Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain

Abstract

Background Functioning of the adult nervous system depends on the establishment of neural circuits during embryogenesis. In vertebrates, neurons that make up motor circuits form in distinct domains along the dorsoventral axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear. Methods We used CRISPR/Cas9 to generate the first germline mutants for prdm12 and employed this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation. Results We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal touch-evoked escape response. Using luciferase reporter assays, we demonstrate that Prdm12b acts as a transcriptional repressor. We also show that the Bhlhe22 TF binds via the Prdm12b zinc finger domain to form a complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1. Conclusions We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in formation of the p1 domain – suggesting that prdm12b is not solely required to repress non-p1 fates, but is specifically n...

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

“…circuits, V1 inhibitory neurons ensure proper swim frequency during fast swim movements; ablation of these neurons [47] or loss through mutation of prdm12b ( [45] and our results) results in reduced swim cycle frequency and prolonged bend duration. Based on Kv1.1's previous shown role in regulating neuron excitability [65][66][67], we predict loss of kcna1a induces hyperactivity in specific neurons, making it unlikely that loss of kcna1a in V1 neurons leads to the exaggerated response phenotype.…”

“…Zebrafish prdm12b was previously identified in a screen for genes that express in the nervous system [ 44 ]. Morpholinos targeting prdm12b [ 27 ] and a prdm12b CRISPR mutation [ 45 ] result in larvae with aberrant, exaggerated touch behaviors, similar to the exaggerated acoustic startle response we observe in the p419 mutants. prdm12b is required for development of engrailed 1b positive, V1 interneurons in the spinal cord [ 27 ].…”

“…While it is possible dolk and kcna1a are solely required in motor neurons to drive correct movement magnitude, we observe Kv1.1 expressed broadly in the spinal cord, including in commissural axons and ipsilateral axon tracts ( Fig 4C ). Within fast motor circuits, V1 inhibitory neurons ensure proper swim frequency during fast swim movements; ablation of these neurons [ 47 ] or loss through mutation of prdm12b ([ 45 ] and our results) results in reduced swim cycle frequency and prolonged bend duration. Based on Kv1.1’s previous shown role in regulating neuron excitability [ 65 – 67 ], we predict loss of kcna1a induces hyperactivity in specific neurons, making it unlikely that loss of kcna1a in V1 neurons leads to the exaggerated response phenotype.…”

The duality of PRDM proteins: epigenetic and structural perspectives

“…(H4K20me3, G9a/H3K9me2) 30,31 , PRDM12 [32][33][34][35] and PRDM14 (EZH2/K27me3), 36,37 , suggesting that PRDMs are able to recruit histone "writers" through protein-protein interactions. Furthermore, PRDMs are able to recruit histone "erasers" (i.e.…”

Modeling Neuronal Diseases in Zebrafish in the Era of CRISPR

“…Another gene implicated in neurogenesis in the neural tube is prdm12b . Using CRISPR/Cas9 to generate two fish lines carrying frameshift mutations in prdm12b, authors revealed that eng1b expression was suppressed in V1 interneurons, leading to defective Mauthner cell-dependent locomotion and embryonic lethality [ 100 ].…”