medaka (Oryzias latipes). Maternal/zygotic Wdr8-null (Wdr8-/-) blastomeres exhibit severe defects in PCM assembly that cause them to divide asymmetrically and develop multipolar mitotic spindles and aneuploidy. We demonstrate that Wdr8 interacts via its WD40 domains with the centriolar satellite protein SSX2IP. Strikingly, exogenously provided Wdr8 fully rescues Wdr8-/- embryos to adulthood, except in variants with mutations in the WD40 domains. This combination of targeted gene inactivation and in vivo reconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal PCM and the stability of the zygotic genome in the early vertebrate embryo.

Cell Biology↗

bioRxiv2014

Interplay of TGFb superfamily members governs optic fissure closure

The optic fissure is a gap in the developing vertebrate eye and must be closed as development proceeds. A persisting optic fissure is referred to as coloboma, a major cause for blindness in children. Multiple factors have been linked to coloboma formation, however, the actual process of fissure closure is only poorly understood.\n\nBased on our findings we propose an important role of TGFb signaling for optic fissure closure. We show active TGFb signaling in the fissure margins, analyzed by a new TGFb signaling reporter zebrafish. We found BMP antagonists regulated by TGFb. These antagonists we also found expressed in the fissure margins. Finally we show a coloboma phenotype in a TGFb KO mouse. Microarray data analysis indicates intense TGFb dependent remodeling of the extracellular matrix (ECM) during optic fissure closure.\n\nWe propose that TGFb is driving optic fissure closure by ECM remodeling. As previously shown, inhibition of BMP signaling is important for such TGFb dependent ECM remodeling. We show that this is achieved by the regulation of BMP antagonists, expressed in the optic fissure margins.

Developmental Biology↗

bioRxiv2014

Epithelial flow into the optic cup facilitated by suppression of BMP drives eye morphogenesis

The transformation of the oval optic vesicle to a hemispheric bi-layered optic cup involves major morphological changes during early vertebrate eye development. According to the classical view, the lens-averted epithelium differentiates into the retinal pigmented epithelium (RPE), while the lens-facing epithelium forms the neuroretina. We find a 4.7 fold increase of the entire basal surface of the optic cup.\n\nAlthough the area an individual RPC demands at its basal surface declines during optic cup formation, we find a 4.7 fold increase of the entire basal surface of the optic cup. We demonstrate that the lens-averted epithelium functions as reservoir and contributes to the growing neuroretina by epithelial flow around the distal rims of the optic cup. This flow is negatively modulated by BMP, which arrests epithelial flow. This inhibition results in persisting neuroretina in the RPE domain and ultimately in coloboma.

Developmental Biology↗