We show here that a mutation in Dnct1, a microtubule-motor associated protein, perturbs INM in a selective fashion and results in an overproduction of early-born neurons in the zebrafish retina. The progenitor nuclei move more quickly and more deeply in the basal direction and more slowly apically. We further established that anti-neurogenic Notch signals are enriched on the apical side of the neuroepithelium in both mutants and wildtype. Combined with previous observations that progenitors whose nuclei migrate deep are more likely to produce postmitotic neuronal daughters following their return to the apical side (Baye and Link, 2007a
), our studies suggest a mechanism by which INM co-operates with an apical-basal Notch gradient to select progenitors for cell cycle exit and apportion cell fates ().
The phenotype of the mok
mutant is reminiscent of manipulations of cell fate determinants, such as overexpression of bHLH proneural genes. We therefore asked if the mok
mutation altered the intrinsic competence of progenitors. The bHLH transcription factor Atoh7 functions as a proneural factor to set neurogenic competency of early retinal cell types and is essential for RGC fate. Disruption of atoh7
eliminates RGCs and increases the number of progenitors that remain in the cell cycle (Brown et al., 2001; Kay et al., 2001
; Wang et al., 2001). As a consequence of this enlarged progenitor pool, bipolar cells and glia are increased in number in the zebrafish lak
) mutant. Conversely, overexpression of atoh7
results in an excess of RGCs, resembling the mok
phenotype (Hatakeyama and Kageyama, 2004; Vetter and Brown, 2001; Yan et al., 2005). In lak
double mutants, glia and bipolar neurons, which are severely reduced in mok
single mutants, develop similarly to lak
single mutants, demonstrating that mok
mutant progenitors retain their potential to generate later-born neurons. Photoreceptors, which develop normally in lak
single mutants, however, are absent in the double mutants, as they are in mok
single mutants, due to a related but independent function of Dnct1 in photoreceptor nuclear positioning (Tsujikawa et al., 2007
). In conclusion, Dnct1 does not affect the neurogenic competence of retinal progenitor cells.
Inhibition of Notch (Austin et al., 1995
) or disruption of GDF11 (Kim et al., 2005
) mimic some aspects of the mok
phenotype. Notch prevents progenitors from leaving the cell cycle and from differentiating prematurely. Its activation during the time progenitors are competent to produce RGCs leads to a depletion of this cell type. GDF11, on the other hand, is secreted by differentiated RGCs to negatively regulate the number of new RGCs produced by suppressing atoh7
. As a consequence, progenitors are more likely to generate RGCs when transplanted into an environment in which RGCs are absent, as in lak
mutants (Poggi et al., 2005a
). We made a complementary observation by transplanting wildtype cells into mok
mutants. These clones tend to form fewer RGCs, suggesting that the negative feedback signals are intact in the mutant environment. Moreover, we show that constitutive activation of Notch in mok
mutants blocks RGC production and leads to an overproduction of glia, as it does in wildtype. Together, these results demonstrate that mok
mutant cells are still able to produce, and respond to, extrinsic regulators of cell fate.
A Notch gradient along the apical-basal axis of the neuroepithelium is likely to play a key role in neurogenesis. Notch mRNA is increased on the apical side, whereas Delta mRNA and protein are enriched basally. This results in a gradient of Notch transcriptional activity, as demonstrated by the higher concentration of Notch ICD in the apical domains and the increased her4 expression in cells whose nuclei move into the high-Notch environment. We have found no evidence that Notch signaling, neuroepithelial polarity, or the Notch gradient itself, are altered in mok mutants. Distribution of the endocytic pathway components Rab11 and Numb, which in other contexts regulate Notch, is unaltered. Moreover, an independent manipulation of the Dynein/Dynactin-dependent component of INM (disruption of the nuclear anchor protein Syne2a) perturbs cell fate decisions similarly to the mok mutation.
Between mitoses, a progenitor nucleus moves twice through a Notch spatial gradient. If the nucleus stays close to the apical side it will encounter high Notch levels throughout the cell cycle, and both of its daughters are likely to remain proliferative. On the other hand, if the nucleus is translocated more basally, Notch activity is reduced, predisposing the progenitor to produce one or two daughter neurons during its subsequent mitosis. It follows that, in mok
mutants, the balance of neurogenic vs. proliferative divisions is shifted by decreasing exposure to Notch across the progenitor population. Given that both INM and Notch signaling compartments are ubiquitous features of CNS neuroepithelia (Frade, 2002
) as well as non-neuronal epithelia (Bort et al., 2006
), it seems likely that this mechanism is widely employed during embryonic development, growth, and regeneration.