Carboxypeptidase A6 in zebrafish development and implications for VIth cranial nerve pathfinding

Abstract

Carboxypeptidase A6 (CPA6) is an extracellular protease that cleaves carboxy-terminal hydrophobic amino acids and has been implicated in the defective innervation of the lateral rectus muscle by the VIth cranial nerve in Duane syndrome. In order to investigate the role of CPA6 in development, in particular its potential role in axon guidance, the zebrafish ortholog was identified and cloned. Zebrafish CPA6 was secreted and interacted with the extracellular matrix where it had a neutral pH optimum and specificity for C-terminal hydrophobic amino acids. Transient mRNA expression was found in newly formed somites, pectoral fin buds, the stomodeum and a conspicuous condensation posterior to the eye. Markers showed this tissue was not myogenic in nature. Rather, the CPA6 localization overlapped with a chondrogenic site which subsequently forms the walls of a myodome surrounding the lateral rectus muscle. No other zebrafish CPA gene exhibited a similar expression profile. Morpholino-mediated knockdown of CPA6 combined with retrograde labeling and horizontal eye movement analyses demonstrated that deficiency of CPA6 alone did not affect either VIth nerve development or function in the zebrafish. We suggest that mutations in other genes and/or enhancer elements, together with defective CPA6 expression, may be required for altered VIth nerve pathfinding. If mutations in CPA6 contribute to Duane syndrome, our results also suggest that Duane syndrome can be a chondrogenic rather than a myogenic or neurogenic developmental disorder.

Figure 1. Members of the A/B subfamily of metallocarboxypeptidases identified in the zebrafish.

Amino acid sequences of identified CPs were aligned using ClustalW. Greater sequence similarity is indicated by darker background, while locations of signal peptide cleavage, as predicted by SignalP, and of propeptide cleavage, based on experimental evidence for other orthologs, are indicated by a ∧ and #, respectively. Critical active site residues are numbered above the alignment according to their corresponding site in bovine CPA1. Sequences were obtained from the NCBI or Ensembl databases and correspond to the following accession numbers: zCPB1, ENSDARP00000066831; zCPB2, BC095834; zCPO, NM001145629; zCPA1, ENSDARP00000024981; zCPA2, ENSDARP00000064203; zCPA4, ENSDARP00000014821; zCPA5, ENSDARP00000034171; zCPA6, XM001342113.

Figure 2. Phylogenetic tree indicating relationships of mammalian and zebrafish A/B-type metallocarboxypeptidases.

Sequences were aligned by ClustalW and .dnd files produced to construct a phylogenetic tree. Scale bar indicates substitutions per site.

Figure 3. Temporal and spatial expression of zebrafish CPA genes.

(A, B) qPCR was performed using cDNA prepared from zebrafish at the indicated developmental time points. All values were normalized to β-actin, and are shown in (A) as expression relative to CPA1 mRNA at 2 dpf, or in (B) as expression of each gene relative to its highest level (100%). (C, D) In situ hybridization was used to determine the spatial distribution of CPA mRNA expression. (C) At 2 dpf CPA5 mRNA (arrows) was detected in a mast cell population and CPA6 mRNA was seen in precursor tissues found in the stomodeum, posterior to the eyes, and in the pectoral fin buds. (D) At 4 dpf, mRNA for CPA1, 2, 4, and 5 were expressed in the pancreas.

Figure 4. Cellular distribution and enzymatic activity of zebrafish CPA6.

(A) EGFP-tagged CPA6 (+) or EGFP only (−) were expressed in HEK293T cells. Media, cell and ECM extracts were resolved by SDS-PAGE and western blotted with an anti-GFP antibody, showing the majority of processed CPA6-EGFP (asterisk) localized to the ECM. (B) ECM from cells transfected with zCPA6 was incubated with fa-FF at the pH values indicated or (C) with a range of commercially available fa substrates at pH 7.5. CP activity was determined by a change in substrate absorbance at 336 nm. Error bars indicate range of duplicate determinations from a representative experiment. Similar results were obtained in two separate experiments.

Figure 5. Detailed analysis of CPA6 mRNA expression throughout zebrafish development.

In situ hybridization indicated CPA6 mRNA (purple) is found in newly formed somites (A–D), ectodermal cells of the tail (C,D), a tissue posterior to the eye (E–H, arrows), the stomodeum (G, asterisk), and the pectoral fins (H, arrowhead). CPA6 expression posterior to both left and right eyes can be seen in F and H. A summary of the spatial (I) and temporal (J) expression of CPA6 throughout zebrafish development is shown.

Figure 6. Distribution of CPA6 mRNA compared with tissue-specific markers at 2 dpf.

(A) A general marker of muscle precursors, myogenin , labels most extraocular muscles (orange) by in situ hybridization, but does not co-localize with CPA6 mRNA (purple). (B) Myogenin mRNA, as well as MyoD mRNA, is also found in the pectoral musculature, unlike CPA6 mRNA which is ectodermal. CPA6 mRNA does not co-localize with Meox1 (C), or Lbx1 (D), both putative markers of the lateral rectus muscle in the chick, but likely not in the zebrafish. (E) A schematic of a 2 dpf zebrafish indicates the relative spatial expression of CPA6, myogenin (in lateral rectus), Lbx1 and Meox1. lr, lateral rectus; mr, medial rectus; sr, superior rectus; ah, adductor hyoideus; cd, constrictor dorsalis; MD, myodome precursors; O.V., otic vesicle.

Figure 7. Distribution of CPA6 mRNA compared with somitogenesis markers.

In situ hybridization was performed with RNA probes specific for (A) CPA6 (purple) and MyoD (orange) at 3 and 8 somite stages (11 and 14 hpf), and (B) CPA6 (purple) and Twist1b (orange) at 24 hpf. Arrows indicate ectodermal cells arranged along the ventral ridge of the tail and expressing CPA6. The regular arrangement of these cells, also found along the dorsal ridge, is illustrated in the inset. PSM, presomitic mesoderm; ys ext, yolk-sac extension.

Figure 8. Knockdown of CPA6 gene function.

(A) Morpholinos were designed to interfere with normal CPA6 mRNA splicing (Ctr-MO: control morpholino; MO-2 and MO-3: CPA6 specific morpholinos; lines: MO binding sites; arrows: primer binding sites). (B) RT-PCR using RNA extracted from either uninjected or morpholino-injected embryos (3 ng) showed CPA6-specific knockdown. (C) 48 hpf embryos injected with control morpholino and CPA6 MO-2. CPA6 morphants exhibited yolk-sac edema. (D) DiI injected into the lateral rectus muscle (LR) of 8 dpf Ctr-MO and MO-2 CPA6-morphant zebrafish larvae retrogradely labeled the VIth cranial nerve (nVI). Scale bar  = 40 µm.

Figure 9. Optokinetic reflex measurement of CPA6 morphants at 5 dpf.

(A) Morphology of CPA6 morphants. (B) Schematic illustration of central neural pathways for either lateral or medial rectus mediated optokinetic tracking and saccades. (C) Representative example of eye position (blue trace; top panel), fast phase saccades (green hatching; top panel) and slow phase tracking velocity (cyan trace; bottom panel) in response to ±20°/s velocity steps optokinetic stimuli (grey trace; bottom panel) at 0.0625 Hz. (D) Quantification of saccadic velocity produced by either the lateral (orange bars) or medial rectus muscle (grey bars) in CPA6 morphants and controls. Data are presented as mean ± S.D. (E) Representative eye tracking, saccades and gain in a MO-2 (3 ng) morphant and corresponding control. (F) Optokinetic reflex in MO-3 (6 ng) morphants and corresponding control.