yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.

The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Folate deficiency in early development leads to disturbance in multiple processes, including neurogenesis during which fibroblast growth factor (FGF) pathway is one of the crucial pathways. Whether folic acid (FA) directly affects FGF pathways to influence neurodevelopment and the possible mechanism remains unclear. In this study, we presented evidence that in human FA-insufficient encephalocele, the FGF pathway was interfered. Furthermore, in Brachyury knockout mice devoid of such T-box transcription factors regulating embryonic neuromesodermal bipotency and a key component of FGF pathway, change in expression of Brachyury downstream targets, activator Fgf8 and suppressor dual specificity phosphatase 6 was detected, along with the reduction in expression of other key FGF pathway genes. By using a FA-deficient cell model, we further demonstrated that decrease in Brachyury expression was through alteration in hypermethylation at the Brachyury promoter region under FA deficiency conditions, and suppression of Brachyury promoted the inactivation of the FGF pathway. Correspondingly, FA supplementation partially reverses the effects seen in FA-deficient embryoid bodies. Lastly, in mice with maternal folate-deficient diets, aberrant FGF pathway activity was found in fetal brain dysplasia. Taken together, our findings highlight the effect of FA on FGF pathways during neurogenesis, and the mechanism may be due to the low expression of Brachyury gene via hypermethylation under FA-insufficient conditions.-Chang, S., Lu, X., Wang, S., Wang, Z., Huo, J., Huang, J., Shangguan, S., Li, S., Zou, J., Bao, Y., Guo, J., Wang, F., Niu, B., Zhang, T., Qiu, Z., Wu, J., Wang, L. The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Antitumor Efficacy of Huqizhengxiao (HQZX) Decoction Based on Inhibition of Telomerase Activity in Nude Mice of Hepatocarcinoma Xenograft.

OBJECTIVE: Huqizhengxiao (HQZX) decoction is a mixture of traditional Chinese medicines comprising 10 herbs, with inhibitory effects on hepatocarcinoma. The aim of the study is to observe the antitumor efficacy and mechanism of HQZX decoction in nude mice with hepatocellular carcinoma xenografts. METHODS: HepG2-luc subcutaneous hepatocarcinoma was established in nude mice. The mice were divided into 5 groups: control, cinobufagin, HQZXS, HQZXM, and HQZXH with doses 13.52, 27.03, and 54.06 g/kg, respectively. HQZX decoction was prepared for intraperitoneal intragastric administration for 3 weeks. Tumor growth was measured with Vernier calipers and in vivo imaging system. α-Fetoprotein (AFP) was determined by radioimmunoassay. Tumor necrosis factor-α (TNF-α) was measured with enzyme-linked immunosorbent assay (ELISA) assay. Telomerase activity was measured with polymerase chain reaction-ELISA. Nuclear mitosis and necrosis were observed with hematoxylin-eosin stain. Apoptotic proteins of caspase-3, Bcl-2, and Bax were examined by Western blot. Signaling molecules of ERK, mTOR, and STAT3 were measured with Luminex assay. RESULTS: HQZX decoction showed good inhibition of HepG2-luc xenografts. Compared with control group, the relative tumor proliferation rate was less than 60% in the HQZXH and HQZXS. The tumor inhibition rate of HQZXH group reached 52% ± 15%. Relative average optical density values of the HQZXS and HQZXH groups decreased significantly. The mitotic index in HQZXS, HQZXM, and HQZXH groups decreased greatly. Telomerase activity of HQZXS was clearly reduced, and, the caspase-3 expression upregulated in HQZXH group. Bcl-2 expression was downregulated in HQZXS and HQZXH. The ratios of p-ERK/ERK and p-STAT3/STAT3 in HQZXS group were significantly downregulated. CONCLUSION: HQZX decoction can clearly inhibit the growth of hepatocellular carcinoma and induce tumor apoptosis. Its antitumor mechanism may be related to reducing telomerase activity and regulating the STAT3 and ERK signal pathway.

An autocrine loop between TGF-ß1 and the transcription factor brachyury controls the transition of human carcinoma cells into a mesenchymal phenotype.

The epithelial-mesenchymal transition (EMT) is a process associated with the metastasis of solid tumors as well as with the acquisition of resistance to standard anticancer modalities. A major initiator of EMT in carcinoma cells is TGF-ß, which has been shown to induce the expression of several transcription factors ultimately responsible for initiating and maintaining the EMT program. We have previously identified Brachyury, a T-box transcription factor, as an inducer of mesenchymal features in human carcinoma cells. In this study, a potential link between Brachyury and TGF-ß signaling has been investigated. The results show for the first time that Brachyury expression is enhanced during TGF-ß1-induced EMT in various human cancer cell lines, and that a positive feedback loop is established between Brachyury and TGF-ß1 in mesenchymal-like tumor cells. In this context, Brachyury overexpression is shown to promote upregulation of TGF-ß1 at the mRNA and protein levels, an effect mediated by activation of the TGF-ß1 promoter in the presence of high levels of Brachyury. Furthermore, inhibition of TGF-ß1 signaling by a small-molecule inhibitor of TGF-ß receptor type I decreases Brachyury expression, induces a mesenchymal-to-epithelial transition, and renders cancer cells more susceptible to chemotherapy. This study thus has implications for the future development of clinical trials using TGF-ß inhibitors in combination with other anticancer agents.

Comparing immobilized kinase inhibitors and covalent ATP probes for proteomic profiling of kinase expression and drug selectivity.

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification in vivo. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼ 100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets.

The beneficial effect of fiber supplementation in high- or low-fat diets on fetal development and antioxidant defense capacity in the rat.

BACKGROUND: There is mounting evidence that an imbalance in oxidant/antioxidant activities plays a pivotal role in fetal development. AIM OF THE STUDY: To determine the effects of maternal intake of fat and fiber on fetal intrauterine development and antioxidant defense systems of rats. METHODS: Virgin female Sprague-Dawley rats were randomly assigned to 4 groups according to diet: the low-fat, low-fiber group (LL); the low-fat, high-fiber group (LH); the high-fat, low-fiber group (HL); and the high-fat, high-fiber group (HH). The diets were fed 4 weeks prior to breeding through day 17.5 of pregnancy. Dietary intakes of fiber (wheat bran and oat) and fat were quantitatively varied, while intakes of energy and essential nutrients were kept constant among the diets. RESULTS: Rats fed a fiber-rich diet had significantly improved fetal numbers, as well as enhanced activity of superoxide dismutase (SOD) and capacity of scavenging free radicals (p < 0.05). Meanwhile, the placental malondialdehyde and protein carbonyl levels were affected by the diet fat and fiber levels (p < 0.05). Compared with the LL group, the mRNA abundance of hypoxia-inducible factor 1α (HIF-1α) and thioredoxin-2 (Trx2) in the maternal liver and glutathione peroxidase 1 (GPx1) in the placenta and fetus were significantly downregulated in the HL group (p < 0.05). Furthermore, rats fed a fiber-rich diet had significantly upregulated mRNA expressions of Cu,Zn-SOD, Mn-SOD, and HIF-1α in the maternal liver (p < 0.05); Cu,Zn-SOD and Mn-SOD in the placenta (p < 0.05); and Cu,Zn-SOD in the fetus (p < 0.05). CONCLUSION: When energy intakes are equivalent, consumption of fiber in high- or low-fat diets benefits fetal development and growth, through improvements in maternal, placental, and fetal antioxidant defense capacities.

ω-3 Polyunsaturated fatty acids prevent pressure overload-induced ventricular dilation and decrease in mitochondrial enzymes despite no change in adiponectin.

BACKGROUND: Pathological left ventricular (LV) hypertrophy frequently progresses to dilated heart failure with suppressed mitochondrial oxidative capacity. Dietary marine ω-3 polyunsaturated fatty acids (ω-3 PUFA) up-regulate adiponectin and prevent LV dilation in rats subjected to pressure overload. This study 1) assessed the effects of ω-3 PUFA on LV dilation and down-regulation of mitochondrial enzymes in response to pressure overload; and 2) evaluated the role of adiponectin in mediating the effects of ω-3 PUFA in heart. METHODS: Wild type (WT) and adiponectin-/- mice underwent transverse aortic constriction (TAC) and were fed standard chow ± ω-3 PUFA for 6 weeks. At 6 weeks, echocardiography was performed to assess LV function, mice were terminated, and mitochondrial enzyme activities were evaluated. RESULTS: TAC induced similar pathological LV hypertrophy compared to sham mice in both strains on both diets. In WT mice TAC increased LV systolic and diastolic volumes and reduced mitochondrial enzyme activities, which were attenuated by ω-3 PUFA without increasing adiponectin. In contrast, adiponectin-/- mice displayed no increase in LV end diastolic and systolic volumes or decrease in mitochondrial enzymes with TAC, and did not respond to ω-3 PUFA. CONCLUSION: These findings suggest ω-3 PUFA attenuates cardiac pathology in response to pressure overload independent of an elevation in adiponectin.

Retinoic acid and Wnt/beta-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus.

A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.

A zinc finger transcription factor, ZicL, is a direct activator of Brachyury in the notochord specification of Ciona intestinalis.

In ascidian embryos, Brachyury is expressed exclusively in blastomeres of the notochord lineage and play an essential role in the notochord cell differentiation. The genetic cascade leading to the transcriptional activation of Brachyury in A-line notochord cells of Ciona embryos begins with maternally provided beta-catenin, which is essential for endodermal cell specification. beta-catenin directly activates zygotic expression of a forkhead transcription factor gene, FoxD, at the 16-cell stage, which in turn somehow activates a zinc finger transcription factor gene, ZicL, at the 32-cell stage, and then Brachyury at the 64-cell stage. One of the key questions to be answered is whether ZicL functions as a direct activator of Brachyury transcription, and this was addressed in the present study. A fusion protein was constructed in which a zinc finger domain of Ciona ZicL was connected to the C-terminus of GST. Extensive series of PCR-assisted binding site selection assays and electrophoretic mobility shift assays demonstrated that the most plausible recognition sequence of Ciona ZicL was CCCGCTGTG. We found the elements CACAGCTGG (complementary sequence: CCAGCTGTG) at -123 and CCAGCTGTG at -168 bp upstream of the putative transcription start site of Ci-Bra in a previously identified basal enhancer of this gene. In vitro binding assays indicated that the ZicL fusion protein binds to these elements efficiently. A fusion gene construct in which lacZ was fused with the upstream sequence of Ci-Bra showed the reporter gene expression exclusively in notochord cells when the construct was introduced into fertilized eggs. In contrast, fusion constructs with mutated ZicL-binding-elements failed to show the reporter expression. In addition, suppression of Ci-ZicL abolished the reporter gene expression, while ectopic and/or overexpression of Ci-ZicL resulted in ectopic reporter expression in non-notochord cells. These results provide evidence that ZicL directly activates Brachyury, leading to specification and subsequent differentiation of notochord cells.

Roles of Eph receptors and ephrins in segmental patterning.

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have key roles in patterning and morphogenesis. Interactions between these molecules are promiscuous, but largely fall into two groups: EphA receptors bind to glycosylphosphatidyl inositol-anchored ephrin-A ligands, and EphB receptors bind to transmembrane ephrin-B proteins. Ephrin-B proteins transduce signals, such that bidirectional signalling can occur upon interaction with the Eph receptor. In many tissues, there are complementary and overlapping expression domains of interacting Eph receptors and ephrins. An important role of Eph receptors and ephrins is to mediate cell contact-dependent repulsion, and this has been implicated in the pathfinding of axons and neural crest cells, and the restriction of cell intermingling between hindbrain segments. Studies in an in vitro system show that bidirectional activation is required to prevent intermingling between cell populations, whereas unidirectional activation can restrict cell communication via gap junctions. Recent work indicates that Eph receptors can also upregulate cell adhesion, but the biochemical basis of repulsion versus adhesion responses is unclear. Eph receptors and ephrins have thus emerged as key regulators that, in parallel with cell adhesion molecules, underlie the establishment and maintenance of patterns of cellular organization.

Glucocorticoid deficiency delays stratum corneum maturation in the fetal mouse.

The stratum corneum (SC) matures during late gestation in man and other mammals. Using the fetal rat as an experimental model, we have previously shown that glucocorticoids given in pharmacologic doses accelerate fetal SC maturation and barrier formation. To determine whether glucocorticoids are required for normal SC maturation, we examined the epidermal morphology of glucocorticoid-deficient (C-) murine pups, derived from matings of mice homozygous for null mutations of the corticotropin-releasing hormone alleles. In control pups on day 17.5 of gestation (term is 19.5 d), a multilayered SC was present and neutral lipid deposition in a membrane pattern was observed using Nile red fluorescence histochemistry. Ultrastructurally, mature lamellar unit structures predominate in the SC intercellular domains. In contrast, in C-pups only a single layer of SC was evident on day 17.5, and secreted lamellar material was not organized into mature lamellar structures. Furthermore, the expression of structural proteins necessary for cornified envelope formation, involucrin, loricrin, and filaggrin, and the activity of the lipid synthetic enzymes beta-glucocerebrosidase and steroid sulfatase, markers of barrier maturation, were reduced in day 17.5 C-pups. C-pups derived from pregnancies supplemented with physiologic amounts of cortisone, however, display normal SC ultrastructure on day 17.5 of gestation. Furthermore, at birth, both control and C-pups exhibit a multilayered SC replete with mature lamellar membrane structures. These data demonstrate that fetal glucocorticoid deficiency delays SC maturation, and suggests that normal levels of glucocorticoids are not absolutely required for SC development.

Expression and activity of hexokinase in the early mouse embryo.

The maximal activity and Michaelis constant, KM, of hexokinase have been measured in the peri-implantation mouse embryo using an ultramicrofluorescence technique. In addition, transcript detection of the predominant isoenzyme hexokinase I has been determined in single preimplantation mouse embryos at successive stages of development using reverse transcriptase-mediated cDNA amplification. Maximal hexokinase activity decreased dramatically peri-implantation, from 0.97 +/- 0.19 nmol/microgram protein/h at the blastocyst stage to 0.31 +/- 0.05 nmol/microgram protein/h on day 6.5. The KM remained relatively low and constant over this period (0.23-0.39 mM), indicating the absence of the hexokinase type IV isoenzyme. The pattern of hexokinase activity resembled that of glucose consumption suggesting a possible regulatory role for the enzyme during this period of development. Hexokinase I mRNA was detected in the oocyte and all preimplantation stages of development. The blastocyst polymerase chain reaction (PCR) product, when cloned and sequenced was found to be 98% homologous with mouse tumour hexokinase I. Taken together, these data suggest that the hexokinase gene is not under transcriptional control during early mouse embryo development but plays a significant role in the regulation of glucose consumption. A role for hexokinase in the phosphate-induced inhibition of early embryo development is also proposed.

Characterisation of the Sek-1 receptor tyrosine kinase.

We present an initial characterisation of the mouse Sek-1 protein, a member of the Eph subfamily of putative receptor tyrosine kinases, which has been proposed to play a role in the segmentation of both the hindbrain and the mesoderm. Antibodies raised against the protein have been used to confirm the early embryonic expression pattern previously established by mRNA in situ hybridisation. These antibodies, together with the expression of the Sek-1 gene in a baculovirus system, were instrumental in demonstrating that the protein carries a tyrosine kinase activity and that it is presented at the cell surface with its N-terminal (putative ligand-binding) domain outside of the cell. Therefore, as expected from its amino acid sequence, Sek-1 conforms to the general model of receptor-type tyrosine kinases.

cDNA cloning and characterization of a Cek7 receptor protein-tyrosine kinase ligand that is identical to the ligand (ELF-1) for the Mek-4 and Sek receptor protein-tyrosine kinases.

We have isolated a murine cDNA encoding a ligand for the Cek7 receptor protein-tyrosine kinase (RPTK), a member of the Eph/Eck RPTK subfamily. Sequence analysis predicts an open reading frame of 209 amino acids with a predicted molecular mass of 24 kDa. The Cek7 ligand shows a 48% sequence identity at the protein level to B61, a ligand for the related Eck RPTK, 30% to the Cek5 ligand, 59% to the recently cloned Ehk1-L, and identity to ELF-1, a recently described ligand for the Mek4 and Sek RPTKs. The expressed Cek7 ligand is functionally active as it induces autophosphorylation of the Cek7 RPTK.

Control of proglucagon-derived peptide synthesis and secretion in fetal rat hypothalamus.

The hypothalamus has the highest concentration of proglucagon-derived peptides (Pgdp's) in the brain, however, the control of the synthesis and secretion of these peptides is not understood. The goal of our studies was to examine in detail the regulation of synthesis and secretion of Pgdp's in the hypothalamus. Hypothalamic cultures were prepared from fetal rats on day 19-21 of gestation and Pgdp's in media and cells were determined by radioimmunoassay after treatment with test agents. Dibutyryl cyclic AMP or forskolin, activators of protein kinase A, markedly stimulated both Pgdp synthesis (by 5-fold) and secretion (by 10-fold) after 24 h of treatment (p < 0.05). The effects of protein kinase A stimulation on Pgdp's in the hypothalamus were greater than seen in our previous studies with the Pgdp-producing pancreatic A and intestinal L cells. Therefore there are tissue-specific differences with regard to the magnitude of the response of Pgdp's to protein kinase A stimulation. Consistent with an involvement of protein kinase A in hypothalamic Pgdp synthesis and secretion, somatostatin-14, an inhibitor of protein kinase A, was found to inhibit Pgdp synthesis and secretion in a dose-dependent fashion (p < 0.05). Phorbol myristate acetate (PMA), a stimulator of protein kinase C, did not significantly affect the synthesis or secretion of Pgdp's at 6 h, but significantly stimulated Pgdp secretion after 24 h (p < 0.05). The inactive phorbol ester, phorbol triacetate was without effect on Pgdp synthesis or secretion after 24 h of incubation (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Further evidence of the presence of rat embryonic hypothalamic factors that induce the differentiation of gonadotrophic hormone-releasing hormone-containing secretory neurons.

We examined the presence of factor(s) in the embryonic medial basal hypothalamus (MBH) that may influence nasal placode (NAP)-derived luteinizing hormone-releasing hormone (LHRH) neurons in determining their secretory phenotype. In this study, we performed organotypic culture and transplantation of the NAP from 12.5-day-old embryos of rats and vomeronasal organ (VNO) from 14.5-day-old embryos. Surgical operations, however, were performed on 16.5-day-old embryos. The NAP and VNO were cultured singly or with the MBH obtained from the embryos of the same age and, further, in a medium with a nerve growth factor or fibroblast growth factors. Although LHRH neurons were derived from the NAP and VNO in all the cultures, judging from numbers and cellular morphologies, the MBH was most effective. The VNO was transplanted into the third ventricle of adult female rats singly or with the cerebral cortex, the mesencephalon-myelencephalon complex, or the MBH from 14.5-day-old embryos. All the grafts gave rise to LHRH neurons, but the number of the neurons was far greater in the grafts cotransplanted with the MBH, in which the neurons projected long processes to blood capillaries and formed neurovascular complexes, the feature of which may suggest the occurrence of the secretory activity in the fibers. The animals were examined 5 days after the surgical operations. In rhinoectomized embryos, LHRH neurons were distributed throughout the brain in the same pattern as found in intact rats, showing normal cellular morphology. In the encephalectomized rats, immunoreactive LHRH cells were present only in the terminal ganglia. These findings indicate that the embryonal MBH has a factor (s) that is essential to the development of secretory LHRH neurons.

In vitro development of the mammalian embryo.

Normal growth and differentiation of mammalian embryos in vitro during the preimplantation period appear to be dependent upon the availability of appropriate metabolic substrates. For preimplantation embryos, defined conditions of culture have been achieved only in a few laboratory species. There is now evidence that differentiation factors isolated from fetal calf serum and human placental cord serum may promote further development of blastocysts. Postimplantation rat and mouse embryos can be cultured during the organogenesis period with rat or human sera in roller bottles. The embryonic differentiation of the rat at this stage of development is progressively retarded in such cultures with male rat serum. The embryonic development is not improved, even in sera obtained from rats at different days of gestation (12, 15-16, and 20-21). Inability to grow placental tissues simultaneously with embryos, accumulation of unfavorable substances, and rapid depletion of nutrients contribute to the retardation of embryonic growth. To improve growth and differentiation of conceptuses, a continuous culture system with the possibility of infusion of increasing concentrations of oxygen in the roller bottle gas atmosphere has been developed. This improved method allows considerable continuous growth and differentiation from the neurula stage with development of numerous primary organs. Utilizing these in vitro culture methods during pre- and postimplantation periods, it is now possible to assess embryotoxic or teratogenic potential of drugs and chemical agents. The postimplantation culture procedure allows a more precise assessment of mechanisms associated with anomalous embryonic differentiation. Bioactivation of teratogens and effects of active toxic metabolites on organ primordium differentiation have been shown by combining embryo culture with a hepatic microsomal activating system. Microinjection of teratogens and cells into conceptus compartments is being used to elucidate specific anomalous differentiation processes.