Photoreceptors are highly polarized sensory neurons that require intense protein trafficking through a narrow connecting cilium to optimize phototransduction in the light sensitive outer segment [1
]. As part of its normal physiology, the outer segment turns over about 10% of its length every day through a process called disc shedding. Disc shedding is compensated for by new outer segment assembly to maintain its length. The synthetic machinery supporting outer segment turnover is in the inner segment. In this compartment, newly synthesized outer segment proteins such as rhodopsin and phospholipids are delivered to the base of the connecting cilium in a vesicular fraction derived from the trans-Golgi [3
]. The inner segment also supports the highly dynamic synaptic compartment via a short axon.
Microtubule based motors are thought to play a significant role in each of the major compartments of the photoreceptor. For example, axonal transport by both dynein and kinesin motors is necessary to support proper ribbon synapse formation and maintenance [5
], and recent observations in zebrafish indicate that Dynactin1 is required for nuclear positioning in zebrafish [7
]. Furthermore, Golgi and endoplasmic reticulum positioning and post-Golgi trafficking generally involve microtubule based motors [8
], and outer segment turnover is now known to depend on intraflagellar transport using kinesin and dynein motors along the axoneme of the outer segment [1
]. Thus, photoreceptors utilize multiple microtubule based motors in diverse cellular processes to facilitate normal development, maintenance, and function.
Dynein1 is a multi-subunit complex that consists of two 530 kDa heavy chains, responsible for force production, a group of 74 kDa intermediate chains, 53 to 57 kDa light intermediate chains, and 8 to 21 kDa light chains [10
]. In photoreceptors, Dynein1 has been implicated in post-Golgi trafficking of rhodopsin because Dynlt1 (formerly Tctex-1), a dynein1 light chain subunit, binds to the carboxy-terminal domain of rhodopsin and can translocate rhodopsin-containing vesicles on microtubules [11
]. However, Dynein1 has also been implicated in multiple cellular functions, including the positioning of the Golgi apparatus, endosomes, lysosomes, nuclei, centrosomes and mitotic spindles as well as retrograde axonal transport in neurons [9
Another multi-subunit protein complex, Dynactin, serves as an adaptor and confers additional functions to Dynein1 by expanding the range of its cargo and increasing its motor processivity [13
]. In a recent report, zebrafish embryos carrying a mutation (mikre oko
) in dynactin1a
; previously called p150) failed to position photoreceptor nuclei to the proper layer without affecting the overall cell morphogenesis or the transport of opsins to the outer segment [7
]. In the same study, the over-expression of another Dynactin component, Dctn2 (formerly p50/dynamitin), a manipulation known to dissociate the Dynein1/Dynactin complex, phenocopied mok
. These results suggest that nuclear positioning in photoreceptor cells is carried out by a Dynein1/Dynactin dependent pathway.
To date, the precise cellular function(s) of Dynein1 in vertebrate photoreceptors remains poorly understood and recent studies have provided limited insight towards defining its role in trafficking and organelle positioning within the inner segment. In addition, potential Dynein1 functions within the outer segment have not been investigated. However, a second cytoplasmic Dynein, Dynein2, has been localized prominently in bovine photoreceptor outer segments along the connecting cilium, with expression also in the inner segment [17
]. Functional insight for Dynein2 comes from knock-down studies in zebrafish where morpholinos directed against Dynein2 subunits resulted in short and disorganized photoreceptor outer segments [18
]. Interestingly, the loss of Dynein2 function did not affect opsin trafficking, suggesting that Dynein2 plays a role in a transport pathway different from that proposed for Dynein1. Previous studies in Chlamydomonas
and Caenorhabditis elegans
, have clearly established a role for Dynein2 in the retrograde translocation of proteins from distal tips of ciliary axonemes to the base near the cell body [19
]. This process, referred to as intraflagellar transport (IFT), requires a complex of at least 17 proteins for movement in the 'retrograde' direction. Indeed, in the study from Krock et al
], one anterograde IFT particle, IFT88, accumulated distally within photoreceptor outer segments of Dynein2 morphants, suggesting a failure in retrograde IFT.
Here we report the identification of a new zebrafish mutant, cannonball (cnb), which affects the dynein cytoplasmic 1 heavy chain 1 (dync1h1) locus. Using cnb, we studied the roles of Dynein1 in developing photoreceptor neurons. This mutant showed severe defects in photoreceptor organelle organization and outer segment formation. With regard to outer segment function, we present evidence that Dynein1 localizes along the connecting cilium and is detected in detergent extracted fractions enriched with axonemes of isolated outer segments. Finally, we used different concentrations of anti-sense morpholinos against dync1h1 to study dosage-dependent loss-of-function phenotypes. Embryos injected with a high dose of dync1h1 morpholino phenocopied cnb defects. Interestingly, lower concentrations of morpholino revealed specific outer segment defects. Overall, we provide support for multiple roles of Dynein1 in photoreceptor development, including organelle positioning, post-Golgi vesicle trafficking, and essential function(s) within the outer segment.