Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways.

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.

EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.

Patient-derived glioblastoma cells show significant heterogeneity in treatment responses to the inhibitor-of-apoptosis-protein antagonist birinapant.

BACKGROUND: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. METHODS: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. RESULTS: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. CONCLUSIONS: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment.

Eph receptors as therapeutic targets in glioblastoma.

The dismal outlook for patients with the most aggressive and common form of adult brain cancer, glioblastoma (GBM), motivates a search for new therapeutic strategies and targets for this aggressive disease. Here we review the findings to date on the role of Eph family receptor tyrosine kinases and their ephrin ligands in brain cancer. Expression of the Eph family of cell surface proteins is generally downregulated to very low levels in normal adult tissues making them particularly attractive for directed therapeutic targeting. Recent Eph targeting studies in pre-clinical models of GBM have been very encouraging and may provide an avenue to treat these highly refractory aggressive tumours.

The Fat1 cadherin is overexpressed and an independent prognostic factor for survival in paired diagnosis-relapse samples of precursor B-cell acute lymphoblastic leukemia.

Improved survival of patients with acute lymphoblastic leukemia (ALL) has emerged from identifying new prognostic markers; however, 20% of children still suffer recurrence. Previously, the altered expression of Fat1 cadherin has been implicated in a number of solid tumors. In this report, in vitro analysis shows that Fat1 protein is expressed by a range of leukemia cell lines, but not by normal peripheral blood (PB) and bone marrow (BM) cells from healthy donors. In silico analysis of expression of array data from clinical leukemias found significant levels of Fat1 transcript in 11% of acute myeloid leukemia, 29% and 63% of ALL of B and T lineages, respectively, and little or no transcript present in normal PB or BM. Furthermore, in two independent studies of matched diagnosis-relapse of precursor B-cell (preB) ALL pediatric samples (n=32 and n=27), the level of Fat1 mRNA expression was prognostic at the time of diagnosis. High Fat1 mRNA expression was predictive of shorter relapse-free and overall survival, independent of other traditional prognostic markers, including white blood cell count, sex and age. The data presented demonstrate that Fat1 expression in preB-ALL has a role in the emergence of relapse and could provide a suitable therapeutic target in high-risk preB-ALL.

Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival.

Aberrant expression of Eph and ephrin proteins has well-established functions in oncogenesis and tumour progression. We describe EphA1 expression in 6 colorectal cancer (CRC) cell lines, 18 controls and 125 CRC specimens. In addition, a well-characterised cohort of 53 paired normal colon and CRCs was also assessed. Expression of EphA1 mRNA was assessed by quantitative real-time PCR and correlated with protein expression by flow cytometry, immunoprecipitation, western blotting and immunohistochemistry. Significant upregulation (2- to 10-fold) of EphA1 was seen in over 50% of cases (P=0.005) whereas many of the remainder showed downregulation of EphA1. Intriguingly, EphA1 over-expression was more prevalent in stage II compared to stage III CRCs (P=0.02). Low EphA1 expression significantly correlated with poor survival (P=0.02). Epigenetic silencing appeared to explain the loss of EphA1 expression as methylation of the EphA1 CpG island strongly correlated with low EphA1 expression (P<0.01). Furthermore, EphA1 re-expression could be induced by treatment with demethylating agents. Our findings identify EphA1 as a potential prognostic marker in CRC. Although therapies targeting high EphA1 expression seem plausible in CRC, the loss of expression in advanced disease suggests a potential risk that targeted therapy, by selecting for loss of expression, might contribute to disease progression.

Hypermethylation of the 5' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis.

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 5' CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.

Signals from Eph and ephrin proteins: a developmental tool kit.

Interactions between Eph receptors and their ligands the ephrin proteins are critically important in many key developmental processes. Emerging evidence also supports a role for these molecules in postembryonic tissues, particularly in pathological processes, including tissue injury and tumor metastasis. We review the signaling mechanisms that allow the 14 Eph and nine ephrin proteins to deliver intracellular signals that regulate cell shape and movement. What emerges is that the initiation of these signals is critically dependent on which Eph and ephrin proteins are expressed, the level of their expression, and, in some cases, which splice variants are expressed. Diversity at the level of initial interaction and in the downstream signaling processes regulated by Eph-ephrin signaling provides a subtle, versatile system of regulation of intercellular adhesion, cell shape, and cell motility.

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y.

OBJECTIVE: The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. MATERIALS AND METHODS: A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. RESULTS: We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1(+) HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PY(lo) and PY(hi) subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the PY(lo)Rh(lo)Ho(lo/sp) PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PY(hi)). We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. CONCLUSION: Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the Rh(lo)Ho(lo/sp) PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population.

Characterization of a human synovial cell antigen: VCAM-1 and inflammatory arthritis.

The contribution of synovial cells to the pathogenesis of rheumatoid arthritis (RA) is only partly understood. Monoclonal antibody (mAb) 1D5 is one of very few mAb ever raised against RA synovial cells in order to study the biology of these cells. Studies on the expression pattern and structural features of the 1D5 Ag suggest that 1D5 recognizes human vascular cell adhesion molecule-1 (VCAM-1), which is an intercellular adhesion molecule. Vascular cell adhesion molecule-1 may be involved in a number of crucial intercellular interactions in RA.

Characterization of the Epha1 receptor tyrosine kinase: expression in epithelial tissues.

The Eph family of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and "native" mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus, kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

A phase I and pharmacokinetic study of subcutaneously-administered recombinant human interleukin-4 (rhuIL-4) in patients with advanced cancer.

PURPOSE: To investigate the pharmacokinetics and tolerability of recombinant human interleukin-4 (rhuIL-4), administered by daily subcutaneous injection, in patients with advanced cancer. PATIENTS AND METHODS: Fourteen patients with advanced cancer treated with rhuIL-4 at escalating dose levels of 0.25, 1.0 and 5.0 microg/kg/day, on days 1, 8-17, and 28-57. The primary endpoints of the study were toxicity of rhuIL-4 and the determination of the pharmacokinetics of rhuIL-4 when given by subcutaneous injection. Secondary endpoints included effects on blood counts, hematopoietic cell precursors, and various immunologic parameters. RESULTS: rhuIL-4 was well tolerated at all three dose levels. Detectable serum levels of IL-4 were found in patients at the 1.0 and 5.0 microg/kg/day dose levels. Peak serum IL-4 levels were achieved about 2 h after injection and IL-4 was still detectable 8 h after injection. No grade 4 toxicities were observed and grade 3 toxicities were confined to fever, headache and raised hepatic alkaline phosphatase. No consistent hematological or immunologic effects were observed. Although therapeutic efficacy was not an endpoint, one complete response (Hodgkin's disease) was observed. One patient with chronic lymphocytic leukemia progressed on therapy. CONCLUSION: rhuIL-4 up to 5.0 microg/kg/day is well tolerated when given by subcutaneous injection. Biologically relevant serum IL-4 levels can be achieved and sustained for at least 8 h after a single injection.

Expression of the Wilms' tumor suppressor gene, WT1, reduces the tumorigenicity of the leukemic cell line M1 in C.B-17 scid/scid mice.

The Wilms' tumor suppressor gene, WT1, encodes a transcription of the Cys2-His2 zinc finger type. Loss of WT1 gene function has been implicated in the development of malignancies including Wilms' tumor and acute leukemias. We have shown previously that ectopic expression of WT1 +KTS isoforms in murine M1 leukemic cells spontaneously induces monocytic differentiation without the requirement for external differentiation-inducing stimuli. To determine whether these observed effects in vitro corresponded to a reduction in tumorigenicity in vivo, parental M1, control M1.Neo, and M1.WT1 +KTS cells were transplanted into C.B-17 scid/scid mice, and the growth and metastatic behavior of the cell lines were monitored for a period of 20 weeks. Mice inoculated either s.c. on the flank or directly into the peritoneal cavity, with M1 cells stably expressing WT1 +KTS isoforms exhibited a marked decrease in tumor formation compared with control groups. Moreover, tumors arising in mice after the injection of M1.WT1 +KTS cells exhibited a loss in ectopic WT1 protein expression. Confirmation that the tumors arose from M1.WT1 +KTS cells was achieved by the amplification of the introduced transgene from tumor samples and indicates that the tumorigenicity of leukemic M1 cells in these animals correlates with a loss in WT1 expression. This investigation is the first to demonstrate the tumor-suppressive effects of WT1 expression in a leukemic cell line, further advancing the notion that WT1 acts as a differentiation-promoting gene during hematopoiesis and that loss of functional WT1 expression may contribute to leukemogenesis in vivo.

Cloning and characterization of EphA3 (Hek) gene promoter: DNA methylation regulates expression in hematopoietic tumor cells.

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal and spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promoter elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the EphA3 gene promoter region. A region of 86 bp located at -348 bp to -262 bp upstream from the transcription start site was identified as the basal promoter. This region was shown to be active in both EphA3-expressing and -nonexpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive restriction enzymes and bisulfite sequencing of genomic DNA, sites of DNA methylation were identified in hematopoietic cell lines which correlated with their levels of EphA3 gene expression. We showed that EphA3 was not methylated in normal tissues but that a subset of clinical samples from leukemia patients showed extensive methylation, similar to that observed in cell lines. These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors.

Expression of the Wilms' tumor suppressor gene, WT1, is upregulated by leukemia inhibitory factor and induces monocytic differentiation in M1 leukemic cells.

The Wilms' tumor gene, WT1, encodes a transcription factor of the Cys2-His2 zinc finger type. The functional significance of WT1 expression in leukemias, in addition to tissues and cell lines of hematopoietic origin, has not been determined. Using the murine myeloblastic leukemia cell line M1 as a model for macrophage differentiation, expression of WT1 is shown to be activated in M1 cells 24 hours after differentiation induction by leukemia inhibitory factor (LIF). Upregulation of WT1 in these cells is associated with cellular differentiation, coinciding with expression of the monocyte/macrophage marker c-fms, and the appearance of mature cells. WT1 isoforms lacking the KTS insert are unable to be ectopically expressed in M1 cells. Stable expression of the WT1 isoforms containing the KTS insert leads to spontaneous differentiation of the M1 myeloblasts through the monocytic differentiation pathway. These cells express c-fms, in addition to the myeloid-specific cell surface marker Mac-1. Exposure of these cells to LIF results in the rapid onset of terminal macrophage differentiation, accompanied by apoptotic cell death. These results show that the WT1 gene is an important regulator of M1 cell monocytic differentiation in vitro, and suggests a potential role for this gene in the molecular control of hematopoiesis.

Ligand for EPH-related kinase (LERK) 7 is the preferred high affinity ligand for the HEK receptor.

HEK is a member of the EPH-like receptor tyrosine kinase family, which appear to have roles in development and oncogenesis. Recently, we purified a soluble HEK ligand which is also a ligand (AL1) for the HEK-related receptor EHK1. Promiscuity appears to be a characteristic feature of interactions between the EPH-like receptors and their ligands, termed ligands for EPH-related kinases (LERKs). This prompted us to analyze the interactions between the HEK exodomain and fusion proteins comprising candidate LERKs and the Fc portion of human IgG1 (Fc) or a FLAGTM-peptide tag by surface plasmon resonance, size exclusion high performance liquid chromatography, sedimentation equilibrium, and transphosphorylation. Our results indicate that AL1/LERK7 is the preferred high-affinity ligand for HEK, forming a stable 1:1 complex with a dissociation constant of 12 nM. As expected the apparent affinities of bivalent fusion proteins of LERKs and the Fc portion of human IgG1 had significantly reduced dissociation rates compared with their monovalent, FLAGTM-tagged derivatives. High-avidity binding of monovalent ligands can be achieved by antibody-mediated cross-linking of monovalent ligands and with LERK7 results in specific phosphorylation of the receptor. By extrapolation, our findings indicate that some of the reported LERK-receptor interactions are a consequence of the use of bivalent ligand or receptor constructs and may be functionally irrelevant.

G-CSF-mobilized peripheral blood progenitor cells: in vitro growth pattern and hematopoietic growth factor receptor profile.

The kinetics of colony formation by granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) were monitored using clone-mapping experiments. Compared with normal resting bone marrow (BM), where the ratio of Day 7:Day 14 granulocyte-macrophage colony-forming cells (GM-CFCs) was 1:0.76-1.9, PB was found to be relatively deficient in progenitor cells with the capacity to form colonies by Day 7 (median ratio Day 7:Day 14 1:21). The most mature Day 7 GM-CFCs, those dispersing or extinguishing before Day 14, were almost absent in PB (< 1% of all GM-CFCs) but comprised 77% of Day 7 GM-CFCs and 32% of all GM-CFCs in BM. The expression patterns of high affinity receptors for G-CSF, GM-CSF, stem cell factor (SCF), and the ligand for flk-2 on CD38hi and CD38-/dim PB CD34+ cells were determined by binding of 125I-labeled ligand and autoradiography. G-CSF receptor (G-CSFR) expression was detected on approximately 25% of CD38-/dim cells (estimated mean 105 receptors per positive cell) and was higher in CD38hi cells (approximately 50% positive, with a mean of 227 receptors per cell). GM-CSFR expression was low (approximately 25% of cells positive, mean of 120 receptor per cell) and did not vary with CD38 expression. c-kit (SCFR) and flk-2 were expressed by > or = 90% and > or = 80% of CD34+ cells, respectively. SCF binding per cell was greater in the CD38hi population, while flk-2 expression did not vary with CD38 expression. These results confirm the heterogeneity of receptor expression by progenitor cells and imply differential regulation of receptor expression during maturation.

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.