Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress

Abstract

Heme oxygenase 1 (HMOX1) degrades heme into biliverdin, which is subsequently converted to bilirubin by biliverdin reductase (BVRa or BVRb) in a manner analogous to the classic anti-oxidant glutathione-recycling pathway. To gain a better understanding of the potential antioxidant roles the BVR enzymes may play during development, the spatiotemporal expression and transcriptional regulation of zebrafish hmox1a, bvra and bvrb were characterized under basal conditions and in response to pro-oxidant exposure. All three genes displayed spatiotemporal expression patterns consistent with classic hematopoietic progenitors during development. Transient knockdown of Nrf2a did not attenuate the ability to detect bvra or bvrb by ISH, or alter spatial expression patterns in response to cadmium exposure. While hmox1a:mCherry fluorescence was documented within the intermediate cell mass, a transient location of primitive erythrocyte differentiation, expression was not fully attenuated in Nrf2a morphants, but real-time RT-PCR demonstrated a significant reduction in hmox1a expression. Furthermore, Gata-1 knockdown did not attenuate hmox1a:mCherry fluorescence. However, while there was a complete loss of detection of bvrb expression by ISH at 24 hpf, bvra expression was greatly attenuated but still detectable in Gata-1 morphants. In contrast, 96 hpf Gata-1 morphants displayed increased bvra and bvrb expression within hematopoietic tissues. Finally, temporal expression patterns of enzymes involved in the generation and maintenance of NADPH were consistent with known changes in the cellular redox state during early zebrafish development. Together, these data suggest that Gata-1 and Nrf2a play differential roles in regulating the heme degradation enzymes during an early developmental period of heightened cellular stress.

Alas1 is essential for neutrophil maturation in zebrafish

“…The data showed that alas2 was not altered compared with that in siblings ( Figure 4F ), suggesting that the erythroid heme accumulation was not due to the compensatory of alas2 , at least at the transcription level. Since heme content is tightly controlled by the homeostasis of heme biosynthesis, degradation, and transport pathways, 35 we then detected the expression of heme oxygenase enzymes ( hmox1a and hmox2a ), 10 , 36 which encode the rate-limiting enzymes for heme degradation. The results showed that both gene expressions were down-regulated in alas1 smu350/smu350 mutants ( Figure 4F ), suggesting the impaired heme degradation in the absence of alas1 .…”

“…As far as we know, the regulatory genes and pathways of heme biosynthesis, degradation and transport are largely conserved between mammals and zebrafish in general. 20 , 23 , 35 , 36 , 43 Vertebrates contain two ALAS isozymes encoded by 2 distinct genes located on different chromosomes; ALAS2 is expressed in erythrocytes, whereas ALAS1 is ubiquitously expressed. 11 By searching the integrated RNA-seq database on BloodSpot, 44 we found that, in humans and mice, ALAS1 is highly expressed in myeloid cells, which is consistent with our zebrafish data and partly explains the importance of alas1 for neutrophils.…”

Combining near-infrared fluorescence with Brainbow to visualize expression of specific genes within a multicolor context

“…This is preferable because of both the relative ease of expression and the minimal disruption to metabolism. In addition to being the rate-limiting enzyme in an important catabolic pathway among eukaryotes, heme oxygenase is also known to specifically function in zebrafish development (Holowiecki et al., 2017) and cardiac function (Tzaneva and Perry, 2016), and thus its manipulation in zebrafish embryos could potentially confound experimental results. Furthermore, expression of the endogenous zebrafish isoforms of heme oxygenase varies with tissue, sex, and age (Holowiecki et al., 2016), potentially complicating the direct relationship between brightness and FP copy number that allows quantification of fusion protein levels.…”

Heme oxygenase limits mycobacterial infection-induced ferroptosis

“…Zebrafish have four Hmox-encoding paralogs: hmox1a, hmox1b, hmox2a, and hmox2b [14][15][16]. To determine which paralog is functional in M. marinum infection, we first infected adult zebrafish by intraperitoneal injection and analysed gene expression changes at 14 days post infection (dpi).…”

“…HMOX1 is a highly evolutionarily conserved enzyme that has been identified in a wide variety of organisms. Zebrafish hmox1a and hmox1b encode paralogs of mammalian HMOX1, and their transcription is responsive to a range of oxidative stress-inducing conditions [14][15][16]. The zebrafish-M. marinum model has been widely used to study conserved host redox perturbations associated with mycobacterial infection [17][18][19].…”