Hcfc1a regulates neural precursor proliferation and asxl1 expression in the developing brain

Abstract

Background: Precise regulation of neural precursor cell (NPC) proliferation and differentiation is essential to ensure proper brain development and function. The HCFC1 gene encodes a transcriptional co-factor that regulates cell proliferation, and previous studies suggest that HCFC1 regulates NPC function. However, the molecular mechanism underlying these cellular deficits has not been completely characterized. Methods: Here we created a zebrafish harboring mutations in the hcfc1a gene (the hcfc1aco60/+ allele), one ortholog of HCFC1, and utilized immunohistochemistry and RNA-sequencing technology to understand the function of hcfc1a during neural development.Results: The hcfc1aco60/+ allele results in an increased number of NPCs, neurons, and radial glial cells. These deficits are associated with the abnormal expression of asxl1, a polycomb transcription factor, which we identified as a downstream effector of hcfc1a using high throughput RNA sequencing technology. Inhibition of asxl1 activity and/or expression in larvae harboring the hcfc1aco60/+ allele completely restored the number of NPCs to normal levels. Conclusion: Collectively, our data demonstrate a novel pathway in which hcfc1a regulates NPCs and neurogenesis.

Missense and nonsense mutations of the zebrafishhcfc1agene result in contrasting mTor and radial glial phenotypes

“…We have previously established that nonsense mutation of hcfc1a (co60 allele) causes an increase in asxl1 expression, which promotes increased proliferation of NPCs (Castro et al, 2020). Asxl1 promotes Akt phosphorylation (Youn et al, 2017) and we established that inhibition of PI3K, an upstream regulator of Akt, is sufficient to reduce asxl1 expression and restore the NPC numbers to normal levels in the co60 allele.…”

Knockdown of hspg2 is associated with abnormal mandibular joint formation and neural crest cell dysfunction in zebrafish

“…In addition, we designed a splice inhibiting morpholino to the 3' splice acceptor of exon 2, but the designed morpholino did not delete exon 3 as predicted, even at the highest concentration injected (2nl of a 0.9mM stock solution). We did, however, utilize a random control morpholino to account for the possibility of morpholino-induced cell death, an endeavor that proved to be rather successful in previous studies (54,55). And, while injection of HS side chains is a potential rescue for the morphant phenotype we observe, there is the possibility that HS/heparin coinjection would fail to rescue because a domain outside of domain I is also essential for regulation of CNCCs.…”

“…For all experiments, the morphant experimental group is compared to either a random control group or wildtype non-injected. The randomized control morpholino has been shown through previous literature to have no associated phenotypes, indicating that it does not in uence nal results and therefore is the appropriate control group for comparison (54,55,57). Thus, for statistical analysis, comparisons were performed using a T-test between the random control group and the morpholino.…”

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

“…While translation-blocking morpholinos are a simple and effective way in which to knockdown genes of interest, they have been associated with off target effects and non-speci c cell death. We did, however, utilize a random control morpholino to account for the possibility of morpholino-induced cell death, an endeavor that proved to be rather successful in previous studies (58,59). And while injection of heparin side chains is a potential rescue for the morphant phenotype we observed, there is the possibility that heparin sulfate co-injection would fail to rescue because a domain outside of domain I is essential for regulation of CNCCs.…”

“…For all experiments, the morphant experimental group is compared to either a random control group or wildtype non-injected. The randomized control morpholino has been shown through previous literature to have no associated phenotypes, indicating that it does not in uence nal results and therefore is the appropriate control group for comparison (58,59,61). Thus, for statistical analysis, comparisons were performed using a T-test between the random control group and the morpholino.…”