Importance of deubiquitinases in zebrafish craniofacial development

Accepted Manuscript Importance of deubiquitinases in zebrafish craniofacial development William Ka Fai Tse PII:

S0006-291X(17)30814-8

DOI:

10.1016/j.bbrc.2017.04.132

Reference:

YBBRC 37690

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 14 April 2017 Accepted Date: 24 April 2017

Please cite this article as: W.K.F. Tse, Importance of deubiquitinases in zebrafish craniofacial development, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.04.132. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Importance of deubiquitinases in zebrafish craniofacial development

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William Ka Fai TSE

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Faculty of Agriculture, Kyushu University, JAPAN

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Center for Promotion of International Education and Research, Faculty of Agriculture,

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Kyushu University, Fukuoka, JAPAN

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Dr. TSE Ka Fai William WKFT: [email protected]

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Tel: +81-092-642-7046

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Fax: +81-092-642-2804

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Keywords: palatogenesis, neural crest cell, ethmoid plate, MAPK pathways

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Correspondence to:

ACCEPTED MANUSCRIPT ABSTRACT:

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Deconjugation of ubiquitin and/or ubiqutin-like modified substrates is essential to

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maintain a sufficient free ubiquitin within the cell. Deubiquitinases (DUBs) play a key

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role in the process. Besides, DUBs also play several important regulatory roles in

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cellular processes. However, our knowledge of their developmental roles are limited.

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The report here aims to study their potential roles in craniofacial development. Based

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on the previous genome-wide study in 2009, we selected 36 DUBs to perform the

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morpholino (MO) knockdown in this study, followed by the Alcian blue cartilage

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staining at 5 days post-fertilization (dpf) larvae to investigate the facial development.

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Results classified the tested DUBs into three groups, in which 28% showed

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unchanged phenotype (Class 1); 22% showed mild changes on the branchial arches

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(Class 2A); 31% had malformation on branchial arches and ethmoid plate (Class 2B);

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and 19% had severe changes in most of the facial structures (Class 3). Lastly, we used

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uchl3 morphant as an example to show that our screening data could be useful for

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further functional studies. To summarize, we identified new craniofacial

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developmental role of 26 DUBs in the zebrafish.

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ACCEPTED MANUSCRIPT Introduction

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Ubiquitylation is an important mechanism in regulating numerous critical cellular

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processes, such as signal transduction, transcriptional control, protein degradation,

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epigenetic modification and intracellular localization. The ubiquitin conjugation

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process includes E1, E2, and E3 enzymes [1]. Discovery of deubiquitylating enzymes

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(DUBs) for deconjugation of ubiquitin modified protein substrates is essential to

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recycle both the substrate proteins and the ubiquitin molecules [2]. In the human

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genome, 95 DUBs were identified and have been classified into five classes [3].

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While in zebrafish, we have identified 85 DUBs, and most of them are highly

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conserved to humans [4].

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In 2009, our group has performed the first genome-wide DUBs in vivo knockdown

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screening by using antisense oligonucleotide morpholino (MO) technology to

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temporarily knock down DUBs in zebrafish embryos. Out of 85 DUBs, 57 DUBs

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(67%) are essential in development. In the knockdown study, DUBs morphants

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showed abnormal phenotypes at the early developmental stage, affecting the

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development of multi-organs and regions, such as head, brain, eyes, body axis,

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notochord, precardial region, gut, and tail. Using the standard in situ hybridization

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method, we further four DUBs are related to the BMP signalling pathway and are

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essential in dorsoventral patterning in zebrafish [4].

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limitation of the DUB studies may be due to the complex regulatory mechanism of

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DUBs. It is generally believed that a single DUB can regulate numerous E3 ligases.

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Up to date, there are about 100 DUBs in different species (human, mouse, zebrafish);

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when comparing to the large number of E3 ligases in the cell, it is reasonable to

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predict that one DUB must regulate several E3 ligases. The suggestion is supported by

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the multi- protein interaction targets in humans DUB via bioinformatics prediction [5]

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and our functional studies in Cosp6 [6]. Recently, more DUB works were published,

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such as USP4, USP15, and USP25, were identified to play important roles in

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regulating Akt signalling, TGF signalling, and IL-17 signalling [7,8,9]. All these lines

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of evidence have shown the functions and importance of DUBs in regulating different

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signalling pathways. However, the developmental roles of DUBs are not well-known.

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Different from previous report in 2009 that focus on the early development, the report

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here extended the investigation up to 5 days post fertilization (dpf) for craniofacial

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development.

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We selected 38 potential DUBs for the study from our previous screening data, and

ACCEPTED MANUSCRIPT performed the MO knockdown studies. The report generated the first craniofacial

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Alcian blue staining profile of 36 selected DUBs. Results showed that 26 DUBs are

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related to craniofacial development, which worth for further biochemical mechanistic

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studies in the future. Lastly, we selected the uchl3 to perform addition biochemistry

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and in situ hybridization studies to convince the readers that our screening result can

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be further investigated.

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ACCEPTED MANUSCRIPT Methods

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Fish strains, maintenance and morpholino (MO) injection

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The wild-type strain used in the screen was AB line. They were raised and staged as

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described [10]. Throughout the experiments, the embryos were incubated at 28oC.

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All MOs were purchased from Gene Tools , resupended in distilled water to make a

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5mM stock and stored at -20oC. Diluted MOs were injected into one-or two-cell stage

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embryos. Embryos from four different pairs of fish were used for each injection. All

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MO sequences and injection concentration were listed on our previous publication;

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and the MO-induced phenotypes were not due to non-specific effects caused by

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treatment with high MO concentrations [4].

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Screening procedure, Alcian Blue cartilage staining and Whole mount in situ

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hybridization (WISH)

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The screening procedure was based on our previous screening protocol [4,11].

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Injected embryos (including control) were collected and fixed by the 4% PFA at 5 dpf,

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and then followed by the Alcian Blue staining protocol as described [12]. The 5dpf

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larvae were examined based on their structure of ethmoid plate and branchial arches

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(Figure 1). All phenotypes were observed in a dominant feature of injected embryos at

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a non-toxic dose. In addition, uchl3 morphant at 2dpf was collected and fixed for the

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sox9a whole mount in situ hybridization (WISH). Single in situ hybridization was

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performed as described [11].

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100 Human Phospho-MAPK Array and Western blotting analysis

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The MAPK array was performed as manufactory protocol (RnDSystems). Briefly, the

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5dpf embryos were homogenized and resuspended by the lysis buffer. Protein

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concentration was measured by the DC Protein Assay Kit (Bio-read). 200µg protein

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was used in the Array. The detailed array content was listed in the Table 1. The

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Western blotting was performed as described [13]. Western blotting analyses were

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conducted using primary antibodies to human UCHL3 antibody (1:1000; Abcam),

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p-MAPK, MAPK, p-ERK, ERK (1:1000; Cell Singling) [14]. β-actin (1:500;

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Hybridoma bank) was used to serve as the loading control [15]. Corresponding

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secondary antibodies (1:4000) conjugated to with horseradish peroxidase was used.

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Specific bands were visualized using chemiluminescent reagent (Western-lightening

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Plus, Perkin-Elmer Life Sciences).

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ACCEPTED MANUSCRIPT Result

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36 DUBs were selected to undergo craniofacial developmental study

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In 2009, our group has identified 85 DUBs in zebrafish genome and performed the

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first large scale DUBs knockdown screening via the morpholino (MO) approach. In

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the screen, we have examined the morphants’ phenotype up to 2dpf, and performed in

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situ hybridization staining to demonstrate several DUBs are involved in the Notch or

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Bmp pathway [4]. Through the study, we confirmed the importance of DUBs during

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early development [4]. However, their roles in later development are not well known.

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From the screening, we found some DUB morphants had strange head phenotype at

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2dpf, thus we suspected that some DUBs may play roles in the craniofacial

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development. 36 DUBs were selected to perform the second round screening in this

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study that focus on the craniofacial development.

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Screening identified DUBs affecting craniofacial development

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36 selected DUB MOs were injected to the embryos. The screening strategy was

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based on the Alcian blue cartilage staining at 5dpf. The pharyngeal skeleton of the

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zebrafish consists of 7 pharyngeal. Based on the staining on these pharyngeal (P1:

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Meckel’s cartilage and palatoquadrate; P2: ceratohyal; P3-P7: ceratobranchial), and

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the ethmoid plate (ep) structure (Figure 1A), we further divided the morphants into 4

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ACCEPTED MANUSCRIPT classes (Table 2). Class 1: No observable change (28%); Class 2A: Mild structural

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changes in P1 to P7 region and ethmoid plate (22%); Class 2B: Structural changes in

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all region, and obvious misshaped ethmoid plate (31%); and Class 3: Severe structural

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changes in all regions with strongly reduced or loss of ethmoid plate (19%). As MO

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generated a wide range of phenotypes, the classification described here was the

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dominant phenotypes (>70%), showing the representative facial skeleton structure. It

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should be noted that the structure of ethmoid plate determinates the classification of

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class 2A and class 2B. Class 2 morphants showed different degree of P1 to P7

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malformation. The sub-classification of class 2A and class 2B was mainly based on

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the ethmoid plate structure in Alcian blue staining.

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Class 1 DUB morphants showed no observable changes in Alcian blue staining

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We selected the DUBs that based on the strange head shape phenotype from our

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previous screening. Results from this second screen showed that some of them did not

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have any defects in their cartilage structures development. Those DUB morphants

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without any observable changes in Alcian blue staining were classified as Class 1

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(Figure 1B).

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Class 2 DUB morphants showed mild structural changes in P1 to P7 region, in which

ACCEPTED MANUSCRIPT the class 2B has extra misshaped ethmoid plate structure

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All class 2 DUB morphants showed a mild malformation of facial skeleton. The P1 to

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P7 region showed different degree of structural abnormities. Morphants in this group

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has shortened jaw structure, rounded Meckel’s cartilage and unclear P3 to P7

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structures. In class 2A morphants, their ethmoid plate structures are relatively normal

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when compared to the class 2B morphants’ (Figure 2). The palatogenesis progress in

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class 2B morphants was clearly affected, showing the gap in the midline ethmoid

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plate (Figure 3).

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Class 3 DUB morphants had severe facial phenotypes with extremely short

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pharyngeal arches and absence of ethmoid plate

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Class 3 DUB includes 7 members, which includes DUBs from all five families (USP:

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Usp24, Usp25, Usp48; UCH: Uchl3; OUT: Vcpip; JAMM: Cops6; and MJD: Josd1.

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All the morphants showed the extreme malformation of the facial skeleton. Missing or

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extremely short of ethmoid plate is the most obvious feature in this class. Furthermore,

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greatly reduced jaw structure was found in these morphants (Figure 4).

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Class 3 uchl3 morphant showed abnormal sox9a expression at 48 hours post

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fertilization

ACCEPTED MANUSCRIPT As class 3 DUB morphants showed sever ethmoid plate defect, which may due to the

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abnormal neural crest cell expression. We selected the Class 3 uchl3 morphant to

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examine a common CNCC in situ marker, sox9a, at 48hpf. sox9a is used to mark the

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neural crest chondrogenesis in cartilage [16]. Results clearly showed that the uchl3

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morphant displayed a disturbed and reduced expression when compared to the control

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(Figure 5A).

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Class 3 uchl3 morphant upregulated the MAPK pathway

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To understand if uchl3 regulates the MAPK pathway, a MAPK array was used to test

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various MAPK phosphorylations (Table 1). Result showed the activation of MAPK

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phosphorylation in the uchl3 morphant (Figure 5B). Western blotting analysis of

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p-p38 and p-ERK were done to confirm the array data (Figure 5C).

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ACCEPTED MANUSCRIPT Discussion

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Deubiquitylating enzymes play multifunctional roles in different signalling pathways.

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Although large scale siRNA screen or overexpression in cell line or yeast had been

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performed [5,17], the developmental functions of DUBs in vertebrate are still remain

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unclear. Zebrafish, as an external fertilization species and excellent developmental

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model, provides a more easy and efficiency way to study the unknown developmental

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function of DUBs. Furthermore, its well-established genome database and the high

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degree of conservation to human, further shape it as an excellent in vivo

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high-throughput, scalable model [18,19]. We have successfully used zebrafish to

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screen for DUBs’ developmental roles by morpholino knockdown in 2009, which

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provided the first evident that DUBs are important in development [4]. After years,

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we here performed the second round screening that focus on the craniofacial

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development. The high occurrence birth defect rate (1/700) of clefts of the lip and/or

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palate (CLP) in the world [20] leads us to investigate if the DUBs have roles in the

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facial development. Vertebrate craniofacial architecture is formal by cranial neural

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crest cells (CNCCs). Migration and morphogenesis of CNCCs are critical for the

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formation of the facial skeleton [21]. In humans, the palate serves to separate the nasal

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and oral cavities. The defect in palate formation will result in CLP [22]. Numerous of

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reports following the craniofacial defect mutants identified from the zebrafish

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ACCEPTED MANUSCRIPT large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screening in 1996 have

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successfully demonstrated that zebrafish is a good model to study craniofacial

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development [23]. Furthermore, the ethmoid plate in zebrafish is embryologically

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analogous to the mammalian palate [24,25], which provides another excellent reason

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to use zebrafish in this study. Recent study showed that the CUL3-KBTBD8 ubiqutin

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ligase are related to the facial malformation disease called the Treacher Collin

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Syndrome [26], which further suggested that the DUB might involve in facial

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development [27].

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Here, based on the published database in 2009, 36 DUBs were chosen for further

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studies.

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point that the basic head structure is formed. Results allowed us to divide the DUB

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morphants into 3 classes, in which 10 DUB morphants showed normal Alcian blue

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staining and 26 morphants showed different degree of malformation of craniofacial

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structure. Class 2 DUBs were further sub-classified into two classes: class 2A and

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class 2B. They all showed strange structure in the lower jaw with missing or fused

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branchial arches (P3 to P7). Class 2A morphants had less severe ethmoid plate defect

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than class 2B. A clear gap can be found in the midline of the ethmoid plate in most of

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the class 2B morphants. In addition, class 2B morphants had a relatively rounded P1

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All the morphants were screened by Alcian blue staining at 5dpf, a time

ACCEPTED MANUSCRIPT and P2 structure. Class 3 DUB morphants were clearly destined from the other classes,

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which they all showed missing or extremely shorten ethmoid plate, together with the

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reduced lower jaw structure. Facial formation required strict signal regulation, in

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which signalling pathways such as sonic hedgehog (Shh) and Wnt, regulate

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palatogenesis [28,29]. Besides typical developmental signalling pathways, some

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studies have shown the miRNAs could affect facial development as well. miR140

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inhibits plate-derived growth factor receptor alpha (pdgfra), which affect the CNCCs

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migration and results in a cleft between the lateral elements of the ethmoid plate [30].

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For the lower jaw development, Edn, Notch, and Bmp pathways showed critical

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functions on the process [31,32] . In order to demonstrate that the screening result can

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be use for further studies, we select the Class3 uchl3 morphant for some functional

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studies. uchl3 was showed to be involved in the Notch signaling [4], which the

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cross-talk between the Notch and Edn signaling is critical in facial formation [31]. We

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then used the sox9a in situ probe to label the CNCCs expression patterns. Results

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showed that the CNCCs expression was altered in the uchl3 morphant. The formation

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of jaw structure and palatogenesis is tightly regulated. Jaw and branchial arches form

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the basic, segmented feature of the vertebrate head, which the migration of neural

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crest cell plays the critical role on such development [24]. Evident have shown that

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migration of CNCCs is critical for palatogenesis and the formation of all components

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ACCEPTED MANUSCRIPT of facial form [21,33]. Thus the original cause of the facial malformation in uchl3

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morphant may be due to the misexpression of CNCCs in the early development. In

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addition, the sox9a probe provided information on the neural crest chondrogenesis in

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cartilage. Chondrocyte differentiation, cartilage formation and maturation are

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regulated by the MAPK signaling during development [34,35] . In order to provide a

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new insight of the craniofacial developmental role in uchl3, we applied the

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Phospho-MAPK array to confirm if MAPK pathway is affected in the morphant.

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Results showed that the reduced of Uchl3 will lead to the activation of various

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MAPKs, and thus might alter the chondrogenesis progress, and finally lead to the

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facial malformation. Nevertheless, the detailed developmental network among Uchl3,

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MAPK, and Notch signaling needs further studies.

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To this end, our primary objective of this study is to provide update information of

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DUB in development. Our screening results clearly showed that some DUBs are

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important in craniofacial development. Although it is highly recommended that the

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researchers should further confirm the phenotype, our further study on the uchl3

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demonstrated that the screening result could be further used in the biochemical studies

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to unfold the detailed mechanism.

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Competing interests

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The authors declare that they have no competing interests.

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266 Acknowledgements

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This craniofacial research work in the laboratory was funded by JSPS Joint Research

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Project.

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ACCEPTED MANUSCRIPT Figure Legends

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Figure 1. Alcian blue staining of class 1 DUB morphants at 5dpf. (A). General

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cartilage structure of a 5dpf zebrafish head showing the criteria for classification. (B)

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Alcian blue staining on AB-wildtype and Class I DUB morphants at 5dpf.

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Abbreviations: P1: Meckel’s cartilage and palatoquadrate; P2: ceratohyal; P3-P7:

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ceratobranchial, and ep: ethmoid plate.

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Figure 2. Alcian blue staining of class 2A DUB morphants at 5dpf. Eight DUB

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morphants showed class 2A phenotypes, which showed a mild malformation of facial

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skeleton. The P1 to P7 region showed different degree of structural abnormities.

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Morphants in this group has shortened jaw structure, rounded Meckel’s cartilage and

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unclear P3 to P7 structures.

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Figure 3. Alcian blue staining of class 2B DUB morphants at 5dpf. 11 DUB

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morphants were classified into the class 2B. In addition to the class 2A phenotype, the

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ethmoid plate structures are malformed, which the midline structure was separated

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(red arrow).

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Figure 4. Alcian blue staining of class 3 DUB morphants at 5dpf. Seven DUB

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morphants were grouped in class 3. They all showed severe facial malformation, with

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an obvious missing or extremely short ethmoid plate phenotype (red dotted circle).

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292 Figure 5. General biochemistry studies of class 3 uchl3 morphants. (A) Decreased

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sox9a expression in the ethmoid plate (red arrow/ red asterisk) and pharyngeal arch

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(red arrow head) in uchl3 morphant, indicating the CNCC localization is affected.

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Abbreviations: ep: ethmoid plate; pa: pharyngeal arches. (B) Phospho-MAPK array

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showed a general activation of different MAPK in uchl3 morphant. (C) Confirmation

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of the array result by the Western blotting in the 5dpf uchl3 morphant. Activation of

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p-p38 and p-ERK was found in the morphant.

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palatogenic gene program in zebrafish, Dev Dyn 240 (2011) 2204-2220. [25] T.F. Schilling, P. Le Pabic, Fishing for the signals that pattern the face, J Biol 8 (2009) 101.

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Restricts Cartilage Formation in the Zebrafish Upper Face, PLoS Genet 12 (2016) e1005967.

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84 (2008) 131-154.

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Target/ Control Reference Spots Reference Spots Akt1 Akt2 Akt3 Akt pan CREB ERK1 ERK2 GSK-3α/β GSK-3β HSP27 JNK1 JNK2 JNK3 JNK pan MKK3 MKK6 MSK2 p38α p38β p38δ p38γ p53 p70 S6 Kinase RSK1 RSK2 TOR PBS Reference Spots

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Coordinate A1, A2 A21, A22 B3, B4 B5, B6 B7, B8 B9, B10 B11, B12 B13, B14 B15, B16 B17, B18 B19, B20 C3, C4 C5, C6 C7, C8 C9, C10 C11, C12 C13, C14 C15, C16 C17, C18 D3, D4 D5, D6 D7, D8 D9, D10 D11, D12 D13, D14 D15, D16 D17, D18 D19, D20 E19, E20 F1, F2

Phosphorylation Site detected / / S473 S474 S472 S473, S474, S472 S133 T202/Y204 T185/Y187 S21/S9 S9 S78/S82 T183/Y185 T183/Y185 T221/Y223 T183/Y185,T221/Y223 S218/T222 S207/T211 S360 T180/Y182 T180/Y182 T180/Y182 T183/Y185 S46 T421/S424 S380 S386 S2448 / /

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Table 1 - Phospho-MAPK Array coordinates

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Table 2 – 36 DUBs were classified into four classes

Members

1

Usp3, Usp4, Usp19, Usp28, Usp33, Usp43, Usp45, Usp53, Eif3hb,

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Class

Mpnd

Usp7, Usp21, Usp36, Usp37, Mysm1, Psmd7, Uchl5, Josd2

2B

Usp5, Usp15, Usp18, Usp20, Usp39, Usp44, Bap1, Cyld, Cyldb,

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Eif3f, Tnafip

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Usp24, Usp25, Usp48, Vcpip, Cops6, Josd1, Uchl3

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3

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A ep P2 P3-7

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P1

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P3-7

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P1

P2

Figure 1

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Lateral

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usp7

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usp21

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usp36

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psmd7

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mysm1

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usp37

uchl5

josd2

Figure 2

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Lateral

Ventral

usp5

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usp15

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usp18

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usp20

usp44

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bap1

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usp39

cyld

cyldb

eif3f tnafip Figure 3

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Lateral

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usp24

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usp25 usp48

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vcpip

cops6

Ventral

josd1

uchl3

Figure 4

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A

sox9a (2dpf)

Ctrl

ep

ep

pa

uchl3 MO

pa

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p-ERK

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*

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uchl3 Ctrl MO p-p38 p38 p-ERK ERK Uchl3

p-p38

β-Act p-ERK

uchl3 MO

p-p38

Figure 5