Quantitative description of Hb neuropil asymmetry
Although asymmetric neurite development has been described qualitatively in several reports (Concha et al., 2000
, Concha et al., 2003
, Bianco et al., 2008
), we developed metrics to quantitatively measure Hb asymmetry. In the zebrafish epithalamus (schematized in ), the L-R asymmetric size ratio of medial and lateral habenular subnuclei can be distinguished based on soma distribution (). During late development, the lateral Hb subnuclei shift dorsal and lateral to the medial subnuclei. Thus, the designation of “medial” and “lateral” Hb subnuclei refers to their relative positions in the adult brain, which is opposite their positions in the 96hpf larva. Antibodies against acetylated tubulin mark all axons and dendrites, collectively referred to as neuropil (). Distribution of neuropil volume is robustly asymmetric in the zebrafish habenulae, with the greatest volume of neuropil contained in a large soma-free region at the core of the left lateral subnucleus (arrow in ). To quantitatively describe Hb neurite development, we used image analysis software (Volocity) to precisely determine the neuropil volume in each individual subnucleus. The left Hb contains significantly more neuropil than the right (p=1.34E-7, n=12), owing largely to the contribution of the left lateral subnucleus.
Asymmetry in the 96 hpf zebrafish habenular nuclei
Asymmetry in subnucleus and neuropil organization is tightly correlated with asymmetric expression of Kctd12 proteins. Kctd12.1 () expression is restricted to neurons of the lateral subnuclei, while Kctd12.2 () is expressed primarily in medial Hb neurons in a pattern largely complementary to Kctd12.1 (). The correlation between asymmetric morphological organization of the Hb and asymmetric expression of Kctd12 proteins lead us to investigate their role in Hb development.
Identification and confirmation of Ulk2 as Kctd12.1 interactor
In order to assess the function of Kctd12.1 protein in Hb neurons, we used the yeast two-hybrid assay to screen a brain cDNA library to identify protein-protein interactions with Kctd12. Because a high-quality zebrafish brain cDNA library is not available, we used the single human homolog of Kctd12.1 and Kctd12.2, HhKCTD12, as bait (fused to the Gal4 DNA binding-domain) and screened a commercially available human fetal brain cDNA library (Clontech). We isolated 66 interaction-positive clones after screening through approximately 2.3×106 library clones. Interacting clones included a-kinase interacting protein 1 (AKIP1), ubiquitin-conjugating enzyme E2N-like (UBE2NL), and several Golgins. Sequence corresponding to Unc-51-like-kinase 2 (Ulk2) was isolated from three independent clones.
First, we wanted to confirm the interaction by testing the zebrafish homologues of Kctd12.1 and Ulk2. After cloning zebrafish Ulk2, we found that it is able to activate interaction-selection cassettes when co-transformed along with Kctd12.1 (). To gain an initial understanding of structure-function relationships, we tested deletions of the two primary domains of Kctd12.1 for their ability to interact with full-length Ulk2. The N-terminal domain is thought to promote oligomerization of Kctd monomers (Dementieva et al., 2009
), and the C-terminal domain has no described function. We found that deletion of either the N-terminal or C-terminal domain of Kctd12.1 abolishes all interaction with full-length Ulk2. .
Kctd12.1 interacts with the proline-serine-rich region of Ulk2
To independently verify this interaction, we generated HA:Kctd12.1 and MYC:Ulk2 fusion proteins by in vitro coupled transcription and translation (). In co-immunoprecipitation experiments, we found that MYC:Ulk2 can be pulled down with HA:Kctd12.1 and that HA:Kctd12.1 can likewise be pulled down along with MYC:Ulk2.
Kctd12.1 and Ulk2 colocalize in Hb processes
Kctd12 proteins and Ulk2 must be co-expressed in Hb neurons to be considered relevant to Hb neuropil development. By in situ
hybridization (), we detect ulk2
mRNA in most neurons of the central nervous system, consistent with previous reports of broad brain expression patterns in other organisms (Yan et al., 1999
). Importantly, we find ulk2
mRNA bilaterally enriched in Hb neurons as early as 48hpf, and this expression continues until at least 96 hpf. A combination of fluorescent in situ
hybridization for ulk2
and immunofluorescence for Kctd12.1 protein confirms that at the level of tissue organization, ulk2
and Kctd12.1 are coexpressed in neurons of the lateral subnucleus of the left Hb (). We also analyzed the expression pattern of the related genes ulk1a
. These two genes are expressed broadly in neurons of the brain, but unlike ulk2, ulk1a
are not enriched in the Hb between 48 and 96hpf (data not shown)..
Ulk2 is expressed in the Hb and colocalizes with Kctd12.1
We next wanted to determine the localization of Kctd12.1 and Ulk2 proteins at the subcellular level in Hb neurons. Ulk2 has previously been reported to localize to cytoplasmic puncta in neuronal processes (Zhou et al., 2007
). Because no suitable antibody against zebrafish Ulk2 is available, we expressed a GFP:Ulk2 fusion protein in small numbers of Hb neurons by Gal4:UAS-based scatter labeling () in the Hb>Gal background. Consistent with previous reports, this fusion protein is present in a punctate pattern in dendritic (, arrows) and axonal (not shown) processes of Hb neurons. Counterstaining with antibodies against Kctd12.1 shows that GFP:Ulk2 and Kctd12.1 colocalize at the subcellular level () (N=7). According to these findings, Ulk2 and Kctd12.1 are present together at a relevant developmental time to affect Hb process development.
Morpholino knockdown of Ulk2 inhibits elaboration of Hb neuropil
Based on previous reports of Ulk protein abrogation in developing neurons, we hypothesized that knockdown of Ulk2 in zebrafish would lead to inhibition of neurite elaboration in the developing habenulae. To test the role of Ulk2 in Hb neuron development, we adopted a knockdown strategy using antisense morpholino oligonucleotide (MO) injection. Though Ulk2 morphants have slightly reduced head size and body length (), general neurogenesis is not perturbed, as both brain organization and axonal extension by motor neurons indicate no overall defects in the development of the central nervous system ( insets). The slight morphological defects associated with morpholino injection can be rescued by co-injection of 150pg ulk2 mRNA ().
Antisense knockdown of Ulk2 inhibits the formation of Hb neuropil
To assay the effectiveness of morpholino injection at depleting ulk2 mRNA, we performed RT-PCR using primers within either the ulk2 or βactin coding region on groups of larvae injected with either 0, 2, or 4ng ulk2MOSPL and collected at 3 dpf (). We found that 2 ng ulk2MOSPL is sufficient to knock down ulk2 mRNA to approximately 50% of the levels found in WT larvae (2ng injection: 50.4% ± 0.05 of WT level, N=3), probably through the process of nonsense-mediated decay.
To gauge habenular defects in populations of morphant larvae, we separated larvae into three groups based on the morphology of Hb neuropil: WT (), reduced (), and absent () for population frequency analysis. Samples were categorized without knowledge of treatment group.
Following treatment with either of two morpholinos complementary to different regions of the ulk2 mRNA (5 ng ulk2MOATG or 2 ng ulk2MOSPL), Hb neuropil is absent (, white bars) in many larvae (Spl: 65% N=40, ATG: 66.67% N=21). This effect was not elicited by injection of half-maximal doses of either morpholino (2.5 ng ulk2MOATG or 1 ng ulk2MOSPL respectively), but co-injection of a mixture of suboptimal morpholino concentrations (2.5 ng ulk2MOATG plus 1 ng ulk2MOSPL) was able to effectively inhibit development of Hb neuropil in most larvae (60% N=20), suggesting that both morpholinos target the same transcript.
To confirm that targeting of the ulk2 transcript is responsible for the Hb neuropil phenotype, we injected pre-spliced ulk2 mRNA along with 2 ng ulk2MOSPL. We found that injection of 150 pg ulk2 mRNA is able to rescue neuropil development in many 2 ng ulk2MOSPL larvae.
In order to discount the possibility that reduction in Hb neuropil in Ulk2 morphants is caused by a loss of Hb neurons, we counted Kctd12.1-positive neurons in larvae treated with 2 ng of ulk2MOSPL. Morphants and uninjected larvae do not have significantly different numbers of Kctd12.1-positive neurons (uninjected: 260±7.76, 2 ng ulk2MOSPL: 234 ± 8.53, p=0.12 n=12), indicating that loss of Hb neurons is not responsible for the morphant phenotype (not shown).
Because measurements of neuropil volume unavoidably include signal from the axons of Hb afferents, we verified that Hb dendrites were specifically affected by Ulk2 knockdown. Using membrane-localized GFP (memGFP) to label small clones of cells in control and ulk2MOSPL–injected embryos, we measured average dendrite volume per Hb neuron ()(see Methods). Average dendritic volume was significantly reduced in ulk2 morphants (uninjected: 173.7μm3 ± 15.6 N=16, 2 ng ulk2MOSPL: 131.8 μm3 ± 11.4 N=16, p=0.028).
Overexpression of Kctd12 proteins inhibits Hb neuropil development
To investigate the impact of Kctd12.1 expression on developing Hb neurons, we used the Gal4/UAS expression system to overexpress Kctd12.1 in the Hb of larvae. Hb:Gal drives expression of Gal4 transcription factor bilaterally in almost all Hb neurons by 4 dpf (Scott et al., 2007
). We generated response lines with transgenes containing a UAS element upstream of a Kctd12.1:Myc tag (MT) fusion protein (Tg[UAS:kctd12.1-mt
]). In Hb:Gal>Kctd12.1-MT larvae, we detected bilateral kctd12.1
mRNA (not shown) and overlapping Myc and Kctd12.1 immunofluorescence (). Semi-quantitative RT-PCR revealed a doubling in the level of kctd12.1
mRNA present in double transgenic samples (WT = 1.0 ± 0.11 arbitrary units [AU], Hb:Gal>Kctd12.1-MT = 2.11 ± 0.17 AU, N=3). At 4 dpf, fusion protein was present in almost all Hb neurons. The pattern of Hb neuropil extension in Kctd12.1 overexpression larvae was then assessed by acetylated tubulin immunofluorescence.
Overexpression of Kctd12.1 inhibits elaboration of Hb neuropil
When Kctd12.1 is overexpressed in all Hb neurons, the large region of dense neuropil in the left Hb is dramatically reduced (). Larvae with normal levels of Kctd12.1 expression have nearly twice the process volume in the left Hb as compared to the right, but in Hb:Gal>Kctd12.1:MT larvae, only a low volume of neuropil is detected in both habenulae (arrows, ). Quantification of neuropil volumes in each subnucleus revealed a significant decrease in neuronal processes in the left lateral (p=0.0013, n=16) and both left (p=0.043, n=16) and right (p=0.046, n=16) medial subnuclei.
Hb neuropil defects are not due to an effect of the Myc epitope tag, as overexpression of untagged Kctd12.1 produces an identical phenotype (not shown). This phenotype is also not due to parapineal asymmetry defects, as parapineal placement and morphology are unaffected (not shown).
We also tested the effect of overexpression of Kctd12.2 (). Based on the high level of conservation between Kctd12.1 and Kctd12.2, we hypothesized that Kctd12.2-MT fusion protein would similarly inhibit the extension of Hb neuropil when overexpressed. Indeed, neuropil volume is dramatically reduced in larvae that overexpress Kctd12.2-MT (arrows, ). Volumetric quantification of neuropil in Hb:Gal>Kctd12.2-MT reveals significant reductions in neuropil volume in all subnuclei when compared to WT larvae (left medial: p=1.25E-5, n=16, left lateral: p=8.21E-8, n=16, right lateral: p=1.8E-4, n=16, right medial: p=1.8E-6, n=16,).
Overexpression of Kctd12.2 inhibits elaboration of Hb neuropil
Mutation of Kctd12 gene leads to excess Hb neuropil
Based on the dramatic neuropil reduction when Hb neurons overexpress Kctd12 proteins, we hypothesized that loss of Kctd12 expression through mutation may lead to an excess of Hb neuropil. To test this hypothesis, we made use of a Kctd12.1 null mutant allele, kctd12.1vu442, which carries a large viral insertion interrupting the kctd12.1 locus, and a Kctd12.2 mutant allele, kctd12.2fh312, which carries an ENU-induced stop codon (L74*). Both mutations are null, with no protein detected by immunofluorescence staining in homozygous mutants ( insets), and are homozygous viable.
Mutation of Kctd12 proteins leads to excess Hb neuropil
We analyzed neuropil volume in single and double homozygous mutant larvae to uncover any subtle differences in neurite volume extension in the absence of Kctd12 proteins. In kctd12.1vu442 homozygotes, we detected a significant increase in neuropil volume in both the left (p=0.0054, N=16) and right (p=0.0039, N=16) lateral Hb subnuclei, as compared to wild type (). The affected subnuclei are those that express Kctd12.1 in wild type larvae (see ). Similarly, in homozygous kctd12.2fh312 mutants, quantification reveals excess Hb neuropil in the left medial, right lateral, and right medial subnuclei (p=0.0023, n=16, p=0.000051, n=16, p=0.048, n=16, respectively) (). Indeed, the only subnucleus unaffected by loss of Kctd12.2 is the left lateral subnucleus, from which Kctd12.2 is normally excluded. Comparison of neuropil between kctd12.1vu442 and kctd12.2fh312 single mutants reveals a significantly greater increase in neuropil in kctd12.2fh312 mutants (p=0.00912, N=16).
Consistent with the hypothesis that Kctd12.1 and Kctd12.2 proteins negatively regulate neuropil formation in different subnuclei, we found neuropil volumes in kctd12.1vu442; kctd12.2fh312 double mutants to be additively greater than either single mutant alone, and this increase in neuropil volume affects all Hb subnuclei ().
Overexpression of both Kctd12.1 and Ulk2 can restore normal habenular development
If Kctd12 proteins and Ulk2 operate in the same pathway, as their interaction suggests, we reasoned that overexpression of Ulk2 should be able to rescue Hb neuropil defects in Hb:Gal>Kctd12.1:MT larvae by restoration of correct relative levels of both proteins in Hb neurons. To test this hypothesis, we overexpressed Ulk2 by exogenous mRNA injection in the background of Kctd12.1 overexpression (). As in previous experiments, overexpression of Kctd12.1-MT leads to dramatic loss of Hb neuropil volume compared to controls (). Conversely, we find that injection of 500pg ulk2 mRNA is able to increase Hb neuropil volumes, particularly in medial subnuclei (), which supports the idea that Ulk2 acts to promote the extension of neurites.
Overexpression of Ulk2 by mRNA injection rescues neuropil reduction caused by Kctd12.1 overexpression
When exogenous ulk2 mRNA is administered to Hb:Gal>Kctd12.1:MT embryos, we find that total Hb neuropil volume is restored to levels statistically indistinguishable from WT (p=0.072, n=8) (). In these experiments, overexpression of Kctd12.1 inhibits endogenous Ulk2 activity to an inappropriate degree, but providing high levels of exogenous Ulk2 can overcome this inhibition and reestablish normal Ulk2-dependent dendrite outgrowth.
Ulk2 depletion is epistatic to Kctd12.1 mutation
Based on reported roles of Ulk2 as a positive regulator of neurite outgrowth and the excessive neurites in kctd12 mutations, we hypothesized that Kctd12 proteins negatively regulate the activity of Ulk2 kinase, which positively regulates neuropil formation. To test this hypothesis, we examined the phenotype of kctd12.1vu442 treated with an Ulk2 morpholino (). If Kctd12 activity is upstream of Ulk2, we expect the Hb neuropil of mutant/morphant larvae to resemble that of Ulk2 knockdown alone. Mutation of Kctd12.1 in the context of Ulk2 morpholino treatment (2 ng of ulk2 MOspl) results in severely reduced neuropil elaboration relative to WT or mutation of Kctd12.1 alone ().
Ulk2 depletion is epistatic to Kctd12.1 mutation