Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish.

CRISPR-mediated knock-in (KI) technology opens a new era of fluorescent-protein labeling in zebrafish, a preferred model organism for in vivo imaging. We described here an optimized zebrafish gene-tagging strategy, which enables easy and high-efficiency KI, ensures high odds of obtaining seamless KI germlines and is suitable for wide applications. Plasmid donors for 3'-labeling were optimized by shortening the microhomologous arms and by reducing the number and reversing the sequence of the consensus Cas9/sgRNA binding sites. To allow for scar-less KI across the genome, linearized dsDNA donors with 5'-chemical modifications were generated and successfully incorporated into our method. To refine the germline screen workflow and expedite the screen process, we combined fluorescence enrichment and caudal-fin junction-PCR. Furthermore, to trace proteins expressed at a low abundance, we developed a fluorescent signal amplifier using the transcriptional activation strategy. Together, our strategies enable efficient gene-tagging and sensitive expression detection for almost every gene in zebrafish.

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation.

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.