New Results

Cdon is required for organ Left-Right patterning via regulating DFCs migration and the sequential ciliogenesis

Zhilin Deng, Wenqi Chang, Chengni Li, Botong Li, Shuying Huang, Jingtong Huang, Ke Zhang, Yuanyuan Li, Xingdong Liu, Qin Ran, Zhenhua Guo, Sizhou Huang
doi: https://doi.org/10.1101/2024.03.12.584572

Abstract

Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they were reported to be involved in muscle and neural development at late developmental stage, while their early roles in embryonic development are unknown. Here we discovered that zebrafish cdon but not boc was expressed in dorsal forerunner cells (DFCs) and epitheliums of Kupffer’s vesicle (KV), implying the possible role of cdon in organ LR patterning. Further data showed that the liver and heart LR patterning was disturbed in cdon morphants and cdon mutants. Mechanically, we found that cdon loss of function led to dispersed DFCs migration, smaller KV and defective ciliogenesis, which resulting in randomized Nodal/spaw signaling and the sequential organ LR patterning defect. Finally, predominant distribution of a cdon MO in DFCs led to defects in DFCs migration, KV morphogenesis/ciliogenesis, Nodal/spaw signaling and organ LR asymmetry, being similar to those in cdon morphants and cdon-/- embryos, indicating a cell-autonomous role of cdon in regulating KV formation and ciliogenesis during LR patterning. In conclusion, our data demonstrated that, during gastrulation stage and early somitogenesis stage, cdon is required for proper DFCs migration, KV formation and ciliogenesis, thus playing an important role in setting up organ LR asymmetry.

1. Introduction

Establishment of left-right patterning (LR Patterning) is an important event in early embryonic development, organ asymmetry defect is closely related to the occurrence of various congenital diseases such as congenital heart disease and schizophrenia (Gabriel and Lo, 2020; Huang et al., 2014). Although the mechanisms of establishment of organ LR patterning are complex and diverse in different animal models (Hamada and Tam, 2020; Onuma et al., 2020; Zhu et al., 2020), the role of Kupffer’s vesicle (KV)/Node-Cilia axis in zebrafish and mice is extremely conservative (Forrest et al., 2022; Grimes and Burdine, 2017; Little and Norris, 2021). In zebrafish, the normal development of KV precursor cells (Named dorsal forerunner cells (DFCs) and KV morphogenesis/Ciliogenesis play crucial roles in initiating left-sided signals in the embryonic lateral plate mesoderm (LPM) (Grimes and Burdine, 2017). In organ LR patterning, DFCs proliferate rapidly during gastrulation movement (Abdel-Razek et al., 2023; Gokey et al., 2015), and after bud stage, DFCs differentiate to perform KV and produce cilia in KV, which oscillating to produce a counter-clockwise liquid flow and initiate the specific asymmetric Nodal/spaw on the LPM (Bakkers et al., 2009; Liu et al., 2019). Further, the left-sided Nodal/spaw and its downstream genes pitx2 and lft2 are amplified in the LPM, and the embryonic Midline (such as shh and ntl) also impedes the expansion of left-sided Nodal signals to the right side of embryo, thus making the left-sided asymmetric Nodal signals more stable (Burdine and Grimes, 2016). Finally at the late stage of organ development, the left-sided Nodal directs a asymmetric migration of organ precursors to establish organ asymmetry patterning (Kajikawa et al., 2022).

During organ LR patterning, the role of zebrafish KV morphogenesis/ciliogenesis and the underlying molecular mechanisms have been received extensive attention. It has been found that most of classical signaling pathways such as Wnt and Fgf (Caron et al., 2012; Neugebauer et al., 2009; Xu et al., 2010; Zhang et al., 2012) and many other genes such as Rab5 (Kuhns et al., 2019) and FOR20 (Xie et al., 2019) participated in KV morphogenesis or ciliogenesis. Our previous studies found that, during zebrafish gastrulation, inhibition of retinoic acid (RA) increased the expression of fgf8 in DFCs, leading to longer cilia and cilia motility disorders (Huang et al., 2011). More recently, we found that the chemokine receptor cxcr4a is highly expressed in DFCs, and the expression of cxcr4a promotes CyclinD1 expression to regulate ciliogenesis via regulating the phosphorylation of ERK1/2 (Liu et al., 2019). Although many genes were reported to play crucial roles during KV morphogenesis and ciliogenesis, the mechanisms underlying this process are far from being elucidated.

Cell-adhension molecule-related/down-regulated by oncogenes (Cdon) and its paralog Boc (Brother of Cdo (Kang et al., 2002)) are two members of cell adhesion molecule subfamily III Ig/fibronectin (Jeong et al., 2014; Sanchez-Arrones et al., 2012), they were reported to synergistically or individually regulate different organs development or disease(Jeong et al., 2014; Jeong et al., 2017; Lencer et al., 2023; Sanchez-Arrones et al., 2012; Zhang et al., 2011). As to Cdon, early reports showed that mouse Cdon regulates skeletal muscle and central nervous system development via Shh and N-cadherin/cell adhesion (Cole et al., 2004; Jeong et al., 2014; Zhang et al., 2006a; Zhang et al., 2006b). More detailed studies have shown that Cdon binds to N-cadherin and induces p38/MAPK signals to guide cell differentiation and apoptosis in the process of muscle formation and neural differentiation (Lu and Krauss, 2010). In zebrafish, cdon was reported to negatively regulate Wnt signaling pathway during forebrain development to promote neural differentiation and ventral cell fate determination via interacting with LRP6 (Jeong et al., 2014). Further, researchers also found that the localization of Cx43 protein in cells was disorganized in Cdon mutants, resulting in cardiac structural and functional lesions (Jeong et al., 2017). As to Boc, early reports showed that it plays a role in axon guidance and neuron growth (Connor et al., 2005; Okada et al., 2006). Recently, Lencer et al found that in zebrafish cdon and boc double mutants display trunck neural crest cells (tNCC) migratory defects and loss of slow-twitch muscle, suggesting a non-cell autonomous role of cdon and boc in regulating tNCC migration (Lencer et al., 2023). Although Cdon and Boc were reported to play important roles in many sections in mouse and zebrafish, their roles in early embryonic development are unknown. During early embryonic development in zebrafish, our data showed that cdon, but not boc, was expressed in DFCs during gastrulation movement and in epithenial cells of KV at early somitogenesis stage (Fig. 1 and Fig. S1). On the other hand, Cdon has also been reported to control cell apoptosis and cell migration in various kinds of cells (Chapouly et al., 2020; Lencer et al., 2023; Sanchez-Arrones et al., 2012). These facts above suggest that cdon may be involved in regulation of clustering movement of DFCs and the sequential organ LR patterning in zebrafish. Here our data identified that cdon regulates organ LR pattering via KV/Cilia-Nodal/spaw cascade in early embryonic development.

Figure 1.
Figure 1. Organ left-right patterning defects in embryos injected with cdon MO at 4-cell stage

(A) The expression of cdon was examined using whole mount in situ hybridization from 128-cell stage to 24hpf. Cdon was expressed at 128-cell stage (Aa1). White arrow head showed the expression of cdon in DFCs at 80% epiboly stage (Aa2). Black arrow head showed the expression of cdon in DFCs at bud stage (Aa3-a4). Cdon was also expressed in midline and presumptive neural crest (Aa5). Black arrow head showed the expression of cdon in epithelial cell in KV at 6ss (Aa6-a7). Expression of cdon was examined at 24hpf (Aa8). (B) Embryos injected with cdon MO displayed liver LR defects. b1, normal liver in Tg(fabp10:GFP) transgenic controls (93.1%, n=102); b2, normal liver in embryos injected with cdon MO (60.8%,n=79); b3, liver bifida in embryos injected with cdon MO (25.3%,n=79); b4, reversed liver in embryos injected with cdon MO (13.9%,n=79). b5, normal liver in wild type controls (93.3%,n=75); b6-b8, embryos injected with cdon MO were examined at 4dpf using in situ experiments (n=84). (C) Percentages of normal liver, liver bifida, and reversed liver in embryos used as control, embryos injected with cdon MO and embryos co-injected with cdon MO and cdon mRNA, in respectively. (D) Embryos injected with cdon MO displayed heart LR defects. d1, normal-loop in Tg(cmlc2:GFP) transgenic controls (97.4%,n=77); d2, normal-loop in embryos injected with cdon MO (56.8%, n=95); d3,no loop in embryos injected with cdon MO (16.8%, n=95); d4, reversed-loop in embryos injected with cdon MO (26.4%, n=95); d5, normal-loop in wild type controls (94.5%,n=73); d6-d8, wild-type embryos injected with cdon MO at 4-cell stage were examined for cardiac looping at 72hpf by WISH against cmlc2 (n=83). (E) Percentages of normal looping, no looping, and reversed looping of the heart in embryos being as control, embryos injected with cdon MO, and embryos co-injcted with cdon MO and cdon mRNA. Cdon mRNA injection partially rescued the heart LR defect in embryos injected with cdon MO (E, the right column showing). Notice: in “C” and “E’, all the transgenic embryos and wild type embryos were used together to calculate the percentage.

2 Methods and materials

2.1 Zebrafish

Zebrafish wild type (AB), Tg (cmcl2 :GFP)(Liu et al., 2022), Tg (fabp10 :GFP)(Liu et al., 2022) and Tg (sox17 :GFP)(Zhu et al., 2019) lines were maintained at 28.5°C. The embryonic stage was determined according to the external morphology as the standard criteria described before (Kimmel et al., 1995).

2.2 Whole-Mount in situ hybridization (WISH)

Embryos used for Whole-Mount in situ hybridization (WISH) were collected at desired stages and fixed in 4% PFA at 4°C overnight, then the embryos were washed with PBST (2 times, 5min each), dehydrated with MeOH (100%) and stored in MeOH at −20°C.The WISH follows the experimental procedure described previously (Huang et al., 2011; Zhu et al., 2019). The antisense probes my17, fabp10, spaw, lefty1, lefty2, sox17, sox32 were prepared as previously described (Zhu et al., 2019). To prepare cdon antisense probe, the CDs of cdon were amplified using PCR, then it was cloned into pCS2+ vector. The detailed process could be find in the section “Plasmid Construction”. Then this construct was used as the template to synthesize cdon antisense probe according to previous protocol (Zhu et al., 2019).

2.3 Immunostaining

The immunostaining was performed as previous report (Zhu et al., 2019). Briefly, the embryos were fixed with 4% PFA at 4□ overnight, then dehydrated with methanol gradiently and stored at -20□. After methanol /PBST gradient rehydration, PBTN (4% BSA, 0.02%NaN3, PT) was added and the embryos were incubated at 4□ for 3 h, then the α-tubulin antibody (Sigma, T7451, diluted with PBTN at 1:100) was added and incubated overnight on a shaker at 4°C. Next day the embryos were washed with PT (0.3% triton-X-100, in 1X PBS) 4 times (30min each), and the second antibody Goat anti-Mouse IgG(H+L) Alexa Fluor 594 (Sigma, SAB4600105) was added (diluted with PBTN at 1:1000) for overnight incubation (in darkness). Finally, the embryos were rinsed with PT for more than 6 times (30min each) and the imaging was performed.

2.4 Plasmids Construction

Total RNA was extracted from zebrafish embryos at 24hpf according to the reagent instructions (TRIzol, Ambion No.15596-026). cDNA was prepared using Revert Aid First Strand cDNA Synthesis Kit (Fermentas No.K1622) according to the manufacturer’s instructions. The CDs of cdon was amplified using PCR (Prim STAR Max Premix Takara NO.R045A) and cloned into the vector PCS2+ to generate the expression constructs (5x In-Fusion HD Enzyme Premix, Takara

NO.639649). The primers for cloning were as following:

PCS2+_F: 5′-CTCGAGCCTCTAGAACTATAGTG-3′,

PCS2+_R: 5′-TGGTGTTTTCAAAGCAACGATATCG-3′,

Cdon_F: 5′-TCTTTTTGCAGGATCCGTGAAACAGCGTCATGGAGGAC-3′

Cdon_R: 5′-GTTCTAGAGGCTCGAGTGTTCAGATCTCCTGCACGGT-3′

2.5 MO and mRNA injection

The antisense cdon morpholino (cdon MOs) was synthesized (Gene Tools) and applied to knock down cdon (cdon MO (MOatg), 5’ -ATAATCTCAGGCCACCGTCCTCCAT-3, 400 uM) (Jeong et al., 2014). Cdon MO was injected into the yolk of zebrafish embryos at 1-4 cell stage to block the translation of cdon in whole embryos, or at 256-512 cell stage to specifically block the translation of cdon mRNA in DFCs. The cdon mRNA was synthesized in vitro using mMESSAGE Kit (AM1340, Ambion). In rescue experiments, the concentration for cdon mRNA injection was 15ng/ul.

2.6 Imaging

Images of the Whole-Mount in situ hybridization (in 100% glycerol) were taken with OLYMPUS SZX16 at room temperature. The live embryos of transgenic line Tg (sox17 :GFP) were placed in 1% Low-Point Melting agrose (LPM agrose) and DFCs were photographed by microscope (OLYMPUS SZX16). To get the images of cilia for immunostaining embryos, the embryos were adjusted with right orientation in 1% LPM agrose and then the cilia were photographed using confocal microscope (OLYMPUS Fluview FV1000).

2.7 Statistical analysis

The data were statistically analyzed by Graphpad Prism 9 and ImageJ software. The length of cilia was measured by confocal microscope (OLYMPUS Fluview FV1000). The statistical results were the mean ± SEM of three independent experiments.

2.8 Ethics Statement

The study was approved by the Institutional Review Board of Chengdu Medical College (SYXK(□)2015-196), and Zebrafish were maintained in accordance with the Guidelines of Experimental Animal Welfare from Ministry of Science and Technology of People’s Republic of China (2006).

3. Results

3.1 cdon loss of function leads to heart and liver LR patterning defect in cdon morphants

In zebrafish, cdon and boc had been reported to being involved in trunk neural crest cells (NCs) migration and correct proximo-distal patterning in eye development (Jeong et al., 2014; Lencer et al., 2023), demonstrating the crucial role of cdon in zebrafish organ development. Since Cdon and Boc synergistically regulate NCs migration (Lencer et al., 2023), facial and eye development (Zhang et al., 2011), to study the role of cdon in more early embryonic development, we examined the detailed expression pattern of cdon and boc from 128-cell stage to 24hpf. The data showed that cdon is a maternal factor which distributing in cells unequally (Fig. 1Aa1). During gastrulation movement, beside its expression in the presumptive neural crest and midline (Fig. 1Aa5, (Jeong et al., 2014)), cdon was also highly expressed in DFCs (Fig. 1a2, a4). At 6 somite stage, cdon was enriched in the epitheliums of KV (Fig. 1A5-6). On the contrary, even though boc was maternally and ubiquitously expressed during gastrulation (Fig. S1A, B), but we did not find boc was enriched in DFCs and epitheliums of KV (Fig. S1D-F). Since disturbing DCFs development and the sequential KV/Ciliogenesis leads to organ LR patterning defects (Xie et al., 2019; Zhu et al., 2019), we proposed the possibility that cdon is involved in organ LR patterning. To preliminarily and rapidly evaluate this hypothesis, an ATG MO for cdon was used to down-regulate the function of cdon (Jeong et al., 2014), then organ LR patterning was examined. The results showed that, in Tg(fabp10:GFP) transgenic embryos, injection of cdon MO did not lead to embryonic defect (Fig. S2), but gave rise to liver LR patterning defect (Fig. 1Bb1-b4, C). Part of embryos injected with cdon MO displayed liver bifida (Fig. 1Bb3) and right-sided liver (Fig. 1Bb4). Being similar, in wild type embryos, injection of cdon MO also led to liver LR patterning defect (Fig. 1Bb5-b8, C). Next, we examined whether injection of cdon MO lead to heart looping defect. The results showed that injection of cdon MO caused heart LR patterning defect (Fig. 1D, E), many of cdon morphants displayed linear heart (Fig. 1Dd3,d7, E) and reversed heart looping (Fig. 1D4,8, E). To further confirm the role of cdon in liver and heart LR patterning, finally we evaluated whether injection of cdon mRNA could rescue the organ LR patterning defect in cdon morphants. The data showed that injection of cdon mRNA partially restored organ LR patterning defect in embryos injected with cdon MO (Fig. S3 and Fig. 1C, E; the right columns showed). These data above indicated that cdon plays a critical role during organ LR patterning in zebrafish.

3.2 left-sided Nodal/spaw cascade is randomized in cdon morphants

The crucial role of Nodal/spaw in LR patterning was proved in previous studies (Kang et al., 2013; Raya and Izpisua Belmonte, 2006; Speder et al., 2007), disruption of Nodal/spaw leads to organ laterality defect (Huang et al., 2011). While in some kinds of mutants with organ LR patterning defect, Nodal/spaw was not disturbed (Huang et al., 2014; Kurpios et al., 2008; Trinh and Stainier, 2004; Yin et al., 2010). To reveal how cdon regulates organ laterality, we examined whether the left-sided Nodal/spaw was disturbed in embryos injected with cdon MO. The data showed that the left-sided Nodal/spaw was disturbed in cdon morphants, displaying both-sided, right-sided and disappeared spaw expression (Fig.2Aa2-a4, B). Furthermore, we sequentially checked lefty1 and lefty2, the downstream genes of spaw in cdon morphants. The data showed that the left-sided expression pattern of lefty2 and lefty1 in the heart field were also perturbed (Fig. 2Cc2-c4,D, Ee2-e4.F). Since cdon was also expressed in midline (Fig. S1Aa5, (Jeong et al., 2014)), we observe whether lefty1 in midline was affected. The data showed that the expression of lefty1 in middle line was not affected (Fig. 2 Cc2-c4, red arrow showed). These data above suggested that the left-sided Nodal/spaw cascade was disturbed in cdon morphants, which may lead to organ LR patterning defect in cdon morphants.

Figure 2.
Figure 2. Expression of left-sided Nodal signaling in controls and embryos injected with cdon MO

(A) Expression of spaw was evaluated in different group of embryos. a1, left-sided spaw in controls (95.9%, n=97); a2, left-sided spaw in embryos injected with cdon MO (37.8%, n=82); a3, bilateral spaw in embryos injected with cdon MO (35.4%, n=82); a4, right-sided spaw in embryos injected with cdon MO (26.8%, n=82).(B) Statistical analysis was performed for the expression of spaw in controls and embryos injected with cdon MO. (C) Lefty1 is expressed in the heart field (black arrow) and trunk midline (red arrow) in controls and embryos injected with cdon MO. c1, left-sided lefty1 in embryos being as controls (96.2%,n=78, black arrow showed); c2, left-sided lefty1 in embryos injected with cdon MO(53.4%, n=73, black arrow showed); c3, absent lefty1 in embryos injected with cdon MO (34.2%,n=73); c4, right-sided lefty1 in embryos injected with cdon MO (12.4%,n=73). (D) Statistical analysis was performed for the expression of lefty1 in controls and embryos injected with cdon MO. (E) Lefty2 is examined in control embryos and embryos injected with cdon MO. e1, left-sided lefty2 in controls (96.1%, n=77); e2, left-sided lefty2 in embryos injected with cdon MO (61.2%, n=67); e3, bilateral expression of lefty2 in embryos injected with cdon MO (26.9%,n=67); e4, right-sided lefty2 in embryos injected with cdon MO (11.8%,n=67). (D) Statistical analysis was performed for the expression of lefty2 in controls and embryos injected with cdon MO.

3.3 Clustering DFCs migration, KV morphogenesis and ciliogenesis are disturbed in cdon morphants

Our current research showed that cdon was expressed in the DFCs at gastrulation stage and in the epithelial cells of KV (Fig. 1Aa2-a7). In zebrafish, DFCs will form the KV at early somite stage, as well the defective KV morphogenesis or defective ciliogenesis will give rise to disturbed left-sided Ndoal/spaw and the subsequent organ LR defect (Essner et al., 2005; Long et al., 2003; Wang et al., 2011). To find out whether cdon regulates DFCs development, KV morphogenesis or ciliogenesis, we analyzed the expression of DFCs marker sox17 and sox32, KV morphogenesis and ciliogenesis in cdon morphants. Compared with that in control embryos, at 80% epiboly stage, the clustering DFCs migration was disturbed in cdon morphants (Fig. 3 Aa2-a3, Aa5-a6, B). At 10-13 SS the KV was smaller in majority of cdon morphants (Fig.3 Cc3, D), and a small part of morphants displayed tiny/absent KV (Fig. 3Cc4, D). Further, we examined the cilia development and found that cdon morphants displayed mild shorter cilia than that in controls (Fig.3 Ee2-e4, F), the cilia number was also decreased in cdon morphants (Fig.3 Ee2-e4, G). These results showed that the normal expression of cdon is required for DFCs migration, KV morphogenesis and ciliogenesis from gastrulation stage to early somitogenesis stage.

Figure 3.
Figure 3. Cdon loss of function disturbs clustering DFCs migration, KV morphogenesis and ciliogenesis

(A) Expression of sox17 and sox32 was examined using WISH at 80% epiboly (white arrow). a1, normal expression of sox17 in controls (96.6%, n=58); a2, normal expression of sox17 in embryos injected with cdon MO at 4-cell stage (46.8%, n=62); a3, dispersed expression of sox17 in embryos injected with cdon MO at 4-cell stage (53.2%, n=62). a4, normal expression of sox32 in controls (100%, n=57); a5, normal expression of sox32 in embryos injected with cdon MO at 4-cell stage (52.2%, n=67); a6, dispersed expression of sox32 in embryos injected with cdon MO at 4-cell stage (47.8%, n=67). (B) Statistical analysis was performed for the expression of sox17 and sox32 in controls and embryos injected with cdon MO. Here all the embryos staining with sox17 or sox32 were used together to calculate the percentage. (C, D) KV morphology was evaluated. c1, normal KV in embryos in controls (94.3%, n=70); c2, normal KV in embryos injected with cdon MO at 4-cell stage (17.8%, n=62); c3, smaller KV in embryos injected with cdon MO at 4-cell stage (71.0%, n=62); c4, tiny/absent KV in embryos injected with cdon MO at 4-cell stage (11.2%, n=62). (E) Number and length of cilia were evaluated in controls and embryos injected with cdon MO. e1, cilia in embryos being as controls; e2-e4, cilia in embryos injected with cdon MO at 4-cell stage. (F) Statistical chart for cilia length in KV. (G) Statistical chart for cilia number in KV. “*”p<0.05,“***”p < 0.001.

3.4 Cdon mutation leads to organ LR patterning defect

To confirm the role of cdon in organ LR patterning, we generated a cdon mutant line using CRISPR-Cas9 method (Shankaran et al., 2017). To generate the cdon mutant, we selected a specific sequence in the exon3 of cdon as the target sequence (Fig. 4A). As the result, in F1 adults we screened out a frame shift mutation line (Fig. 4B, C). In this mutation, the sequence "AAGGGC" in the exon3 of cdon gene was changed to "TTGATGAATGGGGG" (Fig. 4B, C), which resulting in a truncated Cdon protein (only 104 amino acids) (Fig. 4C). In addition, even though we found the expression of cdon mRNA was greatly downregulated in cdon-/-embryos at 8 somite stage and 24hpf (Fig. S4C, D), the cdon-/- embryos have no distinct external phenotype at different stages (Fig. S4 A, B) and could grow up to adults. Then we evaluated whether this frame shift mutation leads to liver and heart LR patterning defect using in situ experiments. The data showed that cdon-/- embryos, but not control embryos (Fig. 4Dd1), displayed liver LR patterning defect: 71.2% of cdon-/-embryos displayed left-sided liver (Fig. 4Dd2, E), 17.8% of cdon-/-embryos displayed bilateral liver (Fig. 4Dd3, E), 11.0% of cdon-/-embryos displayed right-sided liver (Fig. 4Dd4, E). Being similar, there is no heart LR patterning defect was observed in control embryos (97.5%; Fig. 4Ff1, G), but cdon-/- embryos displayed no-loop heart (16.4%; Fig. 4Ff3, G) and reversed loop heart (10.9%; Fig. 4Ff4, G). To further confirm the critical role of cdon in organ LR patterning, we examined whether injection of cdon mRNA could rescue liver and heart LR patterning defect in cdon-/- embryos. The data showed that injection of cdon mRNA partially restored liver and heart LR patterning (Fig. 4E, G). All these data in cdon-/-embryos further demonstrated that cdon is essential for organ LR patterning.

Figure 4.
Figure 4. Organ LR patterning defects in cdon-/-embryos

(A) CRISPR/Cas9 target was showed. The sequence in the exon3 of cdon gene was chose as the target (yellow highlighted). (B) Sequencing results for the genomic DNA in WT embryos, cdon+/- embryos and cdon-/- embryos. (C) Nucleotide sequences highlighted in yellow are CRISPR/Cas9 target. In the cdon mutant, the sequence "AAGGGC" was changed to "TTGATGAATGGGGG". Cdon mutant harbors a frameshift mutation that is predicted to result in the production of truncated Cdon protein (104 amino acids). (D) Cdon-/- embryos were found to display liver LR defects using WISH. d1, left-sided liver in controls (95.5%, n=111); d2, left-sided liver in cdon-/- embryos (71.2%, n=118); d3, liver bifida in cdon-/-embryos (17.8%, n=118); d4, right-sided liver in cdon-/- embryos (11.0%, n=118). (E) Percentages of left-sided liver, liver bifida, and right-sided liver in cdon-/-embryos (n=118), controls (n=111) and cdon-/- embryos injected with cdon mRNA (n=89). (F) Cdon-/- embryos displayed heart LR defects. f1, normal-loop heart in controls (97.5%, n=122); f2, normal-loop heart in cdon-/- embryos (72.7%, n=128); f3, no loop heart in cdon-/-embryos (16.4%, n=128); f4, reversed loop heart in cdon-/- embryos (10.9%, n=128). (G) Percentages of normal looping, no looping, and reversed looping of the heart incdon-/- embryos (n=128), controls (n=122) and cdon-/-embryos injected with cdon mRNA (n=90).

3.5 KV/cilia-Nodal/spaw cascade was also disturbed in cdon mutants

To further confirm the mechanism how cdon regulates organ LR patterning, we also examined whether KV/cilia-Nodal/spaw cascade was affected in cdon-/- embryos. First, we examined the expression of sox17 and sox32 at 80% epiboly stage to evaluate whether the clustering DFCs migration is disturbed in cdon-/- embryos. The data showed that 55.7% of cdon-/ -embryos displayed dispersed expression of sox17 (Fig. 5Aa3, B), 48.0% of cdon-/-embryos displayed dispersed expression of sox32 (Fig. 5Aa6, B). On the contrary, the expression of sox17 and sox32 was normal in control embryos (Fig. 5Aa, Aa4, B). This data indicated that DFCs migration was disturbed in cdon-/ -embryos. Then we evaluated whether KV morphogenesis and ciliogenesis were affected in cdon-/-embryos. The data showed that, in many cdon-/-embryos, the KV became smaller or absent (Fig. 5 Cc3-c4, D). Being similar to that in cdon morphants, in cdon-/-embryos the cilia length is mild shorter (Fig. 5Ee1-e4, F) and cilia number is decreased (Fig. 5Ee1-e4, G). Finally, we examined the expression pattern of spaw in controls and cdon-/- embryos. The data showed that the expression of left-sided spaw was also disturbed in cdon-/- embryos, displaying left-sided spaw (Fig. 5Hh2, I), bilateral spaw (Fig. 5Hh3, I) and right-sided spaw (Fig. 5Hh4, I). In addition, the expression of Nodal/spaw downstream gene lefty1 and lefty2 was also disturbed in cdon-/-embryos (Fig.S5A-D). These data above further confirmed that KV/cilia-Nodal/spaw cascade may mediate cdon to regulate heart and liver LR patterning during early development.

Figure 5.
Figure 5. Cdon mutation gave rise to defects in KV formation and ciliogenesis

(A) Expression of sox17 and sox32 was examined using WISH at 80% epiboly. a1, normal expression of sox17 in controls (96.6%, n = 88); a2, normal expression of sox17 in cdon-/-embryos (44.3%, n = 97); a3, dispersed expression of sox17 in cdon-/-embryos (55.7%, n = 97). DFCs (white arrow). a4, normal expression of sox32 in controls (93.7%, n = 95); a5, normal expression of sox32 in cdon-/-embryos (52.0%, n = 100); a6, dispersed expression of sox32 in cdon-/-embryos(48%, n =100). DFCs (white arrow). (B) Statistical analysis was performed for DFCs clustering migration in controls (n=183) and cdon-/-embryos(n=197). Here all the embryos staining with sox17 or sox32 were used together to calculate the percentage. (C) Morphology of KV in different group of embryos at 10ss. c1,normal KV in controls (98.0%, n =99); c2,normal KV in cdon-/-embryos (41.0%, n =105); c3,smaller KV in cdon-/-embryos (50.5%, n = 105); c4, tiny/absent KV in cdon-/-embryos (8.5%, n =105). (D) Statistical analysis was performed for KV morphogenesis in controls (n=99) and cdon-/-embryos (n=105). (E) Thenumber and the length of cilia were examined at 10ss. e1, cilia in controls; e2-e4, cilia in cdon-/-embryos. (F) Statistical chart for cilia length in KV. (G) Statistical chart for cilia number in KV. “*”p<0.05,“**”p < 0.01. (H) Expression of spaw in controls and cdon-/- embryos. h1, left-sided spaw in controls (96.2%, n=130); h2, left-sided spaw in cdon-/- embryos (71.0%,n=138); h3, bilateral spaw in cdon-/- embryos (18.1%, n=138); h4,right-sided spaw in cdon-/-embryos (10.9%, n=138). (I) Statistical analysis was performed for the expression of spaw in controls (n=130) and cdon-/- embryos (n=138).

3.6 Cdon loss of function in DFCs results in organ LR patterning defects

Our data have showed that cdon was not only expressed in DFCs and KV epithelial cells, but also expressed in some other cells such as cells in midline and PSM (Fig. 1A and Fig. S4 C, D). To confirm whether cdon regulates organ LR patterning via DFCs-KV/cilia-Nodal/spaw cascade specifically, we injected cdon MO at 256-512 cell stage to predominantly block translation of cdon mRNA in DFCs (Amack and Yost, 2004; Zhu et al., 2019), then evaluated whether organ LR patterning was disturbed. Indeed, in the embryos injected with cdon MO at 256-512 cell stage, the liver and heart LR patterning was disturbed (Fig. 6A-D): In both Tg(fabp10:GFP) transgenic embryos (Fig. 6Aa1-a4, B) and wild type embryos (Fig. 6Aa5-a8, B), after predominantly down-regulating the function of cdon in DFCs, the livers were localized in left side, both side and right side, in respectively. In both Tg(cmlc2:GFP) transgenic embryos (Fig. 6Cc1-c4, D) and wild type embryos (Fig. 6Cc5-c8, D), after predominantly down-regulating the function of cdon in DFCs, the hearts displayed normal loop, linear and reversed loop, in respectively. Next, we evaluated whether DFCs-KV/cilia cascade was affected after predominantly down-regulating the function of cdon in DFCs. The data showed that the clustering DFCs migration was disturbed (Fig. S6Aa1-a6, B), the size of KV was smaller (Fig. S6 Cc1-c4, D), the length of cilia was mild shorter (Fig. S6Ee1-e4, F) and the number of cilia was also decreased (Fig. S6Ee1-e4, G). Finally, we continued to evaluate if Nodal/spaw signaling was disturbed in embryos injected with cdon MO at 256-512 cell stage. As result, the expression of left-sided Nodal/spaw and its downstream gene lefty1 and lefty2 was also randomized in embryos injected with cdon MO at 256-512 cell stage (Fig. 6E-J): To the expression of spaw, 44.6% (Fig. 6 Ee2), 40.2% (Fig. 6Ee3) and 15.2%(Fig. 6Ee4) of embryos injected with cdon MO displayed left-sided spaw, both-sided spaw and right-sided spaw, in respectively; while 97.5% of control embryos displayed left-sided spaw (Fig. 6Ee1). To the expression of lefty1, 54.9% (Fig. 6Gg2), 29.7% (Fig. 6Gg3) and 15.4% (Fig. 6Gg4) of embryos injected with cdon MO displayed left-sided lefty1, disappeared lefty1 and right-sided lefty1, in respectively; while 96.5% of control embryos displayed left-sided lefty1. To the expression of lefty2, 60.9.6% (Fig. 6Ii2), 27.6% (Fig. 6Ii3) and 11.5% (Fig. 6Ii4) of embryos injected with cdon MO displayed left-sided lefty2, both-sided lefty2 and right-sided lefty2, in respectively, while 97.2% of control embryos displayed left-sided lefty2 (Fig. 6Ii1). These data indicated that the left-sided Nodal/spaw signaling was randomized after down-regulating the function of cdon in DFCs. In conclusion, all these data above suggested that cdon specifically regulates organ LR patterning via DFCs-KV/cilia-Nodal/spaw cascade.

Figure 6.
Figure 6. Organ left-right patterning defects in embryos injected with cdon MO at the 256-cell stage

(A) Embryos injected with cdon MO at 256-cell stage was found to cause liver LR defects. a1, normal liver in Tg(fabp10:GFP) transgenic controls (96.6%, n=90); a2, normal liver in Tg(fabp10:GFP) transgenic embryos injected with cdon MO at the 256-cell stage (62.2%, n=111); a3, liver bifida in Tg(fabp10:GFP) transgenic embryos injected with cdon MO at the 256-cell stage (27.0%, n=111); a4, reversed liver in Tg(fabp10:GFP) transgenic embryos injected with cdon MO at the 256-cell stage (10.8%,n=111). a5, normal liver in wild type controls (95.1%, n=108); a6-a8,wild-type embryos injected with cdon MO at the 256-cell stage were examined for liver laterality at 4d by WISH against fabp10 probe (n=108). (B) Percentages of normal liver, liver bifida, and reversed liver in control embryos and embryos injected with cdon MO at the 256-cell stage.(C) Heart morphogenesis in Tg(cmcl2:GFP) transgenic embryos and wild-type embryos injected with cdon MO at the 256-cell stage. c1, normal-loop in Tg(cmcl2:GFP) transgenic controls (97.5%, n=80); c2, normal-loop in Tg(cmcl2:GFP) transgenic embryos injected with cdon MO at 256-cell stage (50.5%, n=93); c3, no loop in Tg(cmcl2:GFP) transgenic embryos injected with cdon MO at 256-cell stage (34.4%, n=93); c4, reversed-loop in Tg(cmcl2:GFP) transgenic embryos injected with cdon MO at 256-cell stage (15.1%, n=92); c5,normal-loop in wild type controls (97.2%, n=72); c6-c8,wild-type embryos injected with cdon MO at 256-cell stage were examined for cardiac looping at 72hpf by WISH against cmlc2 (n=110). (D) Percentages of normal looping, no looping, and reversed looping of the heart in all the embryos injected with or without cdon MO at 256-cell stage. (E-F) Expression of Nodal/spaw in controls (n=79) and embryos injected with cdon MO at the 256-cell stage (n=92). (G-H) Expression of lefty1 in controls (n=86) and embryos injected with cdon MO at the 256-cell stage (n=91). (I-J) Expression of lefty2 in controls (n=71) and embryos injected with cdon MO at the 256-cell stage (n=87). Notice: in “B” and “D’, all the transgenic embryos and wild type embryos were used together to calculate the percentage.

4. Discussion

Mouse Node/cilia (referred to as the ‘left-right organizer’ (LRO)) was first identified to play critical role during organ LR patterning (Kajikawa et al., 2022; Schneider et al., 1999; Sulik et al., 1994). Being similar to mouse, the transient structures were identified in other vertebrate embryos such as zebrafish, suggesting a conserved cilia-based mechanism regulates LR patterning in zebrafish (Essner et al., 2002). Indeed, functional studies confirmed the presence of motile cilia and asymmetric fluid flow in Kupffer’s vesicle in zebrafish (Essner et al., 2005) and genetic or embryological perturbation of these ciliated structures disrupted asymmetric Nodal pathway expression and organ laterality (Amack, 2014; Essner et al., 2005; Kuhns et al., 2019; Liu et al., 2019). In zebrafish, the transgenic line Tg(sox17:EGFP) has been developed to label the DFC/KV cells (Liu et al., 2019), and several developmental steps have been identified to build a functional KV/Cilia. DFCs appear at mid-epiboly stages, migrate, proliferate, and then undergo a mesenchymal-to-epithelial transition to form KV in early somite stage (Forrest et al., 2022). KV develops directional fluid flow and establishes LR signaling, and then breaks down around 18 hpf when KV cells undergo a epithelial to mesenchymal transition (Amack, 2021) and migrate away to incorporate into muscle and notochord (Ikeda et al., 2022). In the past decades, much of genes or environmental elements had been reported to involve in DFCs clustering migration (Ablooglu et al., 2010; Gao et al., 2011; Kajikawa et al., 2022; Lai et al., 2012; Liu et al., 2022), proliferation (Abdel-Razek et al., 2023; Gokey et al., 2015; Liu et al., 2019; Zhang et al., 2012) and the final KV formation and ciliogenesis, while the mechanisms underlying this process are from being completely elucidated.

Cdon is cell surface glycoproteins that belong to a subgroup of the immunoglobulin (Ig) super-family of cell adhesion molecules (Sanchez-Arrones et al., 2012). The role of Cdon in organ development and function had been reported in many literatures, including the role in neural differentiation, migration and survival (Jeong et al., 2014; Kim et al., 2023; Powell et al., 2015; Uluca et al., 2022; Wang et al., 2017), cardiac remodeling and fibrosis (Jeong et al., 2017) and myoblast fusion (Castiglioni et al., 2018). More recently, in zebrafish cdon was reported being involved in trunk neural crest cell migration and slow-twitch muscle development (Lencer et al., 2023) and limb growth (Echevarria-Andino et al., 2023). While in more early stage whether it plays a critical role in organ LR patterning is not reported.

Here, we identified cdon was expressed in DFCs during gastrulation movement (Fig. 1Aa2-a5) and in epithelial cells of KV at early somitogenesis stage (Fig. 1Aa6-a7). Further data showed that cdon loss of function led to DFCs clustering movement defect. As the results, KV formation/ciliogenesis and the subsequent organ LR patterning were disturbed. So, during embryonic development, beside the role in regulating trunk neural crest cell migration (Lencer et al., 2023) and defining the correct proximo-distal patterning of the eye development (Jeong et al., 2014), we additionally identified a more early role of cdon in regulating LR patterning. Comparing our data in cdon morphants and cdon mutants, we discovered that the organ LR patterning defect in cdon morphants is stronger than that in cdon mutants, the possible reasons are that: 1, in cdon mutant embryos, there still exists some degree of maternal cdon mRNA or matermal Cdon protein. 2, given the well-known genetic compensation response in zebrafish (Ma et al., 2019), the mutation of cdon possiblely upregulates some other genes to compensate the loss of cdon function. All these possibility need far more work to elucidate.

Supplementary Materials

Figure s1. The expression of boc at different developmental stages

Figure s2. The external phenotype in embryos injected with cdon MO.

Figure s3. The external phenotype in embryos injected with cdon mRNA.

Figure s4. The external phenotype and the expression of cdon in controls and cdon-/-embryos.

Figure s5. The expression of lefty1 and lefty2 in controls and cdon-/ -embryos.

Figure s6. Injection of cdon MO at 256-cell stage disturbed clustering DFCs migration, KV morphogenesis and ciliogenesis.

Author Contributions

Conceptualization, supervision, and funding acquisition, S.H.; methodology, Z.D., Z.G. and S.H.; experiments, Z.D., W.C., C.L., B.L., S.H., K.Z., J.H. and S.H.; software, Z.D., Z.G. and S.H.; validation, Z.D., S.H.; formal analysis, Z.D., W.C., Z.G. and S.H.; data curation, Z.D.; writing-original draft preparation, Z.D., Z.G., Q.R and S.H.; writing—review and editing, S.H., Z.G., Z.D. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32070805) and the Science and Technology Department of Sichuan Province (2021ZYD0074) and the Disciplinary Construction Innovation Team Foundation of Chengdu Medical College (CMC-XK-2102).

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data was in the manuscript and supplementary materials.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the writing of the manuscript, or in the decision to publish the manuscript.

Acknowledgments

We would like to thank Dr. Lingfei Luo for his advice on this work; we also would like to thank the members working in our fish facility for their help taking care of all the fish lines in this study.

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Posted March 13, 2024.
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Cdon is required for organ Left-Right patterning via regulating DFCs migration and the sequential ciliogenesis
Zhilin Deng, Wenqi Chang, Chengni Li, Botong Li, Shuying Huang, Jingtong Huang, Ke Zhang, Yuanyuan Li, Xingdong Liu, Qin Ran, Zhenhua Guo, Sizhou Huang
bioRxiv 2024.03.12.584572; doi: https://doi.org/10.1101/2024.03.12.584572
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