Duplicate dmbx1genes regulate progenitor cell cycle and differentiation during zebrafish midbrain and retinal development

Abstract

BackgroundThe Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome.ResultsUsing gene knockdown experiments we examined the function of the dmbx1 gene paralogs in zebrafish, dmbx1a and dmbx1b in regulating neurogenesis in the developing retina and midbrain. Dose-dependent loss of dmbx1a and dmbx1b function causes a significant reduction in growth of the midbrain and retina that is evident between 48-72 hpf. We show that this phenotype is not due to patterning defects or persistent cell death, but rather a deficit in progenitor cell cycle exit and differentiation. Analyses of the morphant retina or anterior hindbrain indicate that paralogous function is partially diverged since loss of dmbx1a is more severe than loss of dmbx1b. Molecular evolutionary analyses of the Dmbx1 genes suggest that while this gene family is conservative in its evolution, there was a dramatic change in selective constraint after the duplication event that gave rise to the dmbx1a and dmbx1b gene families in teleost fish, suggestive of positive selection. Interestingly, in contrast to zebrafish dmbx1a, over expression of the mouse Dmbx1 gene does not functionally compensate for the zebrafish dmbx1a knockdown phenotype, while over expression of the dmbx1b gene only partially compensates for the dmbx1a knockdown phenotype.ConclusionOur data suggest that both zebrafish dmbx1a and dmbx1b genes are retained in the fish genome due to their requirement during midbrain and retinal neurogenesis, although their function is partially diverged. At the cellular level, Dmbx1 regulates cell cycle exit and differentiation of progenitor cells. The unexpected observation of putative post-duplication positive selection of teleost Dmbx1 genes, especially dmbx1a, and the differences in functionality between the mouse and zebrafish genes suggests that the teleost Dmbx1 genes may have evolved a diverged function in the regulation of neurogenesis.

Neural tube patterning by Ephrin, FGF and Notch signaling relays

“…7B). In zebrafish, Dmbx1a has been shown to promote cell-cycle exit and differentiation of neurons (Wong et al, 2010). It will be interesting to see whether this is occurring in the context of Delta/Notch lateral inhibition and whether it functions to counteract FGF and SoxB1, as seen in the Ciona MG.…”

Neuronal subtype specification in the spinal cord of a protovertebrate

“…Dmbx1 knockout mice show early neonatal lethality owing to diminished milk intake, probably related to its function in the hindbrain (Fujimoto et al, 2007;Ohtoshi and Behringer, 2004). In zebrafish, Dmbx1 paralogs in zebrafish have been shown to regulate cell-cycle exit of neuronal progenitors in the retina and optic tectum (Kawahara et al, 2002;Wong et al, 2010). It is possible that Dmbx genes are involved in a regulatory code for decussating interneuron subtypes in vertebrates, such as the aforementioned Mauthner cells of fish and amphibians.…”

A second visual rhodopsin gene,rh1-2, is expressed in zebrafish photoreceptors and found in other ray-finned fishes

“…In situ hybridizations were performed on 3 dpf (control) and 5 dpf zebrafish embryos (whole mount), and eyes from 21 and 175 dpf zebrafish, as previously described (Wong et al, 2010). DIG-labeled RNA probes 700 bp in length, as well as unlabeled, full-length blocking RNA for rh1-2 (control), were amplified from rh1 and rh1-2 sequences inserted into the pBluescript cloning vector using T3 RNA Polymerase (Fermentas, Waltham, MA, USA).…”