The novel ciliogenesis regulator DYRK2 governs Hedgehog signaling during mouse embryogenesis

Mammalian Hedgehog (Hh) signaling plays key roles in embryogenesis and uniquely requires primary cilia. Functional analyses of several ciliogenesis-related genes led to the discovery of the developmental diseases known as ciliopathies. Hence, identification of mammalian factors that regulate ciliogenesis can provide insight into the molecular mechanisms of embryogenesis and ciliopathy. Here, we demonstrate that DYRK2 acts as a novel mammalian ciliogenesis-related protein kinase. Loss of Dyrk2 in mice causes suppression of Hh signaling and results in skeletal abnormalities during in vivo embryogenesis. Deletion of Dyrk2 induces abnormal ciliary morphology and trafficking of Hh pathway components. Mechanistically, transcriptome analyses demonstrate down-regulation of Aurka and other disassembly genes following Dyrk2 deletion. Taken together, the present study demonstrates for the first time that DYRK2 controls ciliogenesis and is necessary for Hh signaling during mammalian development.


Embryogenesis and patterning of cell differentiation are facilitated by spatiotemporal activation of multiple signaling pathways. The Hedgehog (Hh) signaling is an evolutionarily conserved system that plays a central role in embryogenesis via regulating cell proliferation and differentiation (Ingham and McMahon, 2001). Upon stimulation by ligands, post-translational modification of GLI2 and GLI3 induces the expression of Gli1, which is a key amplifier of Hh signaling. These post-translational and transcriptional activations of three GLIs regulate specific and redundant target genes (Mo et al., 1997; Hui and Angers, 2011). Hence, mutants of Hh components cause typical defects such as skeletal, neural, and retinal abnormalities (Mo et al., 1997).
Unlike other core developmental signaling, vertebrate Hh signaling is uniquely and completely dependent upon primary cilia, which are microtubule-based organelles that are formed during the G0 or G1 phases of the cell cycle (Huangfu et al., 2003). Binding of Hh ligands to Patched 1 (PTCH1) on cilia leads to activation and induction of Seven-spanner smoothened (SMO) to the cilia (Rohatgi et al., 2007). Activated SMO leads to recruitment of GLI2 and GLI3 to the cilia tip via inhibition of protein kinase A (Chen et al., 2009; Kim et al., 2009; Wen et al., 2010). This dynamic ciliary trafficking of Hh components is primarily regulated by intraflagellar transport (IFT) (Haycraft et al., 2005; Eguether et al., 2014). Thus, ciliogenesis is indispensable for tissue development, and defects in this process impact the development of multiple organs to cause human and mouse diseases termed ‘ciliopathies’ (Reiter and Leroux, 2017). Accordingly, the typical phenotype observed in some of ciliopathies such as Joubert syndrome is abnormalities of Hh signaling (Bangs and Anderson, 2017).
Mutations in a number of different ciliopathy-associated genes often result in alterations of ciliary length (Paige Taylor et al., 2016; Reiter and Leroux, 2017). Indeed, genetic screenings of Chlamydomonas, which is a model organism for ciliogenesis, have identified the ciliopathy-associated genes controlling cilia length to generate the optimal length at steady-state (Wemmer and Marshall, 2007). Although these abnormalities in ciliary length are thought to be controlled by the balance of assembly and disassembly via IFT and a postulated length sensor, the mechanisms for maintaining the cell-type-specific ciliary length have not been fully elucidated (Ishikawa and Marshall, 2011). In contrast, the ciliary resorption mechanisms for cell cycle re-entry have been thoroughly investigated by such as an experiment of serum re-addition to starved cells, and these mechanisms include the HEF1-AURKA-HDAC6 pathway (Pugacheva et al., 2007), the PLK-KIF2A pathway (Wang et al., 2013), and the NEK2-KIF24 pathway (Kobayashi et al., 2011; Kim et al., 2015b) that have been observed to induce disassembly of cilia. On the other hand, the ability of these ciliary resorption factors for cell cycle re-entry to control cilia length at steady-state and during ciliogenesis remains to be elucidated. Hence, identification of novel mammalian factors regulating ciliogenesis and ciliary length control will provide insight into the molecular mechanisms underlying embryogenesis and ciliopathy as well as ciliary functions.
Dual-specificity tyrosine-regulated kinase (DYRK) is a family that belongs to the CMGC group that includes cyclin-dependent kinases (CDK), mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK), and CDK-like kinase (CLKs) (Becker and Sippl, 2011). Two isoforms of Dyrk2 have been identified; long and short forms, the latter lacks a 5’ terminal region. In human cancer cells, we have functionally identified DYRK2 as a regulator of p53-induced apoptosis in response to DNA damage (Taira et al., 2007) and of G1/S transition (Taira et al., 2012). During development in lower eukaryotes, MBK2, which is an ortholog of DYRK2 in Caenorhabditis elegans, regulates maternal-protein degradation during the oocyte-to-embryo transition via a ubiquitin-dependent mechanism (Pellettieri et al., 2003; Pang et al., 2004; Lu and Mains, 2007; Yoshida and Yoshida, 2019). While these reports lead us to speculate that DYRK2 must also play important roles in mammalian development, no reports are available regarding the mechanistic role of DYRK2 in vivo.
In the present study, we aim to reveal a function for DYRK2 in mammalian development in vivo. Here, we demonstrate that DYRK2 is a novel regulator of ciliogenesis and is required for normal embryogenesis via activation of Hh signaling during development.


Dyrk2 deficiency cause suppression of Hedgehog signaling during mouse embryogenesis

We generated Dyrk2 knockout mice (Dyrk2-/-) by eliminating the third exon of the Dyrk2 genomic locus (Figure 1—figure supplement 1A–B). The absence of DYRK2 protein in homozygous Dyrk2-/- mice was confirmed (Figure 1—figure supplement 1C). Although the gross morphology of homozygous Dyrk2-/- embryos appeared normal during early development, multiple defects became obvious during later stages of gestation, and the mice died at or close to birth (Figure 1A). Specifically, defects in skeletal development were remarkable, and these included a shorter dorsum of the nose (Figure 1A), cleft palate including hypoplasia of the tongue (Figure 1B–C), loss of the basisphenoid, basioccipital, and presphenoid bones (Figure 1D), shorter limbs (Figure 1E), defects of segmentation of the sternebrae in the sternum (Figure 1F), and vertebra (Figure 1G) at embryonic day (E) 18.5. These skeletal defects that included reduction of bone mineralization were observed until E16.5 (Figure 1H).

Deletion of DYRK2 shows skeletal defects in mouse development.

(A) Whole embryo gross images of wild-type and homozygous Dyrk2-/- embryos at birth. (B, C) Palatal and tongue abnormalities in Dyrk2-/- embryos. Gross images of the palate with mandible removed from wild-type and Dyrk2-/- embryos at E18.5 (B), and HE staining from the coronal plane at E13.5 (C). Dotted lines in (B) and an asterisk in (C) indicate cleft of the secondary palate. (D–H) Arizarin red and alcian blue staining of the craniofacial skeleton (D), forelimbs (E), sternum (F), and vertebra (G) from wild-type and Dyrk2-/- embryos at E18.5, and whole skeleton staining at E16.5 (H). Arrowheads in (H) indicate regions that decreasing bone mineralization. bo, basioccipital bone; bs, basisphenoid; h, humerus; r, radius; p, palatal shelves; ps, presphenoid; s, scapula; st, sternebrae; t, tongue; u, ulna. Scale bars, 5 mm.

As Dyrk2-/- embryos at E18.5 exhibited a similar phenotype to that observed in response to certain defects in Hh signaling (Mo et al., 1997), we assessed Gli1-expression, which is an indicator of Hh signaling activation (Niewiadomski and Rohatgi, 2015). In situ hybridization demonstrated that Gli1-expression was decreased in the craniofacial region in Dyrk2-/- embryos at E14.5 (Figure 2A). Protein levels of GLI1 were also decreased at E13.5 (Figure 2B). Ptch1-expression, which is another indicator of Hh signaling activation (Snouffer et al., 2017), was also decreased in Dyrk2-/- embryos at E13.5, and this was accompanied by a decrease in Gli1; however, Shh-expression remained unchanged (Figure 2C). We also observed a repression of Foxf2-expression, which is a direct target gene of GLI1 (Everson et al., 2017), in the craniofacial region of Dyrk2-/- embryos (Figure 2D–E).

Deletion of DYRK2 affects activation of Hh signaling in mouse development.

(A) In situ hybridization of Gli1 in the craniofacial region in wild-type and Dyrk2-/- embryos from the sagittal plane at E14.5. (B) Immunoblotting of GLI1 in extracts from the limbs of wild-type and Dyrk2-/- embryos at E13.5. GAPDH serves as a loading control. (C) qPCR of Gli1, Ptch1, and Shh in the limbs from wild-type and Dyrk2-/- embryos at E13.5. (D, E) Repression of Foxf2-expression in the craniofacial region of Dyrk2-/- mice. (D) In situ hybridization of Foxf2 in the craniofacial region in wild-type and Dyrk2-/- embryos from the sagittal plane at E14.5. (E) qPCR of Foxf2 in the mandibular arch from wild-type and Dyrk2-/- embryos at E10.5. Hypoxanthine phosphoribosyltransferase (Hprt) in (C and E) was used as an internal standard, and fold change was calculated by comparing expression levels relative to those of wild-type. Data are presented as the means ± SEM (n = 3 biological replicates). The statistical significance between wild-type and Dyrk2-/- was determined by the Student’s t-test. (*) p
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