Cell & Development Biology and Genetics News

Neurogenic Potential of hESC-derived Human Radial Glia is amplified by Human Fetal Cells [Stem Cell Research]

2013-04-03T04:01:00Z

Available online 3 April 2013
Publication year: 2013
Source:Stem Cell Research

The efficient production of human neocortical neurons from human embryonic stem cells (hESC) is the primary requirement for studying early stages of human cortical development. We used hESC to obtain radial glial cells (hESC-RG) and then compared them with RG cells isolated from human fetal forebrain. Fate of hESC-RG cells critically depends on intrinsic and extrinsic factors. The expression of Pax6 (intrinsic factor) has a similar neurogenic effect on hESC-RG differentiation as reported for human fetal RG cells. Factors from the microenvironment also play a significant role in determining hESC-RG cell fate. In contrast to control cultures, wherein hESC-RG generate mainly astroglia and far fewer neurons, in co-cultures with human fetal forebrain cells, the reverse was found to be true. This neurogenic effect was partly due to soluble factors from human fetal brain cultures. The detected shift towards neurogenesis has significance for developing future efficient neuro-differentiation protocols. Importantly, we established that hESC-RG cells are similar in many respects to human fetal RG cells, including their proliferative capacity, neurogenic potential, and ability to generate various cortical neuronal sub-types. Unlike fetal RG cells, the hESC-RG cells are readily available and can be standardized, features that have considerable practical advantages in research and clinics.

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2013-04-03T00:00:00Z

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Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors [Stem Cell Research]

2013-04-03T04:01:00Z

Available online 2 April 2013
Publication year: 2013
Source:Stem Cell Research

Understanding how to specify rapid differentiation of human neural progenitor towards enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5 days of differentiation, with near complete suppression of neuronal differentiation. The resultant cells express major astrocytic markers, are amenable to co-culture with neurons, can be propagated as astrocyte progenitors and are cryopreservable. Although previous reports have generated astrocytes from pluripotent cells, the differentiation required extensive culture or selection based on cell surface antigens. The development of a label free and rapid differentiation process will expedite future derivation of astrocytes from various sources pluripotent cells including, but not limited to, human astrocytes associated with various neurological diseases.

Enhanced derivation of mouse embryonic stem cell-derived cortical interneurons by induced expression of Nkx2.1 [Stem Cell Research]

2013-04-03T04:01:00Z

Available online 1 April 2013
Publication year: 2013
Source:Stem Cell Research

Forebrain GABAergic interneurons are divided into subgroups based on their neurochemical markers, connectivity and physiological properties. Abnormal interneuron function is implicated in the pathobiology of neurological disorders such as schizophrenia, autism, and epilepsy. Studies on interneuron development and their role in disease would benefit from an efficient mechanism for the production and selection of specific interneuron subgroups. In this study, we engineered a mouse embryonic stem cell (mESC) line for doxycycline-inducible expression of Nkx2.1, a required transcription factor for cortical interneurons derived from the medial ganglionic eminence (MGE). This mESC line was modified to express GFP in Lhx6⁺ cells, a marker of newly postmitotic and mature MGE-derived cortical interneurons. Addition of doxycycline to differentiating ESCs efficiently induced Nkx2.1 protein and increased the production of GFP⁺ cells. Transplantation of GFP⁺ putative interneuron precursors resulted in migratory, morphological, and neurochemical features consistent with cortical interneuron fates. To test the hypothesis that Sonic hedgehog (Shh) primarily influences cortical interneuron fate determination through the induction of Nkx2.1, ESCs were grown with doxycycline and the Shh antagonist cyclopamine. We found induced Nkx2.1 renders Shh signaling dispensable for the generation of MGE-derived interneurons. These results demonstrate that inducible expression of fate determining genes in embryonic stem cells can be used to study fate determination of the developing forebrain.

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2013-04-01T00:00:00Z

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2013-04-01T00:00:00Z

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Stage Specific Requirement of Gfrα1 in the Ureteric Epithelium during Kidney Development [Mechanism of Developmentα1 in the Ureteric Epithelium during Kidney Development]

2013-03-31T22:01:00Z

Available online 28 March 2013
Publication year: 2013
Source:Mechanisms of Development

Glial cell line-derived neurotrophic factor (GDNF) binds a coreceptor GDNF family receptor α1 (GFRα1) and forms a signaling complex with the receptor tyrosine kinase RET. GDNF-GFRα1-RET signaling activates cellular pathways that are required for normal induction of the ureteric bud (UB) from the Wolffian duct (WD). Failure of UB formation results in bilateral renal agenesis and perinatal lethality. Gfrα1 is expressed in both the epithelial and mesenchymal compartments of the developing kidney while Ret expression is specific to the epithelium. The biological importance of Gfrα1’s wider tissue expression and its role in later kidney development are unclear. We discovered that conditional loss of Gfrα1 in the WD epithelium prior to UB branching is sufficient to cause renal agenesis. This finding indicates that Gfrα1 expressed in the nonepithelial structures cannot compensate for this loss. To determine Gfrα1’s role in branching morphogenesis after UB induction we used an inducible Gfrα1 -specific Cre-deletor strain and deleted Gfrα1 from the majority of UB tip cells post UB induction in vivo and in explant kidney cultures. We report that Gfrα1 excision from the epithelia compartment after UB induction caused a modest reduction in branching morphogenesis. The loss of Gfrα1 from UB-tip cells resulted in reduced cell proliferation and decreased activated ERK (pERK). Further, cells without Gfrα1 expression are able to populate the branching UB tips. These findings delineate previously unclear biological roles of Gfrα1 in the urinary tract and demonstrate its cell-type and stage-specific requirements in kidney development.

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2013-03-29T00:00:00Z

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2013-03-29T00:00:00Z

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New potential cell source for hepatocyte transplantation: Discarded livers from metabolic disease liver transplants [Stem Cell Research]

2013-04-03T04:01:00Z

Available online 27 March 2013
Publication year: 2013
Source:Stem Cell Research

Domino liver transplantation is a method used to increase the number of liver grafts available for orthotopic liver transplantation (OLT). Reports indicate that livers from patients with metabolic liver disease can be safely transplanted into select recipients if the donor’s defect and the recipient’s metabolic needs are carefully considered. The liver of patients with many types of metabolic liver disease are morphologically and biochemically normal, except for the mutation that characterizes that disease. Other biochemical functions normally performed by liver are present and presumably “normal” in these hepatocytes. Hepatocytes were isolated from the liver of 35 organ donors and 35 liver tissues taken at OLT from patients with liver disease were analyzed for 9 different measures of viability and function. The data indicate that cells isolated from some diseased livers performed as well or better than those isolated from organ donors with respect to viability, cell yield, plating efficiency and in assays of liver function, including drug metabolism, conjugation reactions and ammonia metabolism. Cells from metabolic diseased livers rapidly and efficiently repopulated a mouse liver upon transplantation. Conclusions: As with domino liver transplantation, domino cell transplantation deserves consideration as method to extend the pool of available organs and cells for transplantation.

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2013-03-27T00:00:00Z

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A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.

Highlights

► A novel endoglucanase gene was cloned from DNA isolated from rice straw compost. ► Less than 3000 clones were screened to isolate the RS-EG1 endoglucanase gene. ► RS-EG1 shares about 70% similarity with the closest known bacterial cellulases. ► RS-EG1 is stable over broad temperature and pH ranges. ► RS-EG1 is potentially useful in cellulosic biofuel production.

Association between 1603C>T polymorphism of DBH gene and bipolar disorder in a Turkish population

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

Objectives Dopamine-β-hydroxylase (DBH) is the enzyme responsible for the conversion of dopamine (DA) to norepinephrine (NE, noradrenaline) which is a key neurotransmitter in the central and peripheral nervous systems. Bipolar disorder is a major psychiatric disorder. The present study was designed to explore the associations of polymorphisms of DBH gene in Turkish patients with bipolar disorder. Methods −1021C>T (rs1611115) polymorphism in promoter region, 444G>A (rs1108580) polymorphism in exon 2 and 1603C>T (rs6271; C535R) polymorphism in exon11 of DBH gene were analyzed in 106 patients with bipolar disorder and 106 healthy subjects by using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Results The results showed statistically significant associations for genotypic and allelic distribution between the 1603C>T polymorphism and bipolar disease (p=0.0012 and p=0.034, respectively). There was no association observed between the genotype and allelic frequencies for −1021C>T and 444G>A polymorphisms and bipolar disorder. Conclusions Our data suggests that the 1603C>T polymorphism of the DBH gene is associated with susceptibility to bipolar disorder in a Turkish population.

Highlights

► Decreased DBH plasma activity was observed in patients with bipolar disorder. ► 1603C>T polymorphism is associated with susceptibility to bipolar disorder. ► −1021C>T and 444G>A polymorphisms are not associated with bipolar disorder.

Association of the −1082G/A polymorphism in the interleukin-10 gene with systemic lupus erythematosus: A meta-analysis

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

A great many studies have investigated the −1082G/A polymorphism (rs1800896) in the interleukin-10 gene (IL10) with SLE susceptibility, but the results are inconsistent and inconclusive. The aim of this meta-analysis was in order to more precisely estimate the relationship. The databases of Pubmed and Web of Science updated to Oct, 2012 were retrieved. Odds ratio (OR) and corresponding 95% confidence interval (95%CI.) as effect size were calculated by fixed-effect model. Analysis for allele contrast of stratification by ethnicity in either Asian or Caucasian, as well as in overall population indicated no significant association (Overall: OR 1.096, 95%CI. 0.995–1.207; Asian: OR 1.204, 95%CI.: 0.944–1.535; Caucasian: OR 1.075, 95%CI.: 0.961–1.202). Analysis for recessive model showed no association in overall populations and in Caucasian (Overall: OR 1.135, 95%CI.: 0.945–1.362; Caucasian: OR 1.069, 95%CI.: 0.882–1.296), but significant association in Asian (OR: 2.848; 95%CI.: 1.194–6.791). Analysis for dominant model indicated that the variant G allele carriers (GG+GA) may have increased the risk of SLE when compared with the homozygote AA in overall populations and in Caucasian (Overall: OR 1.203, 95%CI.: 1.029–1.407; Caucasian: OR 1.233, 95%CI.: 1.014–1.499), but not in Asian (OR: 1.154; 95%CI.: 0.856–1.557). Significant association was found by using homozygote contrast model in overall populations and Asian (Overall: OR 1.303, 95%CI.: 1.031–1.648; Asian: OR 3.206, 95%CI.: 1.241–8.284), while no association was found in Caucasian (OR: 1.209; 95%CI.: 0.940–1.556). The results provided evidence for the association between the IL10 −1082G/A polymorphism and the risk of SLE. To confirm these findings, more case–control studies with subtle study design based on adequately sized populations are required.

Highlights

► The IL10 −1082G/A polymorphism was associated with SLE susceptibility. ► IL10 −1082G/A polymorphism may play a role in SLE susceptibility. ► IL10 −1082G/A polymorphism may have ethnicity-specific effects in SLE. ► Further studies on this issue should have a subtle study design.

Association of two ERCC4 tagSNPs with susceptibility to atrophic gastritis and gastric cancer in Chinese

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

Genetic polymorphisms in excision repair cross-complementing group 4 (ERCC4) may contribute to the risk of cancer development. However, there are few reports regarding to susceptibility to gastric cancer (GC) or its precursor, atrophic gastritis (AG). Thereby, we investigated the association between two tag single nucleotide polymorphisms (tagSNPs) rs6498486 and rs254942, which represents the majority of common SNPs of ERCC4 gene, and the risks of GC and AG development in a sex- and age-matched case–control designed study. We found that rs6498486 polymorphism was associated with a reduced AG risk in total population (for AC vs. AA: OR=0.69, 95%CI=0.52–0.94, P=0.016; for AC/CC vs. AA: OR=0.68, 95%CI=0.51–0.92, P=0.010) as well as in the subpopulation of youngers (age<60years) (for AC/CC vs. AA: OR=0.67, 95%CI=0.45–0.99, P=0.048). For the rs254942 polymorphism, compared with the common TT genotype, the genotypes of CT and CT/CC were only observed to reduce AG risk in the subgroups of males (for CT vs. TT: OR=0.64, 95%CI=0.45–0.90, P=0.012; for CT/CC vs. TT: OR=0.66, 95%CI=0.47–0.92, P=0.016) and youngers (for CT vs. TT: OR=0.72, 95%CI=0.53–0.97, P=0.035; for CT/CC vs. TT: OR=0.74, 95%CI=0.55–0.99, P=0.045). However, no significant statistical association of the two SNPs with GC susceptibility was observed in the total population. Only rs6498486 AC and AC/CC genotypes were found to be marginally associated with a reduced GC risk in the subgroup of males (for AC vs. AA: OR=0.69, 95%CI=0.49–0.99, P=0.043; for AC/CC vs. AA: OR=0.71, 95%CI=0.50–0.99, P=0.046). Our findings suggested that the ERCC4 rs6498486 and rs254942 may be associated with AG risk. Further validation of our results in larger populations and additional studies evaluating their molecular function are required.

Highlights

► Two potential functional tagSNPs of ERCC4 were investigated in Chinese population. ► ERCC4 rs6498486 may be associated with a reduced atrophic gastritis risk. ► ERCC4 rs254942 was also associated with atrophic gastritis risk. ► H. pylori infection may enhance ERCC4 genetic effects. ► ERCC4 polymorphisms may be implicated in gastric carcinogenesis process.

Cell cycle arrest in Batten disease lymphoblast cells

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

Batten disease is an inherited neurodegenerative disorder caused by a CLN3 gene mutation. Batten disease is characterized by blindness, seizures, cognitive decline, and early death. Although apoptotic cell death is one of the pathological hallmarks of Batten disease, little is known about the regulatory mechanism of apoptosis in this disease. Since the CLN3 gene is suggested to be involved in the cell cycle in a yeast model, we investigated the cell cycle profile and its regulatory factors in lymphoblast cells from Batten disease patients. We found G1/G0 cell cycle arrest in Batten disease cells, with overexpression of p21, sphingosine, glucosylceramide, and sulfatide as possible cell cycle regulators.

Characterization of Cherax quadricarinatus prohibitin and its potential role in spermatogenesis

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

Prohibitin (PHB) proteins have diverse functions, such as cellular signaling, transcriptional control and mitochondrial biogenesis. In this study, we characterized PHB gene and its protein expression in Cherax quadricarinatus. PHB cDNA comprises 1472 nucleotides with an open reading frame of 828bp, which encodes 275 amino acid residues. The highest transcript levels were found during the spermatogonial developmental phase, with the lowest levels detected during the resting phase in the reproductive cycle. Western blot analysis revealed that PHB is an approximately 30kDa protein, and occurs in a number of unexpected isoforms, ranging from 30kDa to greater than 180kDa in the testes of different developmental phases, which may be the ubiquitinated substrates. The strongest immunolabeling signal was found in spermatogonia, with lower levels of staining in secondary spermatocytes, and weak or absent expression in mature sperm. Immunogold electron microscopy results confirmed the localization of PHB in the inner mitochondrial membranes. The results showed that PHB is a substrate protein for spermatogenesis, with a potential reproductive function involving sperm ubiquitination in invertebrates.

Highlights

► We characterized a novel mitochondrial protein PHB in Cherax quadricarinatus. ► PHB may occur as multiple ubiquitinated substrates. ► IEM results confirmed the localization of PHB in the inner mitochondrial membrane. ► Our data suggest that PHB is an essential substrate protein for spermatogenesis. ► PHB may be involved in sperm ubiquitination and mitochondrial DNA inheritance.

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.

Highlights

► We have characterized and expressed the ram seminal plasma protein RSVP14 gene. ► The cDNA sequence shows numerous biochemical similarities with BSP proteins. ► One CRAC and two FN-2 domains suggest a role for RSVP14 in capacitation. ► The results might explain the RSVP14 protective effect against cold-shock injury. ► The recombinant protein was obtained in insect cell extracts and E. coli.

Characterization of the complete mitochondrial genome of Bombyx mori strain H9 (Lepidoptera: Bombycidae)

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

The complete mitochondrial genome (mitogenome) of Bombyx mori strain H9 (Lepidoptera: Bombycidae) is 15,670base pairs (bp) in length, encoding 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The nucleotide composition of the genome is highly A+T biased, accounting for 81.31%, with a slightly positive AT skewness (0.059). The arrangement of 13 PCGs is similar to that of other sequenced lepidopterans. All the PCGs are initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which is proposed by the TTAG sequence as observed in other lepidopterans. Unlike the other PCGs, the cox1 and cytochrome c oxidase subunit 2 (cox2) genes have incomplete stop codons consisting of just a T. All tRNAs have typical structures of insect mitochondrial tRNAs, which is different from other sequenced lepidopterans. The structure of A+T-rich region is similar to that of other sequenced lepidopterans, including non-repetitive sequences, the ATAGA binding domain, a 18bp poly-T stretch and a poly-A element upstream of transfer RNA M (trnM) gene. Phylogenetic analysis shows that the domesticated silkmoth B. mori originated from the Chinese Bombyx mandarina.

Highlights

► The complete mitochondrial genome of Bombyx mori H9 was firstly determined. ► This mitochondrial genome shows a typical pattern of Lepidoptera insects. ► B. mori H9 has a close evolutionary relationship to B. mori Xiafang.

Distinct function of P63 isoforms during embryonic skeletal development

2013-04-02T22:00:00Z

1 May 2013
Publication year: 2013
Source:Gene, Volume 519, Issue 2

P63 belongs to the P53 family of transcription factors. There are multiple P63 isoforms that play important functions both in cancer and development. The obvious limb defect in p63 null mice and in human skeletal syndromes with P63 mutations suggest its essential role in long bone development. However, how the different P63 isoforms function during long bone development is largely unknown. We have previously shown that TAP63α, the longest P63 isoform, plays a positive role in embryonic skeletal development, since targeting TAP63α expression in hypertrophic chondrocytes accelerates endochondral ossification at both E17.5 and P1 stages. Here, we report transgenic studies of ΔNP63α, another P63 isoform which lacks the N-terminal transactivation domain compared to TAP63α, using the same hypertrophic chondrocyte-specific Col10a1 control element. No skeletal abnormalities were detected in these Col10a1-ΔNP63α transgenic mice at both E17.5 and P1 stages, suggesting less importance of ΔNP63α during late embryonic skeletal development. To further investigate the function of P63 isoforms during early skeletal development, we have generated ΔNP63α and TAP63α transgenic mice using a chondrocyte-specific Col2a1 control element. Surprisingly, while no skeletal defect was shown in the Col2a1-ΔNP63α transgenic mice, reduced ossification was observed in the digit and tail bones of Col2a1-TAP63α transgenic mice at both E17.5 and P1 stages compared to their wild-type littermates. Expression profiling and immunohistochemical analysis detected upregulated expression of Sox9, a major negative regulator of endochondral ossification, in Col2a1-TAP63α transgenic mice. Taken together, our results suggest a distinct function of P63 isoforms, herein, ΔNP63α and TAP63α, during endochondral ossification.

Highlights

► Transgenic mice expressing P63 variants were generated using Col2a1/Col10a1 elements. ► No skeletal abnormalities were observed in Col2a1- or Col10a1-ΔNP63α transgenic mice. ► Delayed ossification and increased Sox9 expression were shown in Col10a1-ΔNP63α mice. ► Conclusion: P63 variants play distinct function during embryonic skeletal development.