FoxD1-driven CCN2 deletion causes axial skeletal deformities, pulmonary hypoplasia, and neonatal asphyctic death

Pulmonary fibrosis is a severely disabling disease often leading to death. CCN2 (Cellular Communication Network factor 2, also known as CTGF) is a known mediator of fibrosis and clinical trials studying anti-CCN2 efficacy in pulmonary fibrosis are currently underway. Fork head box D1 (FoxD1) transcription factor is transiently expressed in several mesenchymal cell types, including those of fetal lungs. Differentiation of FoxD1-progenitor derived pericytes into myofibroblasts involves CCN2 expression and contributes importantly to maladaptive tissue remodeling in e.g. kidney and lung fibrosis models. To generate a model for studying the contribution of CCN2 expression in FoxD1-progenitor derived cells to development of fibrotic tissue remodeling, we set out to establish a FoxD1Cre - CCN2flox/flox mouse colony. However, all double-transgenic mice died soon after birth due to asphyxia. Histopathological examination revealed a reduction in alveolar space and lung weight, and subtle axial (thoracic and cervical) skeletal deformities. Together with the previously reported association of a FoxD1 containing locus with human adolescent idiopathic scoliosis, our data suggest that the development of fatal pulmonary hypoplasia caused by selective deletion of CCN2 from FoxD1-progenitor derived mesenchymal cells was secondary to aberrant axial skeletogenesis.


Introduction
Pulmonary fibrosis is a very severe and life-threatening disease characterized by interstitial fibrosis in the lung parenchyma leading to reduced lung and diffusion capacity, with consequent respiratory failure and often death. Fibrosis is the final common pathway of maladaptive tissue remodeling and loss of organ function. Cellular Communication Network factor 2 (CCN2; also known as Connective Tissue Growth Factor, CTGF) is an important mediator of fibrosis in virtually all organs, including the kidneys and the lungs (Pan et al. 2001;Wang et al. 2019). Myofibroblasts are the main effector cells during tissue fibrosis. Transformation of mesenchyme derived lung pericytes contributes importantly to the increase in myofibroblast numbers during pulmonary and kidney fibrosis a process known to be CCN2 dependent (Shiwen et al. 2009;Hung et al. 2013).
Fork head box D1 (FoxD1) transcription factor expression is transiently expressed in multiple mesenchymal cell types in several organs. FoxD1-derived mesenchymal cells, especially pericytes, contribute importantly to e.g. experimental kidney Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12079-020-00549-4) contains supplementary material, which is available to authorized users. fibrosis and bleomycin induced pulmonary fibrosis (Hung et al. 2013). In the lung CCN2 is also an important mediator of the pericyte-endothelial interface (Hall-Glenn et al. 2012).
In order to generate a tool for the study of CCN2 expression by FoxD1-lineage cells to fibrotic tissue remodeling, we set out to establish a colony of FoxD1cre-CCN2 flox/flox mice. Surprisingly however, FoxD1cre mediated homozygous CCN2 deletion induced an early postnatal fatal phenotype characterized by pulmonary hypoplasia and postnatal asphyxiation. Moreover, subtle axial skeletal defects were observed possibly underlying the pulmonary phenotype.

Animals
The generation processes of FoxD1Cre and CCN2flox mice has been described extensively elsewhere (Liu et al. 2011;Kobayashi et al. 2014). FoxD1Cre and homozygous CCN2flox mice were cross-bred at least 5 generations prior to conduction of experiments. FoxD1Cre -CCN2 flox/flox pups were severely asphyctic and euthanized by decapitation. Heterozygous (FoxD1Cre-CCN2 flox/+ ) littermates showed no abnormalities, consistent with lack of a spontaneous phenotype in heterozygous constitutive CCN2 knockouts (Ivkovic et al. 2003). In subsequent experiments, mothers were killed before delivery on embryonic day (E)18.5 to prevent confounding secondary postnatal pathology. All fetuses in these litters were killed by decapitation immediately after opening the womb.

Whole mount skeletal stain
Whole mount skeletal staining protocol of fetal carcasses is extensively described elsewhere (Rigueur and Lyons 2014). Briefly, wholemounts were rinsed, and fixed in ethanol (95%) and acetone respectively. Cartilage was stained by Alcian blue immersion, after which mounts were rinsed in 70% and 95% EtOH respectively. Using potassium hydroxide (KOH; 1% w/v), mounts were pre-cleared. Alizarin red was used to stain calcified bone. This was followed by immersion in KOH/glycerol (1:1) solution to remove excess red staining before storage in glycerol.

Statistics
Statistical significance between groups was tested using the Student-T test using GraphPad Prism Version 8.0.1 (GraphPad, San Diego, CA). A P value below 0.05 was considered statistically significant. Error bars represented SEM.

Ethics
All experiments were conducted with permission of the animal ethics committee of the University of Utrecht.
The expression of Col1α2 mRNA was not significantly different (Fig. 1e), and also hydroxyproline/proline content was similar in both groups (Fig. 1f). The expression level of Elastin mRNA was significantly reduced in FoxD1Cre/ CCN2 flox/flox mice (P < 0.05; Fig. 1g), but mass spectrometry analysis of isodesmin as a marker for elastin fibrils showed no significant difference (Fig. 1h).
CCN2flox PCR showed a much more pronounced KO band in fetal tail-, than liver DNA, suggesting that FoxD1lineage cells contribute significantly to CCN2-expression during axial skeletal development (Fig. 3e).

Discussion
Here we show that loss of CCN2 from FoxD1-lineage cells leads to aberrant lung morphology with post-natal asphyxiation, and axial skeletal deformities.
In postnatal lungs, CCN2 is mainly expressed in terminal bronchiolar epithelium (Burgos et al. 2010), which does not derive from FoxD1 expressing progenitor cells (Hung et al. 2013). This explains why CCN2 expression levels are not altered significantly in E18.5 FoxD1Cre/CCN2 flox/flox lungs (Fig.  2). The lung hypoplasia in our FoxD1Cre/CCN2 flox/flox mice is very similar to that in constitutive CCN2-knock out mice. CCN2 is expressed in the developing lung (Burgos et al. 2010), and it has been proposed that in constitutive CCN2 -KO mice the absence of pulmonary CCN2 expression in the developing lung itself contributes importantly to pulmonary hypoplasia (Baguma-Nibasheka and Kablar 2008), but the lung hypoplasia in constitutive CCN2-knock out mice has also been interpreted as being secondary to their profound skeletal deformities (Ivkovic et al. 2003;Baguma-Nibasheka and Kablar 2008).
Normal lung development requires a structurally welldeveloped thorax (Inanlou et al. 2005) (Cameron et al. 2009). As an important regulator of enchondral ossification, CCN2 is critically involved in normal skeletal development as evidenced by severe malformations in constitutive CCN2-knockout mice (Kubota and Takigawa 2007) (Ivkovic et al. 2003;Baguma-Nibasheka and Kablar 2008). Similarly, the axial skeletal deformities in our FoxD1Cre/CCN2 flox/flox mice are most likely the direct effect of CCN2 silencing in FoxD1-lineage cells in the developing axial skeleton. This would also be consistent with the reported co-segregation of a human chromosome region spanning 5q13.2 to 13.4 including the FOXD1 gene, as a locus co-segregating with disease in multiple generations of a family with idiopathic scoliosis (Edery et al. 2011).
The similarity of the pulmonary phenotype of constitutive CCN2-knock out mice with the impaired development of fetal lungs of FoxD1Cre-CTGF flox/flox embryos in the current study suggests that also in the latter lung hypoplasia might have developed secondary to the skeletal deformities.
In summary, we report that targeted CCN2 deletion in cells expressing FoxD1 during embryonic development leads to a lethal phenotype associated with axial skeletal deformities and postnatal asphyxiation due to (possibly secondary) pulmonary hypoplasia.
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