Chick Embryo Brain Research Articles

Effect of cocaine, ethanol or nicotine on ornithine decarboxylase activity in early chick embryo brain

Fetal drug exposure causes multiple deficits in the developing child. For both humans and animal models, the single most common drug-related problem is fetal growth suppression. This defect is associated with significant perinatal morbidity and mortality and may also be related to significant behavioral problems appearing later in life. Studies focussed on the molecular mechanism of fetal drug effects in placental models are complicated by multiple interactions of the drug with mother, placenta and fetus. Using early (76–168 h) chick embryos as a non-placental model, and three common drugs of abuse (nicotine, ethanol and cocaine) it was found that each drug suppressed the peak in fetal brain ornithine decarboxylase (ODC) activity that normally occurs at 120 h of development. For each drug, the decrease in ODC activity at 120 h was followed by a small but significant increase in ODC. Thus, although the drug-treated embryos were smaller in size, they appeared to be undergoing compensatory growth and, in fact, became equal in weight to the vehicle-treated animals, if allowed to hatch.

Toxicology of carbaryl and aldicarb on brain and limb cultures of chick embryos.

A variety of carbamates have been developed since the 1960s for use as broad-spectrum insecticides. An easy and inexpensive in vitro assay using chick-embryo derived cells was examined for its capability to screen and test the toxicity of these compounds. Chick embryo brain and limb bud cultures were treated with different concentrations of either carbaryl or aldicarb with or without activation (+/- S-9) for 5 days. Viability and cytotoxicity using the neutral red assay, and carbamate effects on cell migration and colony spread were measured. S-9 decreased the effects of carbaryl and aldicarb on brain cell cytotoxicity at exposures of 15-60 ppm and 40-200 ppm, respectively, as indicated by increased concentrations of neutral red. Viability of brain cell cultures was not altered by aldicarb, but was decreased by carbaryl plus S-9 in concentrations of greater than 40 ppm. In limb cultures, carbaryl without S-9 was significantly toxic at 8-25 ppm, but only concentrations of greater than 25 ppm of carbaryl plus S-9 significantly affected cytotoxicity. In contrast, aldicarb without S-9 caused no effect on limb cell cytotoxicity at concentrations of 40-200 ppm, but aldicarb plus S-9 significantly reduced cellular cytotoxicity at concentrations of greater than 160 ppm. Carbaryl +/- S-9 decreased the spread of both brain and limb colonies; aldicarb +/- S-9 caused a significant increase in the spread of the brain but not limb colonies.(ABSTRACT TRUNCATED AT 250 WORDS)

O6-alkylguanine-DNA alkyltransferase in the chick embryo during development.

In the whole embryo, O6-alkylguanine-DNA alkyltransferase (AT) activity increased until day 9 of development and declined sharply after day 13. AT activity of the liver was greatest between day 12 and day 20 and decreased fast after hatching. In the brain, AT activities reached a maximum at day 17 and 18 and declined sharply after hatching. At two developmental stages with different AT activities (day 10 and day 17) DNA alkylation in the brains was estimated 6, 12, 24 and 48 h after administration of N-methyl-N-nitrosourea in ovo by viscometric measurement of DNA fragmentation. The high AT activities of the chicken embryo brain at the 17th day of development correlated with minor DNA fragmentation following a repair period of 12-24 h. It is suggested that the high basal level of AT in the chick embryo might have a protective function against the persistence of the genotoxic lesion O6-methylguanine during development.

3,5,3′-Triiodothyronine binding sites in synaptosomes from brain of chick embryo. Properties and ontogeny

In this study we have demonstrated the presence of specific 3,5,3′- l-triiodothyronine (T3) binding sites in the synaptosomes of chick embryo cerebral cortex and described their ontogeny. Scatchard analyses of binding data obtained with synaptosomal preparations from 17-day-old embryos revealed two T3 binding sites. The first site (N1) had a high affinity and low capacity since its dissociation constant ( K d) was 68 ± 1.3 nM T3 (mean ± S.D.; n = 3−5) and its maximal binding capacity ( B max) was 8.63 ± 1.59 ng T3/mg of protein, whereas the second site (N2) had a higher K d of 5.04 ± 0.5 μM T3 and a larger B max of 405 ± 49 ng T3/mg of protein. The relative affinity of the synaptosomal fraction for T3 and other analogs was the following: T3 > T4 (thyroxine) > d-T3 (3,5,3′- d-triiodothyronine) = TRIAC (triidothyroacetic acid) > rT3 (reverse T3) . Gel chromatography of the [ 125I]T3 labeled fraction revealed a partially saturable peak with an estimated MW of more than 100 kDa. The ontogenic pattern showed a progressive increase of K d and B max of N1, occuring mainly between the 12 and 19 days of incubation, and a marked fall, particularly of the B max, after hatching. The second site did not show any important variation during the embryogenesis. These data indicate the existence of specific T3 binding sites in synaptosomes from cerebral cortex of chick embryo, whose properties and ontogeny are completely different from those of the nuclear receptor. Furthermore these findings suggest that the synaptosomes could be another site of action of thyroid hormone where T3 could stimulate the development of specialized nervous structures or modulate the activity of various neurotransmitters during the development of chick embryo cerebral cortex.

The cellular environment controls the expression of engrailed-like protein in the cranial neuroepithelium of quail-chick chimeric embryos

We have previously shown that one of two chicken engrailed-like genes, chick En-2, is expressed in a restricted region of the early chick embryo brain: the mes/metencephalon (Gardner et al. 1988). In this study, we examine the role of the cellular environment in regulation of engrailed-like (En) protein expression in quail-chick chimeric embryos. Two types of transplant surgery were performed at the 9-15 somite stage to produce chimeric embryos. In the first, the mid-mesencephalic vesicle or caudal mesencephalic vesicle alar plate (which is En protein-positive) was transplanted from a quail embryo into an En protein-negative region of chick neuroepithelium, the prosencephalon (mMP and cMP grafts, respectively). In the second reciprocal surgery, prosencephalic alar plate which is En protein-negative, was transplanted into the En protein-positive mesencephalic vesicle (PM grafts). A polyclonal antiserum, alpha Enhb-1, which recognizes chick En proteins (Davis et al. 1991) was used to identify En-positive cells 48 h after surgery. In mMP embryos, 71% of integrated grafts had lost En expression (n = 17). In contrast, in cMP grafts, 93% of integrated grafts continued to stain with the antiserum (n = 14). In addition, in 86% of these embryos, the graft induced adjacent chick host diencephalic cells to become En protein-positive as well. All PM grafts contained aEnhb-1-positive cells; such cells never expressed this protein in their normal environment. These early changes in En protein expression correlate well with the morphological changes observed in similar graft surgeries assayed later in development. Thus, our results are consistent with the hypothesis that En genes play a role in the regionalization of the early cranial neuroepithelium.

Pathogenesis of avian encephalomyelitis viruses

The pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmuno-precipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.

T-cadherin, a novel cadherin cell adhesion molecule in the nervous system lacks the conserved cytoplasmic region

We have characterized T-cadherin, a glycoprotein of 95 kd from chick embryo brain by expression cloning and sequencing of corresponding cDNAs. The nucleotide sequence predicts a novel cadherin cell adhesion molecule in the nervous system. Surprisingly, the isolated cDNAs do not encode the cytoplasmic region conserved in other cadherin subclasses. Biochemical analysis revealed that this truncated (T) cadherin is attached to the neuronal plasma membrane through a glycosyl phosphatidylinositol anchor. T-cadherin is a component of different neuronal populations and is expressed in a temporally and spatially restricted pattern during axon growth. These results are consistant with a putative role of T-cadherin in axon growth and guidance.

Monoclonal antibody to chick embryo hyaluronan-binding protein: Changes in distribution of binding protein during early brain development

A monoclonal antibody, MAb IVd4, that recognizes hyaluronan-binding protein (HABP) from chick embryo brain has been produced and characterized. By immunoblotting, MAb IVd4 was shown to recognize three proteins in chick embryo brain of molecular weight 93, 90, and 69 kDa; this interaction was inhibited by addition of hyaluronan hexasaccharides. Overlay of transblots with [ 3H]hyaluronan showed binding to proteins of similar molecular weight. MAb IVd4 blocked binding of [ 3H]hyaluronan to brain HABP and to simian virus-transformed 3T3 cells, indicating a possible relationship with the 85-kDa hyaluronan receptor of these cells. The distribution of HABP during early brain development was analyzed by immunohistochemistry. Immunoreactivity was uniform in newly formed neuroectoderm but became more concentrated in the roof of the brain during the second day of embryonic development. As the neuroectoderm becomes layered, the HABP was increasingly restricted to the forming plexiform layer, an area enriched in neural cell processes. Immunoreactivity was greatly enhanced by pretreatment of tissue with hyaluronidase, presumably due to removal of hyaluronan bound to the HABP, and was abolished on treatment with hyaluronan hexasaccharide, presumably due to inhibition of HABP-antibody interaction. These results suggest that a hyaluronan receptor is involved in early cellular events in brain development.

The chicken tropomyosin 1 gene generates nine mRNAs by alternative splicing

Skeletal muscle beta-tropomyosin, smooth muscle alpha-tropomyosin, and a low molecular weight fibroblast tropomyosin are generated by alternatively splicing RNA transcripts of the chicken tropomyosin 1 (TM 1) gene (Forry-Schaudies, S., Maihle, N. J., and Hughes, S. H. (1990) J. Mol. Biol. 211; 321-330). Two novel tropomyosin cDNAs that derive from mRNAs of the TM 1 gene have been isolated from a chicken embryo brain cDNA library. Brain cDNA BRT-1 is 2.2 kilobases in length and encodes 283 amino acids. It is identical to skeletal muscle beta-tropomyosin from amino acids 1 to 258. The sequence 3' of this point is unique to BRT-1; a comparison to genomic sequence indicates that a new carboxyl-terminal exon is used to generate this sequence. 1.4-kilobase brain cDNA BRT-2 contains sequences found in both fibroblast cDNA FT-beta (5'-end) and skeletal muscle cDNA SKT-beta (3'-end). RNase and S1 nuclease assays using RNA samples from leg muscle, gizzard, fibroblasts, and brain indicate that the TM 1 gene expresses four additional tropomyosin RNAs by alternately splicing previously characterized exons. These results demonstrate that the chicken TM 1 gene encodes nine tropomyosin RNAs through the use of two promoters, two internal exons that are mutually exclusive, and three 3'-exons. Implications for the regulation of alternative splicing are discussed.

Purinergic stimulation of astroblast proliferation: guanosine and its nucleotides stimulate cell division in chick astroblasts.

A highly active fraction that was mitogenic for astroblasts but which contained no amino acids was identified during the purification of peptides from chick embryo brains. This material was purified by ultracentrifugation, ultrafiltration through Diaflo PM-30 and YM-2 membranes and retention on Diaflo YC-05, followed by ion exchange chromatography and reversed phase high performance liquid chromatography (HPLC) on a C18 Deltapak column. On thin layer chromatography and HPLC the material co-chromatographed with authentic commercially-obtained GMP. Its ultraviolet absorption spectrum was also identical with that of GMP. 1H and 31P nuclear magnetic resonance spectra of the isolated material were identical with those of GMP. The close match between the fast atom bombardment (FAB) mass spectra of the unknown material and authentic GMP indicated that the unknown material was GMP of molecular weight 363 Da. Authentic, commercial GMP stimulated the growth of cultured chick astroblasts in the same dose-dependent manner as the material from chick embryo brains; maximal stimulation was at 50 microM. Guanosine, GDP, and GTP also stimulated cell proliferation. The nucleotides were equally as effective as guanosine. 5'-Guanylyl imidodiphosphate, guanosine 5'-O-(2-thiodiphosphate), and guanosine 5'-N-(3-thiotriphosphate), guanine nucleotides which are relatively resistant to enzymatic hydrolysis, were also mitogenic, indicating that the nucleotides do not need to be degraded to nucleosides to be active and that they probably act extracellularly. Guanine nucleosides and nucleotides promoted astroblast growth when other growth factors were removed from the culture medium. The mitogenic effects of guanosine and its nucleotides were inhibited in a dose-dependent fashion by micromolar concentrations of theophylline, a characteristic of phenomena mediated by purinergic receptors. Guanosine and its nucleotides are released in micromolar concentrations by hypoxic or dying cells. Under these circumstances these compounds may stimulate division of adjacent cells in vivo.

Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks

1. 1. The behaviour of total acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of chick embryos between 11th and 21st day of development and of 8 and 180-day-old chicks is described. 2. 2. Total acid soluble carnitine fluctuates around the same levels in the brain, liver and muscle until 18th day of development, whereas it attains a peak on that day in the heart. At hatching compared to 18th day, it suddenly increases three times in the muscle, drops not significantly in the heart and brain, but sharply in the liver (−40%). However the levels are always higher than those of the grown chick in the brain but lower in the other tissues. 3. 3. Free carnitine levels are almost constant in all tissues during the embryonic life; if compared to adult ones, they are very much lower in the liver, heart and muscle, but higher in the brain, even in 8 day-old chick. 4. 4. Short chain esterified, carnitine reaches a maximum on 18th day of egg incubation in the liver, brain and heart; in the muscle it stays on constant levels until this day and then rapidly increases so that at hatching it doubles the values. 5. 5. The short chain esterified to free carnitine percentage ratio peaks in all tissues on 18th day of development, attaining figures which are well above those determined in the grown chick.

Absence of O 6-alkylguanine-DNA alkyltransferase induction in chick embryo liver and brain following X-irradiation or treatment with bleomycin

1. The presence of O 6-alkylguanine-DNA alkyltransferase (AT) in liver and brain of chick embryos, chicks and hens was demonstrated. An induction of AT activity has only been found in the liver of chicks and hens 48 hr after X-irradiation (5, 10 or 12 Gy). 2. The administration of methylmethanesulphonate to the chick embryo resulted 3–24 hr later in strong inhibition of AT activity accompanied by DNA alkylation. Under the same conditions, X-irradiation, dimethylnitrosamine and bleomycin exhibited no effect. 3. The results are compared with those obtained in mouse, rat and human foetal tissues.

Characterization of neuropathy target esterase using trifluoromethyl ketones

Neuropathy target esterase (NTE) is a membrane-bound carboxylesterase activity which is proposed as the target site in nerve tissue for initiation of organophosphate-induced delayed neuropathy. This activity is identified as phenyl valerate hydrolysis which is resistant to treatment with paraoxon and sensitive to co-incubation with paraoxon and mipafox. NTE preparations were obtained, which did not contain paraoxon-sensitive or mipafox-resistant hydrolases, by selective reconstitution of detergent-solubilized NTE from chick embryo brain into asolectin vesicles during gel filtration. The topography of the catalytic site of NTE was then examined by investigating the inhibition of NTE by a series of 3-alkylthio- and 3-arylthio-1,1,1-trifluoro-propan-2-ones. These trifluoromethyl ketones were found to be rapidly reversible, competitive inhibitors of NTE with I 50 values from 1.3 × 10 −4 M to 4.9 × 10 −4M. Correlation of I 50 values with octanol/water partition coefficients ( P), in the range of log P = 1.5 to 5.9, indicated that the optimal lipophilicity for NTE substrates and inhibitors is in the range of log P = 3.0 to 3.4. Electrophilic substitution at the meta position of aromatic rings increased the inhibitory capacity of these inhibitors, whereas substitution at the ortho position reduced inhibitory capacity. These results indicate both that a large hydrophobic pocket is closely associated with the catalytic residue of NTE, and that affinity for the active site is affected by steric and electronic parameters.

Effects of two microtubule-depolymerizing drugs, vincristine and vinblastine, on porphyrin production by primary neural tissue cultures

A porphyrinogenic effect of 10(-5) to 10(-7) M Vincristine (VC) and Vinblastine (VB) was observed on primary neural tissue cultures prepared from chicken embryo brain in the presence of 10(-3) M delta-aminolevulinic acid. This effect is very pronounced in neurocyte cultures, in contrast with the documented neurite elongation defect. The microtubule disassembly by VC and VB changed the quantity of the porphyrins in the cells and medium of glial cell cultures. The correlation was studied between the depolymerization of the microtubules by VC and VB and the porphyrin overproduction of primary neural tissue cultures, the investigation also extending to 10(-7) M taxol. The direct mediation of nucleotide binding proteins of the adenyl-cyclase complex by GTP liberated from beta-tubulin during the disassembly of the microtubules is presumed.

L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells

The intracellular free Ca 2+ concentration, [Ca 2+] i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca 2+] i is partly a function of Ca 2+ influx through voltage-sensitive calcium channels (VSCC), Ca 2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd 2+, Ni 2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca 2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd 2+ (<50 μM), which have been reported to inhibit L-type channels, reduced neurite initiation but not mean neurite length. Ni 2+ (<100 μM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (<300 μM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (>300 μM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 μM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 μM), Cd 2+ (5 μM), and streptomycin (200 μM) reduced neurite initiation, while nickel (50 μM) and neomycin (100 μM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd 2+ inhibited L-type channels but not T-type channels, while Ni 2+ inhibited T-type channels but not L-type channels. Streptomycin slightly inhibited L-type channels but enhanced current flow through T-type channels. Neomycin slightly inhibited both channel types. These data indicate that neurite initiation in these two cell types may be modulated by Ca 2+ influx through L-type channels, but not T- or N-type channels. Neurite length was not significantly influenced by any of the agents tested, suggesting that Ca 2+ influx through VSCC may not affect neurite elongation.

Monoclonal Antibodies Recognizing the Benzodiazepine Receptor of Chick Embryo Brain

The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the 3H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.

Expression of three drebrin isoforms in the developing nervous system

Drebrins are developmentally regulated brain proteins which were first isolated from brains of 10-day chick embryos. They are classified into three forms, drebrins E1, E2 and A. Cloning of drebrin cDNAs revealed that each drebrin isoform is encoded in an independent mRNA. Genomic Southern blot analysis and cloning of a drebrin gene revealed that the mRNAs of three drebrin isoforms are generated by alternative RNA splicing from a single gene. Immunohistochemistry and in situ hybridization analysis of the embryonic cerebellum indicated that drebrin mRNA is first transcribed in postmitotic neurons and that there seem relations between cell migration and expression of drebrin E1.

Effects of DDT and permethrin on neurite growth in cultured neurons of chick embryo brain and Lymnaea stagnalis

The pesticides permethrin and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), dissolved in either ethanol (EtOH) or dimethylsulphoxide (DMSO), were studied to determine their effect on neurite growth from cultured neurons of Lymnaea stagnalis and embryonic chicks. Both of these toxins decreased the percentage of neurons growing neurites, mean neurite length, and number of neurites/cell in a dose-dependent manner. DMSO increased the toxicity of permethrin and DDT in L. stagnalis neurons. EtOH was not used as a solvent with the embryonic chick cultures. Pre-existing neurites of L. stagnalis neurons exposed to permethrin regressed in a dose- and time-dependent manner. These two toxins may affect neurite outgrowth through interference with intracellular calcium regulation.

Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat

Free D-aspartic acid was measured in fertilized chicken eggs, chicken embryos, and neonatal rats. In each tissue examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D at 11 days of incubation; for the retina, 20% D at 13 days of incubation. In the neonatal rat, as in the chicken embryo, D-aspartic acid continued to increase in the retina after that in the brain and other tissues had begun to decline. The maximum, 29% D, was found 7 days after birth. Thus in two phylogenetically distant species, similar developmental patterns of D-aspartic acid change were observed. Some data on similarities between the D/L aspartic acid ratios of adult chicken and rat tissues are also reported. In addition, the total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing that release from a bound form or de novo synthesis is a continuing process during development.