Cdon is essential for organ left-right patterning by regulating dorsal forerunner cells clustering and Kupffer’s vesicle morphogenesis

Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon, but not boc, is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer’s vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon −/− embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry.


Introduction
The establishment of left-right (LR) patterning is a crucial event in early embryonic development.Organ asymmetry defects are closely related to the occurrence of various congenital diseases such as congenital heart disease and schizophrenia (Huang et al., 2014;Gabriel and Lo, 2020).Although the mechanisms for establishing organ LR patterning are complex and diverse across different animal models (Hamada and Tam, 2020;Onuma et al., 2020;Zhu et al., 2020), the role of the Kupffer's vesicle (KV)/Node-Cilia axis in zebrafish and mice is highly conserved (Grimes and Burdine, 2017;Little and Norris, 2021;Forrest et al., 2022).In zebrafish, the normal development of KV precursor cells, called 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).During organ LR patterning, DFCs proliferate rapidly during gastrulation movement (Gokey et al., 2015;Abdel-Razek et al., 2023).After the bud stage, DFCs differentiate to form the KV and produce cilia, which oscillate to generate a counter-clockwise liquid flow and initiate specific asymmetric Nodal/spaw expression on the LPM (Bakkers et al., 2009;Liu et al., 2019).Furthermore, the left-sided Nodal/spaw and its downstream genes pitx2 and lft2 are amplified in the LPM.The embryonic midline (such as shh and ntl) also impedes the expansion of left-sided Nodal signals to the right side of the embryo, thus stabilizing the left-sided asymmetric Nodal signals (Burdine and Grimes, 2016).Finally, at the late stage of organ development, the left-sided Nodal directs an asymmetric migration of organ precursors to establish organ asymmetry patterning (Kajikawa et al., 2022).
The role of zebrafish KV morphogenesis/ciliogenesis and the underlying molecular mechanisms during organ LR patterning have received extensive attention.It has been found that many classical signaling pathways, such as Wnt and Fgf (Neugebauer et al., 2009;Xu et al., 2010;Caron et al., 2012;Zhang et al., 2012) and 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) increases 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 that cxcr4a promotes CyclinD1 expression to regulate ciliogenesis by regulating the phosphorylation of ERK1/2 (Liu et al., 2019).Although many genes have been reported to play crucial roles during KV morphogenesis and ciliogenesis, the mechanisms underlying these processes are far from being elucidated.
More detailed studies have shown that cdon binds to N-cadherin and induces p38/MAPK signaling to guide cell differentiation and apoptosis during muscle formation and neural differentiation (Lu and Krauss, 2010).In mouse, Cdon has been reported to negatively regulate the Wnt signaling pathway during forebrain development, promoting neural differentiation and ventral cell fate determination via interaction with LRP6 (Jeong et al., 2014).Further, researchers 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).Regarding 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 trunk neural crest cell (tNCC) migratory defects and loss of slow-twitch muscle, suggesting a non-cell-autonomous role for cdon and boc in regulating tNCC migration (Lencer et al., 2023).Although cdon and boc have been reported to play important roles in various contexts in mouse and zebrafish, their roles in early embryonic development remain unknown.Our data showed that during early embryonic development in zebrafish, cdon, but not boc, is expressed in DFCs during gastrulation movement and in the epithelial cells of KV at the early somitogenesis stage (Figure 1; Supplementry Figures S1).Additionally, cdon has been reported to control cell apoptosis and migration in various cell types (Sanchez-Arrones et al., 2012;Chapouly et al., 2020;Lencer et al., 2023).These findings suggest that cdon may be involved in regulating the clustering movement of DFCs and the subsequent organ LR patterning in zebrafish.Here, our data identify that cdon regulates organ LR patterning via the KV/ Cilia-Nodal/spaw cascade during early embryonic development.

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.The embryos were then washed with PBST (2 times, 5 min each), dehydrated with 100% MeOH, and stored in MeOH at −20 °C.The WISH procedure followed the experimental protocol described previously (Huang et al., 2011;Zhu et al., 2019).The antisense probes my17, fabp10, spaw, lefty1, lefty2, sox17, and sox32 were prepared as previously described (Zhu et al., 2019).To prepare the cdon antisense probe, the cDNA of cdon was amplified using PCR and then cloned into the pCS2 + vector.The detailed process can be found in the section "Plasmid Construction."This construct was then used as the template to synthesize the cdon antisense probe according to the previously established protocol (Zhu et al., 2019).Organ left-right patterning defects in embryos injected with cdon MO at the 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 arrowhead showed the expression of cdon in DFCs at 80% epiboly stage (Aa2).Black arrowhead 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 24 hpf (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.Embryos injected with cdon MO show a statistically significant difference compared to controls, and embryos co-injected with cdon MO and cdon mRNA show a statistically significant difference compared to those injected with cdon MO alone.(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 the 4-cell stage were examined for cardiac looping at 72 hpf by WISH against my17 (n = 83).The blue dashed line indicates the atrial edge.(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).Embryos injected with cdon MO show a statistically significant difference compared to controls, and embryos co-injected with cdon MO and cdon mRNA show a statistically significant difference compared to those injected with cdon MO alone.Notice: in "C" and "E," all the transgenic embryos and wild type embryos were used together to calculate the percentage.Statistical analysis was performed using Student's t-test."*"p < 0.05, "**" p < 0.01, "***" p < 0.001.Notice: "control" refers to wild-type embryos that were not injected with cdon MO.

Immunostaining
The immunostaining was performed as previously reported (Zhu et al., 2019).Briefly, the embryos were fixed with 4% PFA at 4 °C overnight, then dehydrated with a methanol gradient and stored at −20 °C.After methanol/PBST gradient rehydration, PBTN (4% BSA, 0.02% NaN3, PT) was added, and the embryos were incubated at 4 °C 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.The next day, the embryos were washed with PT (0.3% Triton X-100 in 1X PBS) 4 times (30 min each), and the secondary antibody, Goat anti-Mouse IgG (H + L) Alexa Fluor 594 (Sigma, SAB4600105), was added (diluted with PBTN at 1:1,000) for overnight incubation in darkness.Finally, the embryos were rinsed with PT more than 6 times (30 min each), and imaging was performed.

MO and mRNA injection
The antisense cdon ATG morpholino oligonucleotide (cdon MO atg ) was synthesized (Gene Tools) and applied to block cdon mRNA translation.cdon MO atg : 5′-ATAATCTCAGGCCACCGT CCTCCAT-3′, 400 μM (Cardozo et al., 2014).Cdon MO was injected into the yolk of zebrafish embryos at the 1-4 cell stage to block the translation of cdon in whole embryos, or at the 256-512 cell stage to specifically block the translation of cdon mRNA in DFCs.The cdon mRNA was synthesized in vitro using the mMESSAGE Kit (AM1340, Ambion).In rescue experiments, the concentration for cdon mRNA injection was 15 ng/μL.

Generation of cdon −/− mutants
To generate cdon −/− mutants, we used the CRISPR design tool (http://crispr.mit.edu) to design CRISPR short guide RNAs (sgRNAs) targeting exon 3 of the cdon gene.Injection mixtures contained 500 pg Cas9 mRNA and 120 pg gRNA in each embryo.Embryos injected with sgRNA and Cas9 mRNA generated the F0 line with mosaic mutations.These F0 mosaic adult fish were outcrossed with wild-type fish to produce F1 heterozygous lines.In one F1 adult, the sequence "AAGGGC" in exon 3 of the cdon gene was changed to "TTGATGAATGGGG", resulting in a truncated Cdon protein.This F1 heterozygote was outcrossed with wild-type fish to produce F2 heterozygous fish, then incrossed them to obtain F3 homozygous mutants.The homozygous embryos from this cdon −/− mutant line were used to perform experiments as required.

Imaging
Images of the whole-mount in situ hybridization (in 100% glycerol) were taken with an OLYMPUS SZX16 microscope at room temperature.Live embryos of the transgenic line Tg (sox17: EGFP) were placed in 1% low-point melting agarose (LPM agarose) and DFCs were photographed using a confocal microscope (OLYMPUS Fluview FV1000).To obtain images of cilia for immunostained embryos, the embryos were oriented correctly in 1% LPM agarose, and the cilia were photographed using a confocal microscope (OLYMPUS Fluview FV1000).

Statistical analysis
The data were statistically analyzed using GraphPad Prism 9 and ImageJ software.The length of the cilia was measured using a confocal microscope (OLYMPUS Fluview FV1000), and the OLYMPUS SZX16 microscope was used to measure the KV lumen area.The statistical results are presented as the mean ± SEM of three independent experiments.Statistical comparisons between two groups were performed using Student's t-test.Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Ethics statement
The study was approved by the Institutional Review Board of Chengdu Medical College (SYXK(川) , and zebrafish were maintained in accordance with the Guidelines of Experimental Animal Welfare from the Ministry of Science and Technology of the People's Republic of China (2006).

Heart and liver LR patterning was disturbed in cdon morphants
In zebrafish, cdon and boc have been reported to be involved in trunk neural crest cell (NC) migration and correct proximo-distal patterning in eye development (Cardozo 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 24 hpf.The data showed that cdon is a maternal factor that is distributed unequally in cells (Figure 1Aa1).During gastrulation, besides its expression in the presumptive neural crest and midline [Figure 1Aa5, (Jeong et al., 2014)], cdon was also highly expressed in DFCs (Figure 1Aa2-a4).At the 6-somite stage, cdon was enriched in the epithelium of the KV (Figures 1Aa6, a7).In contrast, although boc was maternally and ubiquitously expressed during gastrulation (Supplementry Figures S1A, B), it was not enriched in DFCs or the epithelium of the KV (Supplementry Figures S1E, F).Since disrupting DFC development and the sequential KV/ciliogenesis leads to organ LR patterning defects (Xie et al., 2019;Zhu et al., 2019), we proposed that cdon is involved in organ LR patterning.To preliminarily and rapidly evaluate this hypothesis, an ATG MO for cdon was used to downregulate the function of cdon (Jeong et al., 2014), and organ LR patterning was examined.The results showed that in Tg (fabp10: GFP) transgenic embryos and wild type embryos, injection of cdon MO did not lead to embryonic defects (Supplementary Figure S2), but gave rise to liver LR patterning defects (Figures 1Bb1-b8, C).Next, we examined whether injection of cdon MO led to heart looping defects.The results showed that injection of cdon MO caused heart LR patterning defects (Figures 1D, E); many cdon morphants displayed linear heart (Figures 1Dd3, d7, E) and reversed heart looping (Figures 1Dd4, d8, E).To further confirm the role of cdon in liver and heart LR patterning, we evaluated whether injection of cdon mRNA could rescue the organ LR patterning defects in cdon morphants.The data showed that injection of cdon mRNA partially restored organ LR patterning defects in embryos Expression of left-sided Nodal signaling in controls and embryos injected with cdon MO (A,B) Expression of spaw was evaluated in different groups 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, p < 0.001); a3, bilateral spaw in embryos injected with cdon MO (35.4%, n = 82, p < 0.001); a4, right-sided spaw in embryos injected with cdon MO (26.8%, n = 82, p < 0.01).(C,D) 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, p < 0.001, black arrow showed); c3, absent lefty1 in embryos injected with cdon MO (34.2%, n = 73, p < 0.001); c4, right-sided lefty1 in embryos injected with cdon MO (12.4%, n = 73, p < 0.01, black arrow showed) (E,F) 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, p < 0.001); e3, bilateral expression of lefty2 in embryos injected with cdon MO (26.9%, n = 67, p < 0.001); e4, right-sided lefty2 in embryos injected with cdon MO (11.9%, n = 67, p < 0.01).Statistical analysis was performed using Student's t-test."**" p < 0.01, "***" p < 0.001.Notice: "control" refers to wild-type embryos that were not injected with cdon MO. injected with cdon MO (Supplementary Figure S3; Figures 1C, E; the right columns show).These data indicate that cdon plays a critical role in organ LR patterning in zebrafish.

Left-sided Nodal/spaw cascade is randomized in cdon morphants
The crucial role of Nodal/spaw in LR patterning has been demonstrated in previous studies (Raya and Izpisua Belmonte, 2006;Speder et al., 2007;Kang et al., 2013).Disruption of Nodal/ spaw leads to organ laterality defects (Huang et al., 2011).However, in some types of mutants with organ LR patterning defects, Nodal/ spaw was not disturbed (Trinh and Stainier, 2004;Kurpios et al., 2008;Yin et al., 2010;Huang et al., 2014).To reveal how cdon regulates organ laterality, we examined whether left-sided Nodal/ spaw was disturbed in embryos injected with cdon MO.The data showed that left-sided Nodal/spaw was disturbed in cdon morphants, displaying both-sided and right-sided(Figures 2Aa2-a4, B).Furthermore, we sequentially examined lefty1 and lefty2, the downstream genes of spaw in cdon morphants.The data showed that the left-sided expression patterns of lefty2 and lefty1 in the heart field were also perturbed (Figures 2Cc2-c4, D, Ee2-e4, F).Since cdon was also expressed in the midline [Figure 1Aa5, (Jeong et al., 2014)], we observed whether lefty1 expression in the midline was affected.The data showed that lefty1 expression in the midline was not affected (Figures 2Cc2-c4, red arrow).These data suggest that the left-sided Nodal/spaw cascade was disturbed in cdon morphants, which may lead to organ LR patterning defects in cdon morphants.

DFCs clustering, KV morphogenesis and cilia are disturbed in cdon morphants
Our current research showed that cdon was expressed in the DFCs during the gastrulation stage and in the epithelial cells of the KV (Figures 1Aa2-a7).In zebrafish, DFCs form the KV at the early somite stage.Defective KV morphogenesis or ciliogenesis can lead to disturbed left-sided Nodal/spaw expression and subsequent organ LR defects (Long et al., 2003;Essner et al., 2005;Wang et al., 2011).To determine whether cdon regulates DFC development, KV morphogenesis, or ciliogenesis, we analyzed the expression of DFC markers sox17 and sox32, as well as KV morphogenesis and ciliogenesis in cdon morphants.Compared with control embryos, at the 80% epiboly stage, the clustering DFC was disturbed in cdon morphants (Figures 3Aa2-a4, Aa6-a8, B), displaying linear expression (Figures 3Aa3, Aa7) and dispersed expression (Figures 3Aa4, Aa8).To further verify whether clustering DFC is affected, we injected cdon MO into Tg(sox17:EGFP) embryos.The results showed that at the 70% epiboly stage, DFCs remained as a tight cluster in the control embryos, while linear (Figure 3Cc3) or fragmented DFC clusters (Figure 3Cc4-c6) were detected in the cdon morphants (Figure 3C).Additionally, there was a significant decrease in the number of DFC cells at the 70% epiboly stage in the embryos injected with cdon MO compared to the control embryos (Figure 3D).Further, at the 10-13 somite stage (SS), the size of the KV lumen was smaller in the majority of cdon morphants (Figure 3Ee3, F, G), and a small portion of morphants displayed tiny or absent KV lumen (Figures 3Ee4, F, G).At the 10 somite stage, we used Tg (sox17:EGFP) transgenic zebrafish to examine the number of epithelium cells in KV.The results showed that, compared to the control, the number of epithelium cells in KV was significantly reduced in embryos injected with cdon MO at the 4-cell stage (Figures 3H, I).Furthermore, we examined cilia development and found that cdon morphants displayed slightly shorter cilia than controls (Figures 3Jj2-j4, K), and the number of cilia was also decreased in cdon morphants (Figures 3Jj2-j4, L).These results showed that normal expression of cdon is required for DFC development and KV morphogenesis, cdon MO injection also led to shorter cilia and decreased the number of cilia.

KV/cilia-Nodal/spaw cascade was also disturbed in cdon mutants
To further confirm the mechanism by which cdon regulates organ LR patterning, we also examined whether the KV/cilia-Nodal/spaw cascade was affected in cdon −/− embryos.First, we examined the expression of sox17 and sox32 at the 80% epiboly stage to evaluate whether the clustering DFC migration is disturbed in cdon −/− embryos.The data showed that 55.7% of cdon -/-embryos displayed dispersed expression of sox17 (Figures 5Aa3, a4, B), and 48.0% of cdon −/− embryos displayed dispersed expression of sox32 (Figures 5Aa7, a8, B).In contrast, the expression of sox17 and sox32 was normal in control embryos (Figures 5Aa1, Aa5, B).This data indicated that clustering DFC was disturbed in cdon −/− embryos.Then we evaluated whether KV morphogenesis and cilia development were affected in cdon −/− embryos.The data showed that in many cdon −/− embryos, the size of the KV lumen became smaller or absent (Figures 5Cc3, c4, D, E).Similar to cdon morphants, in cdon −/− embryos, the cilia length is slightly shorter (Figures 5Ff1-f4, G) and the cilia number is decreased (Figures 5Ff1-f4, H).Finally, we examined the expression pattern of spaw in control and cdon −/− embryos.The data showed that left-sided spaw expression was also disturbed in cdon −/− embryos, displaying left-sided spaw (Figures 5Ii2, J), bilateral spaw (Figures 5Ii3, J), and right-sided spaw (Figures 5Ii4, J).In addition, the expression of the Nodal/spaw downstream genes lefty1 and lefty2 was also disturbed in cdon −/− embryos (Supplementary Figures 5A-D).These data further confirm that the KV/cilia-Nodal/spaw cascade may mediate the regulation of heart and liver LR patterning by cdon during early development.

Cdon loss of function in DFCs results in organ LR patterning defects
Our data have shown that cdon is expressed not only in DFCs and KV epithelial cells but also in other cells such as those in the midline and PSM (Figure 1A; Supplementary Figures S4C, D).To confirm whether cdon specifically regulates organ LR patterning via the DFCs-KV/cilia-Nodal/spaw cascade, we injected cdon MO at the 256-512 cell stage to predominantly block the 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 the 256-512 cell stage, liver and heart LR patterning were disturbed (Figures 6A-D).In liver transgenic embryos Tg (fabp10:GFP), heart transgenic embryos Tg (cmlc2:GFP) and wild-type embryos, after predominantly down-regulating the function of cdon in DFCs, the liver (Figures 6A, B) and heart (Figures 6C, D) LR patterning are all disturbed.Next, we evaluated whether the DFCs-KV/cilia cascade was affected after predominantly down-regulating the function of cdon in DFCs.The data showed that clustering DFC was disturbed (Supplementary Figures S6Aa1-a8, B), the size of the KV lumen was smaller (Supplementary Figures S6Cc1-c4, D-E), the length of cilia was slightly shorter (Supplementary Figures S6Ff1-f4,G), and the number of cilia was also decreased (Supplementary Figures S6Ff1-f4, H).Finally, we evaluated whether Nodal/spaw signaling was disturbed in embryos injected with cdon MO at the 256-512 cell stage.As a result, the expression of left-sided Nodal/spaw and its downstream genes lefty1 and lefty2 was also randomized in embryos injected with cdon MO at the 256-512 cell stage (Figures 6E-J
Cdon is a cell surface glycoprotein that belong to a subgroup of the immunoglobulin (Ig) superfamily of cell adhesion molecules (Sanchez-Arrones et al., 2012).The role of cdon in organ development and function has been reported in many literature, including its role in neural differentiation, migration and survival (Jeong et al., 2014;Powell et al., 2015;Wang et al., 2017;Uluca et al., 2022;Kim et al., 2023), cardiac remodeling and fibrosis (Jeong et al., 2017) and myoblast fusion (Castiglioni et al., 2018).More recently, in zebrafish, cdon was reported to be involved in trunk neural crest cell migration, slow-twitch muscle development (Lencer et al., 2023), and limb growth (Echevarria-Andino et al., 2023).However, whether it plays a critical role in organ LR patterning at earlier stage has not been reported.
Here, we identified that cdon is expressed in DFCs during the gastrulation movement (Figures 1Aa2-a5) and in the epithelial cells of the KV at the early somitogenesis stage (Figures 1Aa6, a7).Further data suggested that cdon loss of function leads to defect in DFC clustering and decreases DFCs number.These defects are correlated with disturbances in KV formation, cilia number and the subsequent organ LR patterning.Therefore, during embryonic development, in addition to its role in regulating trunk neural crest cell migration (Lencer et al., 2023) and defining the correct proximo-distal patterning of eye development (Cardozo et al., 2014), we identified an earlier 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.Given the well-known genetic compensation response in zebrafish (Ma et al., 2019), the possible reason is the mutation of cdon may upregulate other genes to compensate for the loss of cdon function.This possibility needs further work to elucidate.
Even our work identified the role of cdon in organ LR patterning, the detailed mechanism underlying was not elucidated.In our data, we found that the number of DFCs and epithelium cells in KV was decreased (Figures 3C, D, H,  I), as well the cohesive migration was defect in cdon mutants (Figures 3A-D), so the genes being relative to cell migration, cell proliferation/differentiation or cell apoptosis should be affected in cdon mutants.In zebrafish, reduction of Wnt signaling leads to a disruption of LR patterning, shorter and fewer cilia (Caron et al., 2012), depletion of β-catenin 1 or β-catenin 2 in DFCs/KV leads to poor KV cell proliferation, abnormal cilia formation (Zhang et al., 2012).On the contrary, knockdown of Autotaxin/ Lpar3 signaling activates β-catenin and also compromises DFC cohesive migration, KV formation and ciliogenesis (Lai et al., 2012).These reports suggested that both downregulation and upregulation of Wnt signaling would give rise to defect in KV morphogenesis, abnormal cilia formation and the sequential organ LR patterning.Importantly, early reports in mouse demonstrated that Cdon negatively regulates Wnt signaling during neural development and cardiac remodeling (Jeong et al., 2014;Jeong et al., 2017), the Cdo-deficient dorsal forebrain displays stronger Wnt signalling activity, increased cell proliferation and enhanced expression of the dorsal markers and Wnt targets (Jeong et al., 2014).These literature suggested the possibility that in zebrafish, loss of cdon function upregulates Wnt signaling, which compromises DFC cohesive migration, KV formation, ciliogenesis and the sequential organ LR patterning.While far more work is needed to clarify whether Wnt signaling lies downstream of cdon to regulate LR patterning.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.256-cell stage (50.5%, n = 93); c3, no loop in Tg(cmcl2:GFP) transgenic embryos injected with cdon MO at the 256-cell stage (34.4%, n = 93); c4, reversed-loop in Tg(cmcl2:GFP) transgenic embryos injected with cdon MO at the 256-cell stage (15.1%, n = 93); 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 72 hpf by WISH against my17 (n = 110).The blue dashed line indicates the atrial edge.(D) Percentages of normal looping, no looping, and reversed looping of the heart in all the embryos injected with or without cdon MO at the 256-cell stage.Embryos injected with cdon MO at the 256-cell stage show a statistically significant difference compared to controls.(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).Statistical analysis was performed using Student's t-test."**" p < 0.01, "***" p < 0.001, "****" p < 0.0001.Notice: in "B" and "D", all the transgenic embryos and wild-type embryos were used together to calculate the percentage."Control" refers to wild-type embryos that were not injected with cdon MO.

FIGURE 3
FIGURE 3Cdon loss of function disturbs clustering DFCs, KV morphogenesis and cilia (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 the 4-cell stage (46.8%,n = 62); a3, dispersed expression of sox17 (type I) in embryos injected with cdon MO at the 4-cell stage (27.4%, n = 62); a4, dispersed expression of sox17 (type II) in embryos injected with cdon MO at the 4-cell stage (25.8%,n = 62).a5, normal expression of sox32 in controls (100%, n = 57); a6, normal expression of sox32 in embryos injected with cdon MO at the 4-cell stage (52.2%, n = 67); a7, dispersed expression of sox32 (type I) in embryos injected with cdon MO at the 4-cell stage (26.9%,n = 67); a8, dispersed expression of sox32 (type II) in embryos injected with cdon MO at the 4cell stage (20.9%,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.Normal DFC and dispersed DFC show significant differences between control embryos and embryos injected with cdon MO at the 4-cell stage.(C) Using Tg (sox17:EGFP) transgenic zebrafish to detect DFC cell migration at the 70% epiboly stage in both control embryos and embryos injected with cdon MO at the 4-cell stage.c1, the DFCs in control (Continued )

FIGURE
FIGURE 6 (Continued) Figures 6Ii4) of embryos injected with cdon MO displayed left-sided lefty2, both-sided lefty2, and right-sided lefty2, respectively, while 97.2% of control embryos displayed leftsided lefty2 (Figures 6Ii1).These data indicate that left-sided Nodal/ spaw signaling was randomized after down-regulating the function of cdon in DFCs.In conclusion, all these data suggest that cdon specifically regulates organ LR patterning via the DFCs-KV/cilia-Nodal/spaw cascade.