Navin B RamakrishnaDec 5, 2022
Glad and relieved to share on mastodon my first co-first work from one of my PhD projects with Eric Miska & Azim Surani. ☺️ Hope it’s a useful one: we team with Giorgia Battsitoni and Greg Hannon to investigate a prior observation of piRNAs in mPGCs in vitro.
Alas, we do not detect piRNAs in Day 6 mPGCLCs. https://www.cell.com/developmental-cell/fulltext/S1534-5807(22)00783-3
#piRNAs #GermCells #mPGC #mPGCLC #DevCell #MattersArising
Show threadNavin B RamakrishnaDec 5, 2022
By expression analysis of the differentiation from mESCs, we do not detect robust expression of the piRNA pathway in D6 mPGCLCs.
Crucially, from small RNA-seq of 3 more lines (different strain backgrounds) including the E14 line, we do not detect piRNAs.
Show threadNavin B RamakrishnaDec 5, 2022

It seems this is understandable as mPGCLCs share an ~E9.5 mPGC transcriptome & small-RNAome.

With pathway members absent, piRNA biogenesis is unlikely.
Fetal piRNAs emerge later in vivo at E13.5 onwards upon further demethylation and robust upregulation of piRNA pathway members.

Show threadNavin B RamakrishnaDec 5, 2022

Although this was a protracted journey since 2017, I’m very grateful that, Wolf Reik’s lab relooked at the original data in von Meyenn et al 2016 and graciously corrected a mistake with their small RNA data.

Lots went on behind the scenes – but this is an instance of science working well! https://www.cell.com/developmental-cell/fulltext/S1534-5807(22)00809-7

Show threadNavin B RamakrishnaDec 5, 2022

So, will we have a mammalian in vitro fetal piRNA model?
Ishikura et al 2021’s reaggregated rTestis m5/7 cells and gonocyte-like cells (GSCLCs) look v. promising, with marked fetal piRNA pathway upregulation.

https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(21)00342-8?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1934590921003428%3Fshowall%3Dtrue.

Show threadNavin B Ramakrishna

It appears that PGCLCs stall in development likely due to an incorrect physical & signalling environment in EBs.
See Cooke & Moris (2021) for an excellent discussion on the mechanics and extra-cellular cues of this differentiation

https://journals.biologists.com/dev/article/148/23/dev200093/273598/Tissue-and-cell-interactions-in-mammalian-PGC

Tissue and cell interactions in mammalian PGC development

Summary: This Review summarises recent research revealing that primordial germ cell (PGC) development depends on interactions with the surrounding somatic cells and tissues, and that this co-development is crucial for PGC specification, migration and maturation.

The Company of Biologists
Dec 5, 2022 at 4:10pmWeb
Show threadNavin B RamakrishnaDec 5, 2022
Hopefully, once found, this in vitro system can help escalate biochemical dissection of the mammalian fetal piRNA pathway – for example in finding direct bridging links between Miwi2 and Dnmt3c.
Show threadNavin B RamakrishnaDec 5, 2022
In the interest of candour, this publication resulted from my internal PhD examiner Greg Hannon spotting my results tucked in my thesis. He shared that co-first Giorgia Battistoni had experienced the same thing, leading to the collaboration. (Always put every result in your thesis!)
Show threadNavin B RamakrishnaDec 5, 2022
Thanks to Eric, Azim, Greg & PhD labmates for support, other PGC researchers for encouraging us to publish, and Chief-Editor of Developmental Cell J. Ann Le Good for expertly mediating the process.
Looking forward to getting the rest of my PhD work out soon too!