Proliferation and Maturation: Janus and the Art of Cardiac Tissue Engineering.

During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell-derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.

B1 lymphocytes develop independently of Notch signaling during mouse embryonic development.

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.

Multiparametric identification of putative senescent cells in skeletal muscle via mass cytometry.

Senescence is an irreversible arrest of the cell cycle that can be characterized by markers of senescence such as p16, p21, and KI-67. The characterization of different senescence-associated phenotypes requires selection of the most relevant senescence markers to define reliable cytometric methodologies. Mass cytometry (a.k.a. Cytometry by time of flight, CyTOF) can monitor up to 40 different cell markers at the single-cell level and has the potential to integrate multiple senescence and other phenotypic markers to identify senescent cells within a complex tissue such as skeletal muscle, with greater accuracy and scalability than traditional bulk measurements and flow cytometry-based measurements. This article introduces an analysis framework for detecting putative senescent cells based on clustering, outlier detection, and Boolean logic for outliers. Results show that the pipeline can identify putative senescent cells in skeletal muscle with well-established markers such as p21 and potential markers such as GAPDH. It was also found that heterogeneity of putative senescent cells in skeletal muscle can partly be explained by their cell type. Additionally, autophagy-related proteins ATG4A, LRRK2, and GLB1 were identified as important proteins in predicting the putative senescent population, providing insights into the association between autophagy and senescence. It was observed that sex did not affect the proportion of putative senescent cells among total cells. However, age did have an effect, with a higher proportion observed in fibro/adipogenic progenitors (FAPs), satellite cells, M1 and M2 macrophages from old mice. Moreover, putative senescent cells from muscle of old and young mice show different expression levels of senescence-related proteins, with putative senescent cells of old mice having higher levels of p21 and GAPDH, whereas putative senescent cells of young mice had higher levels of IL-6. Overall, the analysis framework prioritizes multiple senescence-associated proteins to characterize putative senescent cells sourced from tissue made of different cell types.

Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors.

[Figure: see text].

Estradiol deficiency reduces the satellite cell pool by impairing cell cycle progression.

The size of the satellite cell pool is reduced in estradiol (E2)-deficient female mice and humans. Here, we use a combination of in vivo and in vitro approaches to identify mechanisms, whereby E2 deficiency impairs satellite cell maintenance. By measuring satellite cell numbers in mice at several early time points postovariectomy (Ovx), we determine that satellite cell numbers decline by 33% between 10 and 14 days post-Ovx in tibialis anterior and gastrocnemius muscles. At 14 days post-Ovx, we demonstrate that satellite cells have a reduced propensity to transition from G0/G1 to S and G2/M phases, compared with cells from ovary-intact mice, associated with changes in two key satellite cell cycle regulators, ccna2 and p16INK4a. Further, freshly isolated satellite cells treated with E2 in vitro have 62% greater cell proliferation and require less time to complete the first division. Using clonal and differentiation assays, we measured 69% larger satellite cell colonies and enhanced satellite cell-derived myoblast differentiation with E2 treatment compared with vehicle-treated cells. Together, these results identify a novel mechanism for preservation of the satellite cell pool by E2 via promotion of satellite cell cycling.

Pluripotent stem cell-derived myogenic progenitors remodel their molecular signature upon in vivo engraftment.

Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSCs) allows the generation of myogenic progenitors endowed with enhanced regenerative capacity. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro-generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared with fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple reinjuries, and contribute to long-term regeneration. Upon engraftment, the transcriptome of reisolated Pax3/Pax7-induced PSC-derived myogenic progenitors changes toward a postnatal molecular signature, particularly in genes involved in extracellular matrix remodeling. These findings demonstrate that Pax3/Pax7-induced myogenic progenitors remodel their molecular signature and functionally mature upon in vivo exposure to the adult muscle environment.

Persistent Fibroadipogenic Progenitor Expansion Following Transient DUX4 Expression Provokes a Profibrotic State in a Mouse Model for FSHD.

FSHD is caused by loss of silencing of the DUX4 gene, but the DUX4 protein has not yet been directly detected immunohistologically in affected muscle, raising the possibility that DUX4 expression may occur at time points prior to obtaining adult biopsies for analysis, with consequent perturbations of muscle being responsible for disease progression. To test the extent to which muscle can regenerate following DUX4-mediated degeneration, we employed an animal model with reversible DUX4 expression, the iDUX4pA;HSA mouse. We find that muscle histology does recover substantially after DUX4 expression is switched off, with the extent of recovery correlating inversely with the duration of prior DUX4 expression. However, despite fairly normal muscle histology, and recovery of most cytological parameters, the fibroadipogenic progenitor compartment, which is significantly elevated during bouts of fiber-specific DUX4 expression, does not return to basal levels, even many weeks after a single burst of DUX4 expression. We find that muscle that has recovered from a DUX4 burst acquires a propensity for severe fibrosis, which can be revealed by subsequent cardiotoxin injuries. These results suggest that a past history of DUX4 expression leads to maintained pro-fibrotic alterations in the cellular physiology of muscle, with potential implications for therapeutic approaches.

Dux facilitates post-implantation development, but is not essential for zygotic genome activation†.

Double homeobox genes are unique to eutherian mammals. It has been proposed that the DUXC clade of the double homeobox gene family, which is present in multicopy long tandem arrays, plays an essential role in zygotic genome activation (ZGA). We generated a deletion of the tandem array encoding the DUXC gene of mouse, Double homeobox (Dux), and found it surprisingly to be homozygous viable and fertile. We characterize the embryonic development and ZGA profile of knockout (KO) embryos, finding that zygotic genome activation still occurs, with only modest alterations in 2-cell embryo gene expression, no defect in in vivo preimplantation development, but an increased likelihood of post-implantation developmental failure, leading to correspondingly smaller litter sizes in the KO strain. While all known 2-cell specific Dux target genes are still expressed in the KO, a subset is expressed at lower levels. These include numerous genes involved in methylation, blastocyst development, and trophectoderm/placental development. We propose that rather than driving ZGA, which is a process common throughout the animal kingdom, DUXC genes facilitate a process unique to eutherian mammals, namely the post-implantation development enabled by an invasive placenta.

DNA aptamers against the DUX4 protein reveal novel therapeutic implications for FSHD.

Aberrant expression of the transcription factor double homeobox protein 4 (DUX4) can lead to a number of diseases including facio-scapulo-humeral muscular dystrophy (FSHD), acute lymphoblastic leukemia, and sarcomas. Inhibition of DUX4 may represent a therapeutic strategy for these diseases. By applying Systematic Evolution of Ligands by EXponential Enrichment (SELEX), we identified aptamers against DUX4 with specific secondary structural elements conveying high affinity to DUX4 as assessed by fluorescence resonance energy transfer and fluorescence polarization techniques. Sequences analysis of these aptamers revealed the presence of two consensus DUX4 motifs in a reverse complementary fashion forming hairpins interspersed with bulge loops at distinct positions that enlarged the binding surface with the DUX4 protein, as determined by crystal structure analysis. We demonstrate that insertion of specific structural elements into transcription factor binding oligonucleotides can enhance specificity and affinity.

Correction to: Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

The article Tet3 regulates cellular identity and DNA methylation in neural progenitor cells, written by Miguel R. Branco and C. Joana Marques, was originally published electronically on the publisher's internet portal.

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.

Mouse models of two missense mutations in actin-binding domain 1 of dystrophin associated with Duchenne or Becker muscular dystrophy.

Missense mutations in the dystrophin protein can cause Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) through an undefined pathomechanism. In vitro studies suggest that missense mutations in the N-terminal actin-binding domain (ABD1) cause protein instability, and cultured myoblast studies reveal decreased expression levels that can be restored to wild-type with proteasome inhibitors. To further elucidate the pathophysiology of missense dystrophin in vivo, we generated two transgenic mdx mouse lines expressing L54R or L172H mutant dystrophin, which correspond to missense mutations identified in human patients with DMD or BMD, respectively. Our biochemical, histologic and physiologic analysis of the L54R and L172H mice show decreased levels of dystrophin which are proportional to the phenotypic severity. Proteasome inhibitors were ineffective in both the L54R and L172H mice, yet mice homozygous for the L172H transgene were able to express even higher levels of dystrophin which caused further improvements in muscle histology and physiology. Given that missense dystrophin is likely being degraded by the proteasome but whole body proteasome inhibition was not possible, we screened for ubiquitin-conjugating enzymes involved in targeting dystrophin to the proteasome. A myoblast cell line expressing L54R mutant dystrophin was screened with an siRNA library targeting E1, E2 and E3 ligases which identified Amn1, FBXO33, Zfand5 and Trim75. Our study establishes new mouse models of dystrophinopathy and identifies candidate E3 ligases that may specifically regulate dystrophin protein turnover in vivo.

Oestradiol affects skeletal muscle mass, strength and satellite cells following repeated injuries.

NEW FINDINGS: What is the central question of this study? Oestradiol (E2 ) plays an important role in regulating skeletal muscle strength in females. To what extent does E2 deficiency affect recovery of strength and satellite cell number when muscle is challenged by multiple injuries? What is the main finding and its importance? E2 deficiency impairs the adaptive potential of skeletal muscle following repeated injuries, as measured by muscle mass and strength. The impairment is likely multifactorial with our data indicating that one mechanism is reduction in satellite cell number. Our findings have implications for ageing, hormone replacement and regenerative medicine in regards to maintaining satellite cell number and ultimately the preservation of skeletal muscle's adaptive potential. ABSTRACT: Oestradiol's effects on skeletal muscle are multifactorial including the preservation of mass, contractility and regeneration. Here, we aimed to determine the extent to which oestradiol deficiency affects strength recovery when muscle is challenged by multiple BaCl2 -induced injuries and to assess how satellite cell number is influenced by the combination of oestradiol deficiency and repetitive skeletal muscle injuries. A longitudinal study was designed, using an in vivo anaesthetized mouse approach to precisely and repeatedly measure maximal isometric torque, coupled with endpoint fluorescence-activated cell sorting to quantify satellite cells. Isometric torque and strength gains were lower in ovariectomized mice at several time points after the injuries compared to those treated with 17ß-oestradiol. Satellite cell number was 41-43% lower in placebo- than in oestradiol-treated ovariectomized mice, regardless of injury status or number of injuries. Together, these results indicate that the loss of oestradiol blunts adaptive strength gains and that the number of satellite cells likely contributes to the impairment.

The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by inappropriate expression of the double homeodomain protein DUX4. DUX4 has bimodal effects, inhibiting myogenic differentiation and blocking MyoD at low levels of expression, and killing myoblasts at high levels. Pax3 and Pax7, which contain related homeodomains, antagonize the cell death phenotype of DUX4 in C2C12 cells, suggesting some type of competitive interaction. Here, we show that the effects of DUX4 on differentiation and MyoD expression require the homeodomains but do not require the C-terminal activation domain of DUX4. We tested the set of equally related homeodomain proteins (Pax6, Pitx2c, OTX1, Rax, Hesx1, MIXL1 and Tbx1) and found that only Pax3 and Pax7 display phenotypic competition. Domain analysis on Pax3 revealed that the Pax3 homeodomain is necessary for phenotypic competition, but is not sufficient, as competition also requires the paired and transcriptional activation domains of Pax3. Remarkably, substitution mutants in which DUX4 homeodomains are replaced by Pax7 homeodomains retain the ability to inhibit differentiation and to induce cytotoxicity.

miR-125b promotes MLL-AF9-driven murine acute myeloid leukemia involving a VEGFA-mediated non-cell-intrinsic mechanism.

The hematopoietic stem cell-enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9 By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9-driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non-cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2 Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non-cell-intrinsic leukemia-promoting effects by an oncogenic miRNA.

A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated.

KEY POINTS: The female hormone oestrogen may protect muscle from injury by reducing inflammation but this is debatable. In this study, the inflammatory response of injured muscle from oestrogen-replete mice was comprehensively compared to that from oestrogen-deficient mice. We show that oestrogen markedly promotes movement of neutrophils, an inflammatory white blood cell type, into muscle over the first few days after injury but has only a minor effect on the movement of macrophages, another inflammatory cell type. Despite the enhancement of inflammation by oestrogen in injured muscle, we found strength in oestrogen-replete mice to recover faster and to a greater extent than it does in oestrogen-deficient mice. Our study and others indicate that lower doses of oestrogen, such as that used in our study, may affect muscle inflammation and injury differently from higher doses. ABSTRACT: Oestrogen has been shown to protect against skeletal muscle injury and a reduced inflammatory response has been suggested as a possible protective mechanism. There are, however, dissenting reports. Our objective was to conduct an unbiased, comprehensive study of the effect of oestradiol on the inflammatory response following muscle injury. Female C57BL6/J mice were ovariectomized and supplemented with and without oestradiol. Tibialis anterior muscles were freeze injured and studied primarily at 1-4 days post-injury. Oestradiol supplementation increased injured muscle gene expression of neutrophil chemoattractants (Cxcl1 and Cxcl5) and to a lesser extent that of monocyte/macrophage chemoattractants (Ccl2 and Spp1). Oestradiol markedly increased gene expression of the neutrophil cell surface marker (Ly6g) but had less consistent effects on the monocyte/macrophage cell surface markers (Cd68, Cd163 and Cd206). These results were confirmed at the protein level by immunoblot with oestradiol increasing LY6G/C content and having no significant effect on CD163 content. These findings were confirmed with fluorescence-activated cell sorting counts of neutrophils and macrophages in injured muscles; oestradiol increased the proportion of CD45+ cells that were neutrophils (LY6G+ ) but not the proportion that were macrophages (CD68+ or CD206+ ). Physiological impact of the oestradiol-enhanced neutrophil response was assessed by strength measurements. There was no significant difference in strength between oestradiol-supplemented and -unsupplemented mice until 2 weeks post-injury; strength was 13-24% greater in supplemented mice at 2-6 weeks post-injury. In conclusion, a moderate level of oestradiol supplementation enhances neutrophil infiltration in injured muscle and this is associated with a beneficial effect on strength recovery.

Generation of functional thyroid from embryonic stem cells.

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes.

Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation.

Germ Cell Nuclear Factor (GCNF) Represses Oct4 Expression and Globally Modulates Gene Expression in Human Embryonic Stem (hES) Cells.

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes, GCNF down-regulated 36% of the genes, and up-regulated 64% in undifferentiated hES cells. In addition, GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process.