Mouse model of congenital heart defects, dysmorphic facial features and intellectual developmental disorders as a result of nonfunctional CDK13

fcell-07-00155 August 6, 2019 Time: 17:21 # 1 ORIGINAL RESEARCH published: 07 August 2019 doi: 10.3389/fcell.2019.00155 Edited by: Eleonora Napoli, University of California, Davis, United States Reviewed by: Junichi Iwata, University of Texas Health Science Center at Houston, United States Julie Siegenthaler, University of Colorado Denver, United States *Correspondence: Marcela Buchtová [email protected] Jiˇ rí Kohoutek [email protected] Specialty section: This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Cell and Developmental Biology Received: 13 February 2019 Accepted: 23 July 2019 Published: 07 August 2019 Citation: Nováková M, Hampl M, Vrábel D, Procházka J, Petrezselyová S, Procházková M, Sedlá ˇ cek R, Kavková M, Zikmund T, Kaiser J, Juan H-C, Fann M-J, Buchtová M and Kohoutek J (2019) Mouse Model of Congenital Heart Defects, Dysmorphic Facial Features and Intellectual Developmental Disorders as a Result of Non-functional CDK13. Front. Cell Dev. Biol. 7:155. doi: 10.3389/fcell.2019.00155 Mouse Model of Congenital Heart Defects, Dysmorphic Facial Features and Intellectual Developmental Disorders as a Result of Non-functional CDK13 Monika Nováková1, Marek Hampl2,3, Dávid Vrábel1, Jan Procházka4,5, Silvia Petrezselyová4,5, Michaela Procházková4,5, Radislav Sedlá ˇ cek4,5, Michaela Kavková6, Tomáš Zikmund6, Jozef Kaiser6, Hsien-Chia Juan7, Ming-Ji Fann7, Marcela Buchtová2,3*and Jiˇ rí Kohoutek1* 1Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czechia, 2Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czechia, 3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czechia, 4Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czechia, 5Czech Centre for Phenogenomics, Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czechia, 6Central European Institute of Technology, Brno University of Technology, Brno, Czechia, 7Department of Life Sciences, Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan Congenital heart defects, dysmorphic facial features and intellectual developmental disorders (CHDFIDD) syndrome in humans was recently associated with mutation in CDK13 gene. In order to assess the loss of function of Cdk13 during mouse development, we employed gene trap knock-out (KO) allele in Cdk13 gene. Embryonic lethality of Cdk13-deficient animals was observed by the embryonic day (E) 16.5, while live embryos were observed on E15.5. At this stage, improper development of multiple organs has been documented, partly resembling defects observed in patients with mutated CDK13. In particular, overall developmental delay, incomplete secondary palate formation with variability in severity among Cdk13-deficient animals or complete midline deficiency, kidney failure accompanied by congenital heart defects were detected. Based on further analyses, the lethality at this stage is a result of heart failure most likely due to multiple heart defects followed by insufficient blood circulation resulting in multiple organs dysfunctions. Thus, Cdk13 KO mice might be a very useful model for further studies focused on delineating signaling circuits and molecular mechanisms underlying CHDFIDD caused by mutation in CDK13 gene. Keywords: cyclin-dependent kinase (CDK), cyclin, transcription regulation, development, mouse, cyclindependent kinase 13, cyclin K INTRODUCTION Recently, de novo missense variants in Cyclin-dependent kinase 13 (CDK13) gene have been identified as an emerging factor involved in the onset of congenial heart defects (CHD) in humans (Sifrim et al., 2016). Documented CHD cases were characterized by ventral and atrial septal defects accompanied by pulmonary valve abnormalities. CHD patients had syndromic facial gestalt, and two patients had agenesis of the corpus callosum. Additional mutations within the CDK13 gene were recognized in humans resembling at clinical level many symptoms previously associated Frontiers in Cell and Developmental Biology | www.frontiersin.org 1August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 2 Nováková et al. Function of Cdk13 During Mouse Development with loss of function of CDK13 or newly described symptoms, such as autism spectrum disorder, seizures, feeding difficulties and craniofacial dysmorphism including short upslanting palpebral fissures, hypertelorism or telecanthus, medial epicathic folds, low-set, posteriorly rotated ears and small mouth with thin upper lip vermilion (Hamilton et al., 2018). In addition, genome wide search for de novo mutations responsible for developmental disorders in patients in Great Britain and Republic of Ireland led to identification of 14 genes that previously lacked compelling evidence of involvement in developmental disorders, among them the CDK13 gene (Deciphering Developmental Disorders Study, 2017). However, heart defects do not seem to be the key feature of this disorder since patients with heterozygous constitutional mutation in CDK13 lacking cardiac anomalies were reported by two groups (Carneiro et al., 2018;Uehara et al., 2018). Spectrum of clinical phenotypes of patients with CDK13 mutations varies from mild to severe with the ubiquitous intellectual disability and developmental delay (ID/DD) (van den Akker et al., 2018). Based on these observations, congenital heart defects, dysmorphic facial features and intellectual development disorder (CHDFIDD) have been recognized as novel syndrome caused by de novo variants of CDK13 gene (Sifrim et al., 2016;Bostwick et al., 2017;Carneiro et al., 2018; van den Akker et al., 2018). Human CDK13 protein consists of 1512-amino acids with a conserved kinase domain surrounded by Nand C-terminal arms of undefined function (Kohoutek and Blazek, 2012). In order to be active, CDK13 binds cyclin K (CycK) and forms enzymatically active complex (Ko et al., 2001;Even et al., 2006; Bartkowiak et al., 2010;Blazek et al., 2011;Cheng et al., 2012; Dai et al., 2012;Kohoutek and Blazek, 2012;Liang et al., 2015). CDK13 belongs to the family of transcription-associated cyclin-dependent kinases phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII). In particular, the CDK13 phosphorylates serine 2 (Ser2) and to a lesser extend also serine 5 (Ser5) within Y1S2P3T4S5P6S7heptapeptides within the CTD of RNAPII in vitro (Greifenberg et al., 2016). Nevertheless, downregulation of CDK13 in tumor derived cell lines had a very small, if any, effect on level of Ser2 within CTD of RNAPII (Blazek et al., 2011;Greifenberg et al., 2016). In parallel to CDK13, there is CDK12 in mammalian cells able to associate with CycK as well (Blazek et al., 2011;Dai et al., 2012;Liang et al., 2015). Even though CDK13 shares high amino acid similarity with CDK12, both kinases appear to function in mutually exclusive complexes in mammalian cells (Blazek et al., 2011;Kohoutek and Blazek, 2012;Greenleaf, 2018). In comparison to CDK12, there is a limited number of papers envisioning the likely function of CDK13 in various biological processes. For instance, the CDK13 was proposed to be involved in oncogenesis; yet, its precise function is still under examination (Kim et al., 2012;Pan et al., 2012). Even though factors involved in RNA processing, RNA splicing, polyadenylation and RNA cleavage were demonstrated to bind CDK13 as a result of global protein-protein interactions, truly associating partners of this kinase are still unknown (Davidson et al., 2014;Bartkowiak and Greenleaf, 2015;Liang et al., 2015). In addition to involvement of CDK13 in diverse cellular processes, this protein participates in regulation of alternative splicing of HIV-1 or influenza virus replication, thus suppressing viral production (Berro et al., 2008;Bakre et al., 2013). In developing mouse embryos and murine cells, CDK13 regulates hematopoiesis, stemness and axonal elongation, suggesting an important function in neuronal development (Pan et al., 2012;Chen et al., 2014). To this date, the impact of complete loss of Cdk13 function during mammalian development has not been investigated. Therefore, we employed a Cdk13 knock-out (KO) mouse model to explore a novel role of Cdk13 during mouse embryonic development. We observed embryonic lethality of Cdk13 KO animals at the embryonic day 16.5. At this stage, improper development of multiple organs has been observed (heart, brain, kidney, liver, and palate formation) resembling phenotype of human patients with de novo missense variants of CDK13 gene. Therefore, our Cdk13-deficient mice may become an important model to study dysregulation of developmental processes occurring in human patients. RESULTS Disruption of Cdk13 Gene Leads to Embryonic Lethality in Mice To examine the role of CDK13 during mouse development, the mice carrying Cdk13tm1a allele were generated at the Transgenic and Archiving Module CCP (Institute of Molecular Genetics of the CAS, Prague). The Cdk13tm1a allele of Cdk13 gene enables cessation of transcription due to presence of two strong poly A sites leading to production of the aberrant transcript of Cdk13 mRNA resulting in non-functional truncated form of CDK13 protein harboring only N-terminal part of this protein, without kinase domain and C-terminal part (Figure 1A). Heterozygous Cdk13tm1a/+mice were intercrossed to obtain Cdk13tm1a/tm1a offspring. Newborn mice were genotyped with specific sets of primers able to distinguish inserted cassette (257 bp PCR product, KO) and wild-type (179 bp PCR product, WT) alleles of Cdk13 gene (Figure 1B). Although the offspring with Cdk13 WT and heterozygous alleles was born at the expected Mendelian ratio, appeared normal and fertile, Cdk13tm1a/tm1a mice were not born at all. This finding suggested that a homozygous deficiency in Cdk13 gene leads to embryonic lethality in mice. To determine the precise stage, when the embryonic lethality occurs, mouse embryos were collected at various gestation time points (Table 1). There were no living Cdk13tm1a/tm1a embryos after the embryonic stage 15.5 (E15.5) judging by the lack of their heart beating. Moreover, from E13.5 to E16.5, we observed increased number of absorbed embryos as a reflection of empty decidua (Table 1). Based on these finding, we concluded that the deficiency of Cdk13 causes severe adverse developmental defects and consequent death from E14.5 resulting in total lethality before E16.5. To confirm the loss of CDK13 protein in Cdk13tm1a/tm1a mice, developing brain from WT and Cdk13tm1a/tm1a homozygous embryos at E14.5 were collected and western blot analyses were carried out with specific antibodies recognizing CDK13 protein. As expected, the WT form of CDK13 was present in Cdk13+/+ Frontiers in Cell and Developmental Biology | www.frontiersin.org 2August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 3 Nováková et al. Function of Cdk13 During Mouse Development FIGURE 1 | Generation of Cdk13tm1a mice. (A) Scheme of Cdk13tm1a allele. The Cdk13tm1a allele consists of strong splicing acceptor (SA), β-galactosidase gene (lacZ) and neomycine resistance gene (neo) both with poly A sites (pA), being surrounded by two FRT sites within intron 2, having two and one lox P sites within intron 2 and 4. (B) PCR genotyping from E12.5 embryos using specific primers distinguishing Cdk13tm1a, mutant (KO, upper bands) and Cdk13+, wild-type, allele (WT, lower bands). (C) Brain extracts were prepared from E14.5 embryos of Cdk13+/+,Cdk13tm1a/+and Cdk13tm1a/tm1a mice and protein levels of Ser2 and Ser5 of RNAPII, CDK13 and γ-catenin (CTNNG) were evaluated by Western blotting. The short and long expositions for CDK13 are presented to demonstrate residual expression of CDK13 in Cdk13tm1a/tm1a embryos. and Cdk13tm1a/+mice, but surprisingly, corroborated residual expression of CDK13 protein was detected in the embryonic brain of Cdk13tm1a/tm1a homozygous embryos (Figure 1C), suggesting that Cdk13tm1a/tm1a mice represent a hypomorphic TABLE 1 | Genotypes of offspring from Cdk13tm1a/+intercross. Empty Stage Cdk13+/+Cdk13tm1a/+Cdk13tm1a/tm1aLitters decidua (25%) (50%) (25%) E9.5 4 (21.1%) 12 (63.2%) 3 (15.8%)∗3 2 E10.5 4 (20%) 10 (50%) 6 (30%)∗2 0 E11.5 14 (31.8%) 23 (52.3%) 7 (15.9%)∗6 4 E12.5 31 (30.7%) 52 (51.5%) 18 (17.8%)∗,#15 13 E13.5 6 (16.2%) 18 (48.6%) 13 (35.1%)∗5 5 E14.5 48 (27.6%) 95 (54.6%) 31 (17.8%)∗,#26 9 E15.5 21 (27.5%) 34 (58%) 10 (14.5%)∗11 5 E16.5 8 (25%) 18 (56.3%) 6 (18.7%)∗,#4 0 P0 39 (33.1%) 79 (66.9%) 0 28 ∗Growth retardation, #dead embryo. mutant phenotype. Because the anti-Cdk13 antibody used in western blot recognizes its N-terminal part of CDK13, we were curious if the truncated form of CDK13, as a result of terminated transcription within intron 2, will be expressed in mice bearing the Cdk13tm1a allele. Indeed, the truncated form of CDK13 was detected in Cdk13tm1a/+and Cdk13tm1a/tm1a animals (Supplementary Figure S1). Since CDK13 was demonstrated to phosphorylate CTD of RNAPII in vitro, we decided to evaluate phosphorylation status of CTD in hypomorphic mice. Thus, the effect of CDK13 downregulation on Ser2 was evaluated in the animal tissue. Even though expression of CDK13 was significantly lowered in Cdk13tm1a/tm1a mice, no effect on either Ser2 or Ser5 phosphorylation within CTD of RNAPII was detected (Figure 1C). The Ser5 phosphorylation was checked in parallel since it is phosphorylated by other CDK kinase, CDK7 in particular (Kohoutek, 2009). Cdk13 Loss Causes Growth Retardation, Developmental Delay and Its Failure To examine deficiency of CDK13 protein in Cdk13tm1a/tm1a mice, morphology at different embryonic stages compared to the WT animals was examined and various abnormalities were Frontiers in Cell and Developmental Biology | www.frontiersin.org 3August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 4 Nováková et al. Function of Cdk13 During Mouse Development FIGURE 2 | Comparison of gross morphology of wild-type, Cdk13tm1a/+and Cdk13tm1a/tm1a embryos at various stages. Cdk13tm1a/tm1a embryos display significant growth retardation compared to wild-type and heterozygous embryos. (D–F) Detailed images of Cdk13tm1a/tm1a at relevant developmental stages. (A) Heterozygous and Cdk13tm1a/tm1a embryos at E12.5. (D) Occasional chest wall deformities manifest at hypomorphs. (B) Wild-type and Cdk13tm1a/tm1a embryos at E13.5. Cdk13tm1a/tm1a embryo exhibits nuchal edema (black arrow). (E) Hypervascularization of the peripheral vessels capillaries (black arrow). (C) Wild-type and Cdk13tm1a/tm1a embryos at E14.5. (F, top) Wild-type embryo demonstrates deep indentations between the developing fingers of embryos E14.5, although not yet separated. (F, bottom) In contrast, Cdk13tm1a/tm1a embryo appears to be 1 day delayed in development as evidenced by the shallow indentation of the footpad, which is characteristic of embryos E13.5. observed in Cdk13tm1a/tm1a embryos at each stage of gestation (Supplementary Figure S2). Observed growth retardation of Cdk13-deficient mice is presented in detail (Figure 2); however, the severity of the developmental delay was variable. On average, the Cdk13tm1a/tm1a embryos appeared to be one embryonic day behind in comparison to their littermate controls as evidenced by the shallow indentation of the footpad, which is characteristic of embryos at E13.5 (Figure 2F, bottom). Frontiers in Cell and Developmental Biology | www.frontiersin.org 4August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 5 Nováková et al. Function of Cdk13 During Mouse Development In contrast, Cdk13+/+littermates exhibited deep indentations between the developing toes (not yet separated), what is characteristic of embryos at 14.5 (Figure 2F, top). In addition, the retarded embryo exhibited nuchal edema (black arrow, Figure 2B), which correlates with the presence of cardiovascular phenotypes. Occasionally, the pericardial effusion were detected in developing Cdk13tm1a/tm1a embryos, most likely caused by dysfunction of the heart (Figure 2D). Summary of various developmental defects associated with hypomorphic Cdk13tm1a allele is presented in Supplementary Table S1. Cdk13 Is Indispensable for the Development of Several Organs To narrow down possible cause of embryonic lethality, embryos at E14.5 were contrasted with Lugol’s solution to visualize gross morphology of individual soft tissues by microCT (Figure 3). Indeed, several developmental abnormalities were detected within developing embryos. The heart wall of both ventricles in Cdk13tm1a/tm1a embryos (Figures 3B,D) appeared thinner in comparison to Cdk13+/+littermate controls (Figures 3A,C). In addition, lung, liver and kidney in Cdk13tm1a/tm1a embryos were smaller and undeveloped (Figures 3D,F,H) in comparison to Cdk13+/+littermates (Figures 3C,E,G). However, detailed 3D reconstruction of liver and kidney (Figures 3I–P) with movable display of E14.5 embryos of Cdk13tm1a/tm1a and Cdk13+/+ genotypes (Supplementary Figures S3A,B) revealed no defect in general gross morphology of these organs. To uncover possible discrepancies in developmental speed of individual organs, the volume analysis was performed with normalization to total body volume of given embryo. Liver size of Cdk13tm1a/tm1a embryos represented only about 46% in comparison to Cdk13+/+littermates (Figures 3,11). Similarly, kidney size of Cdk13tm1a/tm1a animals comprised only about 52% in comparison to Cdk13+/+. In parallel, histological sections of selected organs were analyzed at stages between E14.5 and E16 (Figure 4). Decelerated development of kidneys was identified in Cdk13tm1a/tm1a embryos at E14.5 and E16 (Figures 4D,F) including nephron differentiation as shown by altered proportional representation of individual nephron stages at E14.5 (Figure 4G). Moreover, statistically significant reduction in the number of S-shaped bodies and glomeruli was observed in Cdk13tm1a/tm1a embryos (Figure 4H). At lethality stage E16.5, kidney tissue exhibited tissue abrogation with only few, much reduced tubules visible (data not shown), very likely caused by general pre-necrotic changes. Brains of Cdk13tm1a/tm1a embryos appeared developmentally delayed as demonstrated by reduced size as compared to littermate controls. Depicted in Figure 5 are E14.5 controls and Cdk13tm1a/tm1a mutant samples from two separate litters. The two litters were developmentally at different stages, pre-palatal fusion in the control embryo depicted (Figures 5A,B) and postpalatal fusion in the control embryo depicted (Figures 5E,F). In order to assess the developmental delay in Cdk13tm1a/tm1a embryos the cell proliferation was examined by Ki67 staining, a marker of proliferating cells (Figures 5A’–H’, embryos A and C, as well as E and G are littermates, representative pictures of two embryos are shown to display variability in mutant phenotype). As evident from performed quantification, there was decrease in number of proliferating cells in Cdk13tm1a/tm1a embryos in comparison to Cdk13+/+littermate controls, but this decrease was not statistically significant (Figure 5I). The analysis of craniofacial area revealed also defective palatal shelves development in several Cdk13tm1a/tm1a embryos resulting in their insufficient horizontal growth and the formation of the cleft palate at E15.5 (Figure 6) in Cdk13tm1a/tm1a mouse. Incomplete secondary palate formation exhibited variability in severity among Cdk13-deficient animals. Observed penetrance of secondary cleft palate was 2/4 animals at E15.5. In addition, Cdk13tm1a/tm1a mouse at E15.5 had smaller number of initiated nasal glands in comparison to the controls (Supplementary Figure S4, compare A–D and B–E). Embryonic Lethality in Cdk13tm1a/tm1a Mice Is Due to Heart Failure The heart is the one of the first organs to form during mammalian development. During heart development, significant changes in organ morphology and cardiomyocyte differentiation and organization reflect the increasing needs of growing embryos for nutrition and oxygen supply. Any of these developmental steps are critical for further development of whole embryos. Therefore, we analyzed the microscopic structure of the heart and found that the heart wall of Cdk13tm1a/tm1a mice embryos was less compact in comparison to the heart wall of Cdk13+/+ mice (Figures 7C,D). Further, apparent disruption of tissue architecture was detected in Cdk13tm1a/tm1a embryos with the reduction of myocardium (Figure 7, compare C, E and G to D, F and H). The heart volume was slightly increased to 103% (organ ratio to total body volume) in Cdk13tm1a/tm1a mice compared to Cdk13+/+animals. However, the total volume of heart tissue to the organ volume was significantly lower in case of Cdk13tm1a/tm1a embryos (62.5%) in comparison to WT littermates (80%) suggestive of the thinner heart wall at E15.5 in Cdk13tm1a/tm1a embryos. Percent soft tissue volumes were measured by microCT using Bruker-microCT CT-analyzer, where the object volume representing soft tissues was divided by total VOI volume. The VOI area referring to the heart volume was selected from the dataset manually. Also, decreased expression of myosin was detected in ventricle myocardium of 14.5 hearts (Supplementary Figure S5, compare B and B’ to E and E’). Since the heart mass of Cdk13tm1a/tm1a E14.5 – E16 embryos is significantly reduced with hypomorphic muscular layers of myocardium compared with Cdk13+/+mice (heart/body ratio), we presume that heart developmental defect is the cause of embryonic lethality. In order to evaluate cardiac circulatory physiology, we performed non-invasive ultrasound Doppler imaging to quantitatively assess the hemodynamic function in E14.5 and E15.5 embryos. Out of nine Cdk13tm1a/tm1a embryos dissected at E14.5 stage, only one was found dead (Table 2). All the other Cdk13tm1a/tm1a embryos at this stage exhibited comparable blood flow velocities and velocity-time integral (VTI) in dorsal aorta (Figures 8A,B). However, this situation changed Frontiers in Cell and Developmental Biology | www.frontiersin.org 5August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 6 Nováková et al. Function of Cdk13 During Mouse Development FIGURE 3 | MicroCT analysis of wild-type and Cdk13tm1a/tm1a embryos. High-contrast differentiation resolution by X-ray computed microtomography, where Lugol’s staining was used to visualize the soft tissues. Sagittal sections through body midline (A,B) and more lateral plane at E15.5 (C,D). Horizontal sections through lung (E,F) and liver (G,H). 3D reconstruction of kidney and liver in the right side view of embryo (I,J), left side view (K,L) and caudal view (M,N) with embryo outlined in gray where segmentation of serial sections was used for the liver and kidney reconstruction. (O,P) High power of 3D imaging for liver and kidney. Horizontal view (Q,R) and sagittal detailed view (S,T) on kidney and suprarenal gland. Abbreviation used for individual organs: ag, adrenal gland; d, diencephalon; h, heart; hb, hindbrain; k, kidney; l, lung; li, liver; mb, midbrain; t, tongue; te, telencephalon; s, stomach; sc, spinal cord. Scale bar = 1 mm. dramatically at embryonic stage E15.5 (Figures 8C,D), where only few Cdk13tm1a/tm1a embryos retained normal blood flow parameters (5/16), while the rest of the embryos heart functions declined (standard measurements were not possible due to irregular or spare heart beating 11/16); moreover, an increased portion of embryos were already dead (Table 2). The assessment of cardiac function in developmental interval E14.5 – E15.5 embryos showed dramatic failure in heart function, probably corresponding to increasing needs of embryos at E15.5 for blood supply in growing organ systems, which is very challenging for the defective heart to achieve. The preserved blood flow in few Cdk13tm1a/tm1a embryos was probably due to the observed variability of the embryo size. Our findings suggest that the heart failure appears in most cases during the transition from E14.5 to E15.5. Frontiers in Cell and Developmental Biology | www.frontiersin.org 6August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 7 Nováková et al. Function of Cdk13 During Mouse Development FIGURE 4 | Gross anatomy and microscopic structure of kidney in wild-type and Cdk13tm1a/tm1a embryos. (A,B) High power view on segmented kidneys from Lugol’s stained sections and visualized by X-ray computed microtomography. Growth retardation of kidney is visible in Cdk13tm1a/tm1a embryos at E14.5 (D) and E16 (F) in contrast to littermate wild-type, Cdk13+/+, controls (Cfor E14.5 and Efor E16). (E,F) Only very few just forming glomeruli (gl) were found in Cdk13tm1a/tm1a embryos. (G) Relative quantification of individual developmental stages of nephrogenesis in Cdk13+/+and Cdk13tm1a/tm1a embryos. (H) Increased amount of renal vesicles together with the reduction of S-shaped bodies and glomeruli (gl) was found in Cdk13tm1a/tm1a embryos. The graph values denote median ±s.d., ∗p<0.05, by unpaired t-test. Scale bar (A,B) = 0.3 mm, scale bar (C–H) = 100 µm. Frontiers in Cell and Developmental Biology | www.frontiersin.org 7August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 8 Nováková et al. Function of Cdk13 During Mouse Development FIGURE 5 | Cell proliferation in brain area in wild-type and Cdk13tm1a/tm1a embryos. (A,A’,B,B’) Cdk13+/+and (C,C’,D,D’) Cdk13tm1a/tm1a embryos before palatal shelves fusion. (E,E’,F,F’) Cdk13+/+and (G,G’,H,H’) Cdk13tm1a/tm1a embryos after palatal shelves fusion. Immunohistochemical nuclear labeling of Ki67-positive cells in the frontal head sections in the lower power view (A–H) and in detail (A’–H’).(I) Mitotic index was counted as the ratio between Ki67-positive cells and total amount of prosencephalon cells in three biological triplicates for each group. The graph values denote mean ±s.d, difference is not statistically significant according to unpaired t-test (p-value: 0.1047). Ki67-positive cells - brown nuclei, Ki67-negative cells - blue nuclei (hematoxylin). Scale bar (A–C) = 1 mm; scale bar (A’–C’) = 100 µm. Cdk13 Is Expressed in Affected Organs in the Prenatal and Also Postnatal Period Currently, there is limited information about protein expression pattern of CDK13 either in developing or adult organs; therefore, we decided to evaluate expression of CDK13 protein in developing organs. First, the western blot of CDK13 protein was carried out in selected developing organs (Figure 9). As expected, expression of CDK13 was detected in organs with abnormal embryonic development. The highest protein level of CDK13 was detected in the brain, then lung, kidney and heart (Figure 9). In case of detection of CDK13 in the developing heart, four times concentrated protein lysates had to be used to detect any reproducible signal. To explore gene expression of Cdk13 in adult tissues and organs, the particular organs were isolated and the expression of Cdk13 was examined by activity of β-galactosidase (Supplementary Figure S6). Cdk13 was strongly expressed in the retina of the eye, testes, ovary, uterus, gall bladder (Supplementary Figures S6A,F–I). To a lesser extent, the Cdk13 was detected in the urinary bladder (Supplementary Figure S6D). Interestingly, rather localized, yet strong expression Frontiers in Cell and Developmental Biology | www.frontiersin.org 8August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 9 Nováková et al. Function of Cdk13 During Mouse Development FIGURE 6 | Transversal sections of the head in control Cdk13+/+and Cdk13tm1a/tm1a embryos at E15.5. Rostro-caudal view in Cdk13+/+animal (A) and four Cdk13tm1a/tm1a mutant mice to show variability in the secondary palate morphology (B–E). Palatal shelves do not meet each other in the midline (B”’,E”’) and cleft of secondary palate is visible. Abnormal shape of palatal shelves was observed also caudally with cleft expanding into the soft palate area. (A’–A””, B’–B””, C’–C””, D’–D””) are transversal sections of head in individual embryos in rostrocaudal direction. Scale bar = 100 µm. within organ structure was observed in renal pelvis in kidney, thyroid gland and heart atrium, with substantial expression in the heart ventricle (Supplementary Figures S6B,C,E). Cdk13tm1d/tm1d Mice Exhibits More Severe Phenotype and Earlier Lethality As we observed residual expression of Cdk13 in analyzed organs of hypomorphic Cdk13tm1a/tm1a mice, we decided to cross Cdk13tm1a mice with Flp-deleter mice and Cre-deleter (detailed description of the utilized transgenic strains is provided at the Section “Experimental Procedure”) mice to generate Cdk13tm1d allele with deleted exons 3 and 4 (Figure 10A). As in case of Cdk13tm1a mice, the expression of CDK13 protein was investigated in the Cdk13tm1d mice. High expression of CDK13 was detected in the developing brain of WT Cdk13+/+embryos (Figure 10B). Greatly downregulated expression of CDK13 was detected in heterozygous Cdk13tm1d/+brain with undetectable expression of CDK13 protein in homozygous Cdk13tm1/tm1d brain (Figure 10B). As in the case of Cdk13tm1a mice, no significant reproducible downregulation of Ser2 phosphorylation was observed in Cdk13tm1d/tm1d brain extract (Figure 10B). Interestingly, expected Mendelian ratios were reflected in the portion of Cdk13tm1d/tm1d mice. High number of empty decidua at E12.5 were detected reflecting increased mortality before this stage (Table 3). Critically, only 19 litters out of 42 contained Cdk13tm1d/tm1d embryos suggesting homozygous mice carrying Cdk13/tm1d alleles exhibited more severe defects in phenotype than Cdk13tm1a/tm1a mice, especially in craniofacial area with midline facial cleft (Figure 10C). The prevalence of the midline orofacial deficiency and pericardial effusion was 60.5% in Cdk13tm1d homozygous mice at E12.5-E14.5. Out of 94 cases, only two cases of Cdk13tm1a/tm1a mice had externally visible orofacial clefting (Supplementary Table S1). The prevalence of the pericardial effusion (PE) in Cdk13tm1a/tm1a embryos at E12.5E14.5 was less than 20% in comparison to high occurrence in Cdk13tm1d/tm1d embryos (Supplementary Table S1). No PE or orofacial cleft was recorded in any WT, though 6 embryos from 262 heterozygotes exhibited PE. Frontiers in Cell and Developmental Biology | www.frontiersin.org 9August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 16 Nováková et al. Function of Cdk13 During Mouse Development embedded in paraffin, and sectioned at 5 µm. Hematoxylineosin (H&E) staining was performed. Images were taken under bright field using a Leica compound microscope (DMLB2) with a Leica camera (DFC480) attached (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry Proliferating cells were visualized on E14.5 brain sections by labeling of Ki67 (positive cells are brown). Sections were pretreated in Citrate buffer, pH6, 10 mM, 20 min/97◦C in water bath and were labeled with Ki67 primary antibody (RBK027-05, Zytomed Systems). For primary antibody detection, specific secondary antibody and avidin-biotin complex were used (Vectastain kit, PK-6101, Vector laboratories). The signal was developed by DAB chromogen system (K3468, DAKO). Nuclei were counterstained by hematoxylin (blue). Mitotic index was counted as the ratio between Ki67-positive cells and total amount of cells in three biological samples for each group (three Cdk13+/+embryos, three Cdk13tm1a/tm1a embryos). Statistical significance in cell number differences between control and deficient mice were evaluated by unpaired t-test. E14.5 heart sections were pretreated in DAKO Target Retrieval (32367, DAKO) solution for 15 min/97◦C in water bath and labeled using primary antibodies against actin (sc-1615-R, Santa Cruz Biotechnology) and myosin (sc-32732, Santa Cruz Biotechnology). To detect primary antibodies, secondary Alexa Fluor antibodies (A11004, A11008, Invitrogen) were used. Nuclei were counterstained by DRAQ5TM (62251, Thermo Scientific). Images were captured on fluorescence confocal microscope Leica SP8 (Leica). Micro-Computed Tomography (micro-CT) The embryos at E13.5 or E15.5 were contrasted with Lugol’s solution to visualize gross morphology of individual soft tissues by microCT. For the purpose of motion stabilization during the micro CT scan, mouse embryo was embedded in 1% agarose gel in Falcon conical centrifuge tube. The micro-CT scan was performed using - laboratory system GE Phoenix v| tome| x L 240 (GE Sensing & InspectionTechnologies GmbH, Germany), equipped with a 180 kV/15W maximum power nanofocus X-ray tube and high contrast flat panel detector DXR250 2048 px ×2048 px with 200 µm×200 µm pixel size. The measurement was carried out in the airconditioned cabinet (21◦C) at acceleration voltage of 60 kV and X-ray tube current of 200 µA. Thousand nine hundred projections were taken over 360◦with exposure time 900 ms resulting in 5.5 µm voxel resolution. The tomographic reconstruction was realized by software GE phoenix datos| x 2.0 (GE Sensing & Inspection Technologies GmbH, Germany). Reconstructed slice data were processed using VG Studio MAX 3.1 software (Volume Graphics GmbH, Germany) and segmentation of liver, kidneys and heart was completed manually. Embryonic Ultrasound Imaging and Doppler Echocardiography Pregnant females were anesthetized on gestational day 14.5 or 15.5 (before noon) with an isoflurane/oxygen mixture (anesthesia was initiated with 3–4%, and maintained with 1–2% isoflurane), and maintained on a temperature-controlled mouse platform (with sensors for monitoring of maternal electrocardiogram, respiration and core body temperature). Maternal temperature was maintained at 34–37◦C, and maternal heart beat at ∼400 beats/minute by adjusting the level of anesthesia. An incision of about 2–3 cm in the lower abdomen was made, and a uterine horn was externalized through the incision to provide imaging access. The number of fetuses in right and left uterine horns was counted from the mother’s bladder and later during echocardiographic imaging, labeled as R1, R2, R3, etc (right side) and L1, L2, L3, etc (left side). Pre-warmed ultrasound gel (37◦C) without bubbles was applied between the individual fetuses and the transducer. B-mode imaging scanning of the whole embryo with the focus on the heart and Color Doppler and PW Doppler measurements for heart aorta and dorsal aorta were obtained using a high-frequency ultrasound system (Vevo 2100, FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) equipped with a MS550S transducer operating at a center frequency of 44 MHz. At the end of imaging of all fetuses, the mother was sacrificed by cervical dislocation and a small piece of yolk sac of each fetus was taken for genotyping. DATA AVAILABILITY All datasets generated for this study are included in the manuscript and/or the Supplementary Files. ETHICS STATEMENT All animal procedures were performed in strict accordance to the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (Veterinary Research Institute, Brno, Czechia). AUTHOR CONTRIBUTIONS JKo conceptualized and supervised the project, acquired the funding, wrote the original draft, and administrated the project. MN conceptualized the project, contributed to formal analysis, investigation, and visualization, and wrote, review, and edited the manuscript. DV contributed to investigation and visualization. MH contributed to formal analysis, investigation, and visualization. JP contributed to conceptualization, formal analysis, supervision, and investigation and wrote, review, and edited the manuscript. SP and MP contributed to investigation and visualization. RS supervised the project. MK, TZ, and JKa contributed to software development and visualization. H-CJ contributed to investigation, and wrote, review, and edited the manuscript. M-JF and MB contributed to formal analysis, supervision, writing, review, and editing. Frontiers in Cell and Developmental Biology | www.frontiersin.org 16 August 2019 | Volume 7 | Article 155 fcell-07-00155 August 6, 2019 Time: 17:21 # 17 Nováková et al. Function of Cdk13 During Mouse Development FUNDING This research was supported by grants n. 16-24043J of the Czech Science Foundation to JKo, MN, and DV. The research for this manuscript was financially supported by the Ministry of Agriculture n. MZE-RO0518, by the Ministry of Education, Youth and Sports, n. LM2015040 and LQ1604 by, and by the Ministry of Education, Youth and Sports and European Fund for Regional Development n. OP RDE CZ.02.1.01/0.0/0.0/16_013/0001789 and n. OP RDI CZ.1.05/1.1.00/02.0109 and OP RDI CZ.1.05/2.1.00/19.0395 to JP, MP, SP, and RS. Work by MB and MH was supported by the Ministry of Education, Youth and Sports of the Czech Republic (CZ.02.1.01/0.0/0.0/15_003/0000460). The cooperation between MB and TZ labs is supported by the Czech Science Foundation (17-14886S). MicroCT analyses performed by MK, TZ, and JKa were carried out under the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Program II. Work by H-CJ and M-JF was supported by Ministry of Science and Technology, Taiwan (MOST 106-2811-B-010-046 and MOST 105-2923-B-010 -002 -MY3). ACKNOWLEDGMENTS We would like to thank Assoc. Prof. S. Sevcikova for a critical comments to the manuscript and other members of the laboratory for helpful inputs. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2019.00155/ full#supplementary-material FIGURE S1 | Truncated form of CDK13 protein. (A) Predicted amino acid sequence of truncated CDK13 protein due to transcriptional block mediated by two Poly A sites introduced into the intron 2 as part of trap vector in mice bearing Cdk13tm1a allele. Black and Blue capital letters represent amino acids encoded within Exon 1 and Exon 2 of Cdk13 gene, respectively. Predicted Molecular weight of truncated CDK13 protein is 66.22 KDa (https://www.bioinformatics.org/sms/ prot_mw.html). (B) Brain extracts were prepared from E14.5 embryos of given genotype and protein levels of wild-type and truncated form of CDK13 was evaluated by Western Blotting. The antibody used throughout our study recognizes N-terminal part of CDK13, which is expressed in both forms of Cdk13, wild-type as well as truncated one. The Cdk13+/+mice contained only the wild-type CDK13 protein (WT-CDK13). The heterozygous Cdk13tm1a/+mice included wild-type (WT-CDK13) and truncated (TR-CDK13) forms of CDK13 protein. The Cdk13tm1a expressed the truncated form of CDK13 and minor wild-type CDK13 as demonstrated by immunoblot. FIGURE S2 | Comparison of gross morphology of Cdk13-deficent mice phenotype. (A–H) Wild-type Cdk13+/+,(I–P) heterozygous Cdk13tm1a/+, and (Q–X) Cdk13tm1a embryos at various stages. Growth retardation of Cdk13-deficient mice was apparent from early developmental stages (Q,R). Some embryos exhibited nuchal edema and peripheral hypervascularization (U) and abnormal craniofacial shape (S,T). Developmental delay was observed at later stages (V,W). At E16.5, each of the Cdk13tm1a/tm1a embryos was undergoing resorption (X). No morphological differences between wild-type and heterozygous embryos were observed. Scale bar = 0.1 mm. FIGURE S3 | The 3D reconstruction of selected organs in micro-CT of control Cdk13+/+and Cdk13tm1a/tm1a embryos. Overall view on embryos of Cdk13+/+ (A) and Cdk13tm1a/tm1a (B) with 3D reconstruction of heart, liver and kidney (left). Gross morphology is well visible on movable model on the right side. To visualize only one organ, click on the lower row and select heart (orange), liver (pink) or kidney (purple) organ only. Scale bar = 1 mm. FIGURE S4 | Transversal sections of the head of control Cdk13+/+and Cdk13tm1a/tm1a embryos. Transversal sections through the mouse head with details of the nasal cavity area. At E14.5, no obvious differences were observed and only one epithelial protrusion (marked by arrow) were developed close to each nasal cavity (nc) (compare Ato D). Later at E15.5, the number of nasal glands was reproducibly smaller in Cdk13tm1a/tm1a mice in comparison to control animals (compare Bto E). (C,F) Coronal section through the head of embryos at the level of the eyes. White arrows depict different complexity of the nasal cavity in wild-type, Cdk13+/+, and Cdk13tm1a/tm1a animals. Abbreviations nc – nasal cavity, arrowhead – nasal gland. Scale bar (A,B,D,F) = 100 µm and scale bar (C,F) = 1 mm. FIGURE S5 | Immunohistochemical detection of myosin and actin in the heart of Cdk13+/+and Cdk13tm1a/tm1a embryos. Sagittal sections through the mouse heart were prepared and immunohistochemical detection of myosin and actin was carried out with specific antibodies. Decreased expression of myosin was detected in ventricle myocardium, compare B and B’ to E and E’. Scale bar (A,D) = 500 µm; Scale bar (B–F, without D) −100 µm. FIGURE S6 | The expression of Cdk13 in adult mouse organs visualized by β-galactosidase activity. The expression of Cdk13 was examined by activity of β-galactosidase in adult mouse organs (A) eye, (B) heart, (C) thyroid gland, (D) urinary bladder, (E) kidney, (F) uterus, (G) gall bladder, (H) testes, and (I) ovary. FIGURE S7 | Sagittal sections through heads of Cdk13tma1 and Cdk13tm1d and Cdk13tma1 and Cdk13tm1d mice. High-contrast differentiation resolution by X-ray computed microtomography where Lugol’s staining was used to visualize the soft tissues. The Cdk13tm1a/tm1a,Cdk13tm1d/tm1d and their littermate controls are displayed at E13.5 to show overall developmental delay. FIGURE S8 | Horizontal sections through heads of Cdk13tma1 and Cdk13tm1d mice. High-contrast differentiation resolution by X-ray computed microtomography where Lugol’s staining was used to visualize the soft tissues. The Cdk13tm1a/tm1a, Cdk13tm1d/tm1d and their littermate controls are displayed at E13.5. TABLE S1 | The prevalence of developmental defects in Cdk13tma1 and Cdk13tm1d transgenic mice. REFERENCES Al-Balool, H. H., Weber, D., Liu, Y., Wade, M., Guleria, K., Nam, P. L., et al. (2011). Post-transcriptional exon shuffling events in humans can be evolutionarily conserved and abundant. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 19 August 2019 | Volume 7 | Article 155