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1.
Figure 6

Figure 6. Overexpression of Kctd12.2 inhibits elaboration of Hb neuropil. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) Kctd12.2 (green) is normally expressed in neurons of the medial subnuclei. (B) In Hb:Gal>Kctd12.2-MT larvae, expression of Kctd12.2-MT fusion protein is driven at high levels in nearly all Hb neurons. (C) WT larvae have an elaborate network of neuropil that segregates within each Hb subnucleus, (D) but the presence of high levels of ectopic Kctd12.2-MT fusion inhibits the elaboration of Hb neuropil. (E) Volumetric quantification of Hb neuropil reduction. Overexpression of Kctd12.2-MT causes significant reduction of total Hb neuropil volume compared to WT (18,103±789μm3 vs 30,147±2,588μm3 , p= 7.7E-8, n=16). All subnuclei are significantly affected (asterisks indicate statistical difference compared to WT). Scale bars = 50 μm. *=p<0.05, **=p<0.01, two tailed T-test.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
2.
Figure 2

Figure 2. Kctd12.1 interacts with the proline-serine-rich region of Ulk2. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) Kctd12.1 and Ulk2 interact in a yeast 2-hybrid assay. Kctd12.1 is composed of two domains: an N-terminal domain that promotes oligomerization, and a C-terminal domain of undefined function. Ulk2 is composed of 3 domains: an N-terminal serine-threonine kinase domain (K), an internal proline-serine-rich region (PS rich), and a C-terminal domain (CTD) involved in protein-protein interactions. Neither N- nor C- terminal deletions of Kctd12.1 are able to interact with full-length Ulk2. Full-length Kctd12.1 can interact with full-length Ulk2 as well as the proline-serine-rich (PS) domain of Ulk2 alone. (B) The interaction between Ulk2 and Kctd12.1 is confirmed by in vitro co-immunoprecipitation. After incubation of fusion proteins to allow binding, MYC:Ulk2 could be detected via Western blotting in samples immunoprecipitated with antibodies against HA (white arrow) and likewise, HA:Kctd12.1 was detected upon immunoprecipitation with antibodies against MYC (yellow arrow).

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
3.
Figure 8

Figure 8. Overexpression of Ulk2 by mRNA injection rescues neuropil reduction caused by Kctd12.1 overexpression. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) The large volume of neuropil present in the left Hb of wild type larvae (white arrow), (B) is reduced (yellow arrow) by overexpression of Kctd12.1-MT (green in insets) and (C) is slightly increased by injection of 500pg ulk2 mRNA alone. (D) Injection of 500pg ulk2 mRNA restores relatively normal neuropil volume in the left Hb (red arrow) of larvae overexpressing Kctd12.1-MT. (E) Volumetric quantification of neuropil phenotype. Overexpression of Kctd12.1-MT significantly reduces neuropil volume in the left lateral and both medial Hb subnuclei. Injection of ulk2 mRNA slightly increases neuropil volume in the medial subnuclei. Injection of ulk2 mRNA into Kctd12.1-MT overexpression larvae restores wildtype neuropil volumes to all subnuclei with the exception of the right medial subnucleus (asterisks indicate statistical difference compared to WT). Scale bar = 50 μm. *=p<0.05, **=p<0.01.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
4.
Figure 5

Figure 5. Overexpression of Kctd12.1 inhibits elaboration of Hb neuropil. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) Kctd12.1 (green) is normally expressed only in Hb neurons of the lateral subnuclei. (B) In Hb:Gal>Kctd12.1-MT larvae, Kctd12.1-MT fusion protein (red) is expressed at high levels in nearly all Hb neurons. (C) WT larvae have an elaborate network of neuropil that segregates within each Hb subnucleus, (D) but the presence of high levels of ectopic Kctd12.1-MT fusion inhibits the elaboration of Hb neuropil. (E) Volumetric quantification of Hb neuropil reduction. Overexpression of Kctd12.1-MT causes significant reduction of total Hb neuropil volume compared to WT (19,693±1,664μm3 vs 29,602±1,426μm3, p=0.002, n=16) All subnuclei are significantly affected with the exception of the right lateral subnucleus (asterisks indicate statistical difference compared to WT). Scale bars = 50 μm. *=p<0.05, **=p<0.01, two tailed T-test.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
5.
Figure 3

Figure 3. Ulk2 is expressed in the Hb and colocalizes with Kctd12.1. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A-C) ulk2 transcript is detected by in situ hybridization in many neurons of the CNS, but is enriched in the paired Hb (white outlines) at 48, 72, and 96 hours post fertilization. (D-F) A combination of fluorescent in situ for ulk2 transcript and Kctd12.1 immunofluorescence reveals coexpression in the left lateral Hb. (G-H) Transgenic expression of GFP:Ulk2 fusion protein in a single neuron of the left lateral Hb shows localization of GFP:Ulk2 to puncta in Hb dendrites (arrows). (I-I”) GFP:Ulk2 colocalizes with Kctd12.1. The image in (I) is an optical section through the left Hb in (G-H) with medial to the right and dorsal to the top; dashed lines in (I) indicate the relative orientation of optical sections shown in (I’) and (I”). Scale bars in A and D = 50 μm, in G, H, and I = 15 μm.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
6.
Figure 7

Figure 7. Mutation of Kctd12 proteins leads to excess Hb neuropil. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) By 4 dpf, WT larvae display elaborate extension of neuropil in both Hb and express both Kctd12.1 and 12.2 (green, inset). (B) The kctd12.1 coding sequence is disrupted by a large viral insertion in kctd12.1vu442 mutants (note lack of Kctd12.1 staining in lateral subnuclei in inset). Hb neuropil in Kctd12.1-negative larvae is slightly expanded in lateral subnuclei. (C) An ENU-induced stop codon in the coding sequence of kctd12.2 in kctd12.2fh312 mutants leading to loss of Kctd12.2 protein expression (note lack of Kctd12.2 staining in medial subnuclei in inset). Kctd12.2-negative larvae also display excess elaboration of Hb neuropil, particularly in the medial subnuclei. (D) kctd12.1vu442; kctd12.2fh312 double mutants are negative for both Kctd12 proteins (green, insert) and Hb neuropil is expanded in both the lateral and medial subnuclei. (E) Volumetric quantification of neuropil expansion in kctd12 mutants. Neuropil expansion is restricted to lateral subnuclei in Kctd12.1-negative larvae, consistent with the expression pattern of Kctd12.1. Neuropil expansion in Kctd12.2-negative larvae affects medial subnuclei as well as the right lateral subnucleus. Double mutant larvae display neuropil expansion in all subnuclei (asterisks indicate statistical difference compared to WT). Scale bars = 50 μm. *=p<0.05, **=p<0.01.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
7.
Figure 9

Figure 9. Ulk2 depletion is epistatic to Kctd12.1 mutation. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) Compared to normal Hb, (B) neuropil development (white arrow) is slightly expanded in homozygous null kctd12.1 mutant animals (note absence of Kctd12.1 [green] in insets at right). (C) Reduced neuropil (yellow arrow) in the left Hb as a result of Ulk2 antisense depletion (D) also occurs (red arrow) when Ulk2 is depleted in a Kctd12.1 homozygous mutant, indicating that Ulk2 depletion is largely epistatic to mutation of Kctd12.1. (E) Volumetric quantification of Hb neuropil phenotype. Total neuropil volume is significantly reduced following injection of ulk2 MOspl in either a wild type or Kctd12.1 mutant background (asterisks indicate statistical difference compared to WT). Scale bar = 50 μm. *=p<0.05, **=p<0.01. (F) Model of proposed regulatory system resulting in asymmetric habenular neuropil extension. Ulk2 kinase acts bilaterally to promote elaboration of Hb neuropil, but is negatively regulated by Kctd12 proteins. Asymmetry in neuropil arises from the greater relative potency of Kctd12.2 acting in medial subnuclei.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
8.
Figure 1

Figure 1. Asymmetry in the 96 hpf zebrafish habenular nuclei. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A) The paired habenular nuclei (Hb) receive input via the stria medullaris (sm) and send efferents to the midbrain target the interpeduncular nucleus (IPN). The asymmetric parapineal organ innervates the left lateral subnucleus (green arrow) and is instrumental in establishing Hb laterality. Each subnucleus is asymmetrically subdivided into medial (blue) and lateral (red) subnuclei, with the lateral subnucleus much larger on the left and the medial subnucleus larger on the right. The nomenclature distinguishing the medial from lateral subnuclei is based on their position in the adult brain, which is the opposite of their position in the 96 hpf larvae shown here. (B) An optical slice through a ToPro3 (nuclear marker)-stained larvae demonstrates asymmetrical subnucleus organization. (C) The soma-free regions inside each Hb subnucleus are filled with neuronal processes (acetylated tubulin {AcTub}immunofluorescence) including afferent axons and Hb dendrites. The greatest volume of neuropil is found in the large left lateral subnucleus (arrow). (D) Volumetric quantification of neuropil volume in each Hb subnuclei. The left lateral subnucleus contains much more neuropil volume than the right, so the total neuropil volume is greater in the left Hb. (E) Kctd12.1 protein (green) is primarily expressed in lateral Hb subnuclei. (F) Kctd12.2 (red) is expressed in most neurons of the medial Hb subnuclei, (white dashed line indicates signal from dendritic processes, not Kctd12.2-positive soma) (G) in a largely complementary pattern to Kctd12.1. Scale bars: 50 μm.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.
9.
Figure 4

Figure 4. Antisense knockdown of Ulk2 inhibits the formation of Hb neuropil. From: Asymmetric inhibition of Ulk2 causes left-right differences in habenular neuropil formation.

(A-B) Injection of 2 ng ulk2MOspl at the one-cell stage produces a mild decrease in head size and body length at 4dpf. The general organization of the central (top insets) and peripheral (bottom insets) nervous system as revealed by acetylated tubulin immunofluoresence is intact in morphant larvae (D: diencephalon, T: telencephalon, MN: motor neurons), but Hb neuropil development (white boxes) is disrupted. (C) Injection of pre-spliced Ulk2 mRNA at the one-cell stage rescues all the phenotypes of Ulk2 morpholino treatment. (D) The ulk2 transcript is depleted by morpholino injection. RT-PCR with ulk2 primers (top panel) shows reduction of ulk2 mRNA relative to βactin (bottom panel) (E) Quantification of band intensity in RT-PCR replicates. 0ng = 1 ± .07 arbitrary units (AU), 2ng = 0.5 ± 0.05 AU, 4ng = 0.43 ± 0.02 AU, N=3. (F-H) Morphant larvae can be assigned to one of three categories based on the degree of Hb neuropil elaboration. (I) 100% of uninjected larvae have fully elaborated, asymmetric Hb neuropil (“WT” black bars) When injected with either 2 ng ulk2MOspl or 5 ng ulk2MOATG, the formation of neuropil in the Hb is inhibited in most larvae (white bars), a phenotype not observed in injections of half-maximal dosage (1 ng and 2.5 ng respectively). Hb neuropil inhibition was also observed when half-maximal doses of each MO were combined, indicating that both morpholinos target the same transcript. Injection of 150pg in vitro transcribed ulk2 mRNA was able to rescue Hb neurite formation when coinjected with 2 ng ulk2MOspl. (J-K) Mosaic scatter-labeling of Hb neurons with memGFP, followed by average dendrite volume quantification highlights changes in average dendrite volume between uninjected (J) and ulk2MO-treated (K) larvae Scale bars: (A) 100 μm, (F, J) 50 μm.

Robert W. Taylor, et al. J Neurosci. ;31(27):9869-9878.

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