Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real-time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration-free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.

Zelda and the maternal-to-zygotic transition in cockroaches.

In the endopterygote Drosophila melanogaster, Zelda is an activator of the zygotic genome during the maternal-to-zygotic transition (MZT). Zelda binds cis-regulatory elements (TAGteam heptamers), making chromatin accessible for gene transcription. Zelda has been studied in other endopterygotes: Apis mellifera and Tribolium castaneum, and the paraneopteran Rhodnius prolixus. We studied Zelda in the cockroach Blattella germanica, a hemimetabolan, short germ-band, and polyneopteran species. B. germanica Zelda has the complete set of functional domains, which is typical of species displaying ancestral features concerning embryogenesis. Interestingly, we found D. melanogaster TAGteam heptamers in the B. germanica genome. The canonical one, CAGGTAG, is present at a similar proportion in the genome of these two species and in the genome of other insects, suggesting that the genome admits as many CAGGTAG motifs as its length allows. Zelda-depleted embryos of B. germanica show defects involving blastoderm formation and abdomen development, and genes contributing to these processes are down-regulated. We conclude that in B. germanica, Zelda strictly activates the zygotic genome, within the MZT, a role conserved in more derived endopterygote insects. In B. germanica, zelda is expressed during MZT, whereas in D. melanogaster and T. castaneum it is expressed beyond this transition. In these species and A. mellifera, Zelda has functions even in postembryonic development. The expansion of zelda expression beyond the MZT in endopterygotes might be related with the evolutionary innovation of holometabolan metamorphosis. DATABASES: The RNA-seq datasets of B. germanica, D. melanogaster, and T. castaneum are accessible at the GEO databases GSE99785, GSE18068, GSE63770, and GSE84253. In addition, the RNA-seq library from T. castaneum adult females is available at SRA: SRX021963. The B. germanica reference genome is available as BioProject PRJNA203136.

Isolation functional characterization of allatotropin receptor from the cotton bollworm, Helicoverpa armigera.

Insect allatotropin (AT) plays multi-functions including regulation of juvenile hormone synthesis, growth, development and reproduction. In the present study, the full-length cDNA encoding the AT receptor was cloned from the brain of Helicoverpa armigera (Helar-ATR). The ORF of Helar-ATR exhibited the characteristic seven transmembrane domains of the G protein-coupled receptor (GPCR) and was close to the ATR of Manduca sexta in the phylogenetic tree. The Helar-ATR expressed in vertebrate cell lines can be activated by Helar-AT and each Helar-ATL in a dose-responsive manner, in the following order: Helar-ATLI > Helar-ATLII > Helar-AT > Helar-ATLIII. Helar-ATLI and Helar-ATLII represented the functional ligands to Helar-ATR in vitro, while Helar-AT and Helar-ATLIII behaved as partial agonists. The in vitro functional analysis suggested that the Helar-ATR signal was mainly coupled with elevated levels of Ca2+ and independent of cAMP levels. Helar-ATR mRNA in larvae showed the highest level in the brain, followed by the thorax ganglion, abdomen ganglion, fat body and midgut. Helar-ATR mRNA levels in the complex of the brain-thoracic-abdomen ganglion on the 2nd day of the larval stage and during later pupal stages were observed to be relatively higher than in the wandering and early pupal stages.

The Dachsous/Fat/Four-Jointed Pathway Directs the Uniform Axial Orientation of Epithelial Cells in the Drosophila Abdomen.

The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. We found that the Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. We suggest that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair.

Fetal and neonatal outcomes of preterm infants born before 32 weeks of gestation according to antenatal vs postnatal assessments of restricted growth.

BACKGROUND: Fetal growth restriction is defined using ultrasound parameters during pregnancy or as a low birthweight for gestational age after birth, but these definitions are not always concordant. OBJECTIVE: The purpose of this study was to investigate fetal and neonatal outcomes based on antenatal vs postnatal assessments of growth restriction. STUDY DESIGN: From the EPIPAGE 2 population-based prospective study of very preterm births in France in 2011, we included 2919 singleton nonanomalous infants 24-31 weeks gestational age. We constituted 4 groups based on whether the infant was suspected with fetal growth restriction during pregnancy and/or was small for gestational age with a birthweight <10th percentile of intrauterine norms by sex: 1) suspected with fetal growth restriction/small for gestational age 2) not suspected with fetal growth restriction/small for gestational age 3) suspected with fetal growth restriction/not small for gestational age and 4) not suspected with fetal growth restriction/not small for gestational age. We estimated relative risks of perinatal mortality and morbidity for these groups adjusting for maternal and neonatal characteristics. RESULTS: We found that 22.2% of infants were suspected with fetal growth restriction/small for gestational age, that 11.4% infants were not suspected with fetal growth restriction/small for gestational age, that 3.0% infants were suspected with fetal growth restriction/not small for gestational age, and that 63.4% infants were not suspected with fetal growth restriction/not small for gestational age. Compared with infants who were not suspected with fetal growth restriction/not small-for-gestational-age infants, small-for-gestational-age infants suspected and not suspected with fetal growth restriction had higher risks of stillbirth or termination of pregnancy (adjusted relative risk, 2.0 [95% confidence interval, 1.6-2.5] and adjusted relative risk, 2.8 [95% confidence interval, 2.2-3.4], respectively), in-hospital death (adjusted relative risk, 2.8 [95% confidence interval, 2.0-3.7] and adjusted relative risk, 2.0 [95% confidence interval, 1.5-2.8], respectively), and bronchopulmonary dysplasia (adjusted relative risk, 1.3 [95% confidence interval, 1.2-1.4] and adjusted relative risk, 1.3 [95% confidence interval, 1.1-1.4], respectively), but not severe brain lesions. Risks were not increased for infants suspected with fetal growth restriction but not small-for-gestational-age. CONCLUSION: Antenatal and postnatal assessments of fetal growth restriction were not concordant for 14% of very preterm infants. In these cases, birthweight appears to be the more relevant parameter for the identification of infants with higher risks of adverse short-term outcomes.

Apoptotic forces in tissue morphogenesis.

It is now well established that apoptosis is induced in response to mechanical strain. Indeed, increasing compressive forces induces apoptosis in confined spheroids of tumour cells, whereas releasing stress reduces apoptosis in spheroids cultivated in free suspension (Cheng et al., 2009). Apoptosis can also be induced by applying a 100 to 250MPa pressure, as shown in different cultured cells (for review, see (Frey et al., 2008)). During epithelium development, the pressure caused by a fast-growing clone can trigger apoptosis at the vicinity of the clone, mediating mechanical cell competition (Levayer et al., 2016). While the effect of strain has long been known for its role in apoptosis induction, the reciprocal mechanism has only recently been highlighted. First demonstrated at the cellular level, the effect of an apoptotic cell on its direct neighbours has been analysed in different kinds of monolayer epithelium (Gu et al., 2011; Rosenblatt et al., 2001; Kuipers et al., 2014; Lubkov & Bar-Sagi, 2014). More recently, the concept of a broader impact of apoptotic cell behaviours on tissue mechanical strain has emerged from the characterisation of tissue remodelling during Drosophila development (Toyama et al., 2008; Monier et al., 2015). In the present review, we summarize our current knowledge on the mechanical impact of apoptosis during tissue remodelling.

Aumento mamário por meio da incisão da abdominoplastia: estudo prospectivo de 100 casos / Breast augmentation via the abdominoplasty incision approach: a prospective study of 100 cases

INTRODUÇÃO: A gravidez e a obesidade causam distensão da parede abdominal e também produzem mudanças na forma e no tamanho das mamas. Assim, não é incomum a necessidade de melhoria estética da área abdominal, coincidindo com o desejo de aumento de mama. A mamoplastia utilizando a mesma incisão da abdominoplastia foi descrita pela primeira vez em 1976. Em decorrência da falta de estudos prospectivos empregando essa abordagem, os autores realizaram uma série de dermolipectomias usando a incisão abdominal para inserir o par de implantes mamários de silicone gel. MÉTODO: Cem pacientes consecutivas foram selecionadas, com média de idade de 33 ± 2 anos. A abdominoplastia clássica foi realizada e, em seguida, confeccionados 2 túneis sobre os hipocôndrios direito e esquerdo. Após colocação dos implantes, foi realizada reconstrução do sulco mamário com pontos simples usando fios absorvíveis, fixando o subcutâneo à aponeurose. RESULTADOS: Não houve nenhuma das seguintes complicações: trombose venosa profunda, complicações cardiorrespiratórias ou anestésicas, necrose de pele, sangramento visível, e hematoma ou infecção detectáveis clinicamente. O volume dos implantes variou de 280 ml a 450 ml (mediana de 350 ml). O tempo médio de operação foi de 116 minutos. Em nenhum caso foi necessária reoperação. O período de acompanhamento mínimo foi de 9 meses e máximo, de 84 meses (média de 36 meses). CONCLUSÕES: A técnica de aumento mamário por meio da incisão da abdominoplastia se mostrou confiável e simples, constituindo uma nova opção para a cirurgia mamária sem cicatriz nas mamas.

INTRODUCTION: Pregnancy and obesity cause distension of the abdominal wall and produce changes in the shape and size of the breasts. Thus, the need of aesthetic improvement of the abdominal area is not uncommon, coinciding with the desire for breast augmentation. Performing mammoplasty via the abdominoplasty incision approach was first described in 1976. Because of the lack of prospective studies using this approach, we performed a series of dermolipectomy procedures using the abdominal incision to insert a pair of silicone gel breast implants. METHODS: In total, 100 consecutive patients were selected, with a mean age of 33 ± 2 years. Classic abdominoplasty was performed, and 2 tunnels were then made in the right and left hypochondria. After implant placement, the mammary fold was reconstructed using simple sutures with absorbable threads to attach the subcutaneous tissue to the aponeurosis. RESULTS: None of the following complications were observed: deep-vein thrombosis, cardiorespiratory or anesthetic complications, skin necrosis, visible bleeding, hematoma, or clinically detectable infection. The volume of the implants ranged from 280 to 450 mL (median, 350 mL). The mean operation time was 116 minutes. Reoperation was not necessary in any of the cases. The monitoring period ranged from 9 to 84 months (mean, 36 months). CONCLUSIONS: Breast augmentation via the abdominoplasty incision approach was demonstrated to be a reliable and simple technique, providing a new, scar-free alternative to mammary surgical procedures.

Sexually dimorphic regulation of the Wingless morphogen controls sex-specific segment number in Drosophila.

Sexual dimorphism is widespread throughout the metazoa and plays important roles in mate recognition and preference, sex-based niche partitioning, and sex-specific coadaptation. One notable example of sex-specific differences in insect body morphology is presented by the higher diptera, such as Drosophila, in which males develop fewer abdominal segments than females. Because diversity in segment number is a distinguishing feature of major arthropod clades, it is of fundamental interest to understand how different numbers of segments can be generated within the same species. Here we show that sex-specific and segment-specific regulation of the Wingless (Wg) morphogen underlies the development of sexually dimorphic adult segment number in Drosophila. Wg expression is repressed in the developing terminal male abdominal segment by the combination of the Hox protein Abdominal-B (Abd-B) and the sex-determination regulator Doublesex (Dsx). The subsequent loss of the terminal male abdominal segment during pupation occurs through a combination of developmental processes including segment compartmental transformation, apoptosis, and suppression of cell proliferation. Furthermore, we show that ectopic expression of Wg is sufficient to rescue this loss. We propose that dimorphic Wg regulation, in concert with monomorphic segment-specific programmed cell death, are the principal mechanisms of sculpting the sexually dimorphic abdomen of Drosophila.

Targeted expression of p35 reveals a role for caspases in formation of the adult abdominal cuticle in Drosophila.

Programmed cell death is a fundamental aspect of metazoan development associated with the elaboration of disparate tissues and structures. Specialized cysteine proteases, the caspases, are mediators of cell death; once activated they cleave substrate proteins to dismantle doomed cells. Caspase activity is regulated by several cellular and viral inhibitors. The baculovirus p35 protein blocks the action of a wide range of caspases and inhibits cell death in divergent species. Here, we utilize the Gal4/UAS system to target p35 expression and analyze the requirements of caspase activity for development in Drosophila. We confirm that cell death is essential for proper morphogenesis of the adult male external genitalia and distal portions of the legs. In addition, we find that caspases are also required for elimination of larval epidermal cells and normal elaboration of the adult abdominal cuticle by histoblast derivatives. In particular, rescued p35-expressing larval epidermal cells accumulate along the abdominal midline and are associated with corresponding splits in both dorsal and ventral cuticle structures. This study reveals a novel role for cell death in a specific morphogenetic processes.

Changing gears in the cell cycle: histoblasts and beyond.

Although the molecular elements controlling cell cycle progression are well established, the mechanisms regulating how cell proliferation is triggered in response to extrinsic stimuli and how cell divisions change speed, particularly in stem or tumor cells or regenerative tissues, are poorly understood. One exceptional model system in which these events are precisely defined is Drosophila abdominal morphogenesis, in which stem-like histoblasts build the adult epidermis at metamorphosis by undergoing a series of sequential transitions from a non-proliferative to a growing, and finally to an invasive epithelium. We have recently uncovered in histoblasts an internal logic modulating cell cycle transitions that should constitute a reference paradigm for the study of other equivalent processes in stem cell, cancer or developmental biology.

Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.

During morphogenesis, cell movements, cell divisions and cell death work together to form complex patterns and to shape organs. These events are the outcome of decisions made by many individual cells, but how these decisions are controlled and coordinated is elusive. The adult abdominal epidermis of Drosophila is formed during metamorphosis by divisions and extensive cell migrations of the diploid histoblasts, which replace the polyploid larval cells. Using in vivo 4D microscopy, we have studied the behaviour of the histoblasts and analysed in detail how they reach their final position and to what extent they rearrange during their spreading. Tracking individual cells, we show that the cells migrate in two phases that differ in speed, direction and amount of cellular rearrangement. Cells of the anterior (A) and posterior (P) compartments differ in their behaviour. Cells near the A/P border are more likely to change their neighbours during migration. The mitoses do not show any preferential orientation. After mitosis, the sisters become preferentially aligned with the direction of movement. Thus, in the abdomen, it is the extensive cell migrations that appear to contribute most to morphogenesis. This contrasts with other developing epithelia, such as the wing imaginal disc and the embryonic germband in Drosophila, where oriented mitoses and local cell rearrangements appear to direct morphogenesis. Furthermore, our results suggest that an active force created by the histoblasts contributes to the formation of the adult epidermis. Finally, we show that histoblasts occasionally undergo apoptosis.

Dynamic control of cell cycle and growth coupling by ecdysone, EGFR, and PI3K signaling in Drosophila histoblasts.

Regulation of cell proliferation has been extensively studied in cultured cell systems that are characterized by coordinated growth and cell-cycle progression and relatively uniform cell size distribution. During the development of multicellular organisms, however, growth and division can be temporally uncoupled, and the signaling pathways that regulate these growth programs are poorly understood. A good model for analyzing proliferation control in such systems is the morphogenesis of the Drosophila adult abdominal epidermis by histoblasts. These cells undergo a series of temporally regulated transitions during which neither cell size nor division rate is constant. The proliferation of histoblasts during metamorphosis is uniquely amenable to clonal analysis in combination with live imaging. Thereby, we show that abdominal histoblasts, which grow while in G2 arrest during larval stages, enter a proliferative stage in the pupal period that is initiated by ecdysone-dependent string/Cdc25 phosphatase transcription. The proliferating histoblasts have preaccumulated stores of Cyclin E, which trigger an immediate S phase onset after mitosis. These rapid cell cycles lack a G1 phase and result in a progressive reduction of cell size. Eventually, the histoblasts proceed to a stage of slower proliferation that, in contrast to the preceding, depends on epidermal growth factor receptor (EGFR) signaling for progression through the G2/M transition and on insulin receptor/PI3K-mediated signaling for growth. These results uncover the developmentally programmed changes coupling the growth and proliferation of the histoblasts that form the abdominal epidermis of Drosophila. Histoblasts proceed through three distinct stages: growth without division, division without growth, and growth-coupled proliferation. Our identification of the signaling pathways and cell-cycle regulators that control these programs illustrates the power of in vivo time-lapse analyses after clone induction. It sets the stage for the comprehensive understanding of the coordination of cell growth and cell-cycle progression in complex multicellular eukaryotes.

Neuropeptides in perisympathetic organs of Manduca sexta: specific composition and changes during the development.

We used a combination of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and immunocytochemistry to investigate the peptides from abdominal perisympathetic organs of Manduca sexta. Altogether three mass peaks, detected in mass spectra from single abdominal perisympathetic organs were identical with already known neuropeptides, namely CAP(2b), CCAP, and Manduca-allatotropin. Only CAP(2b) was found throughout the postembryonic development. In larvae, perisympathetic organs of the abdominal ganglia 1 and 7 do not accumulate neuropeptides. During the metamorphosis, the number of putative hormones stored in the abdominal perisympathetic organs, increases dramatically. Not a single substance, however, obtained in mass spectra of larval perisympathetic organs disappeared in the respective adult neurohemal organs. Peptides from abdominal perisympathetic organs are different from those of thoracic perisympathetic organs and the retrocerebral complex. Manduca-FLRFa-2 and -3 are enriched in thoracic perisympathetic organs; FLRFa-1, corazonin and adipokinetic hormone are abundant peptides of the retrocerebral complex. The majority of ion signals, however, represent unknown substances. An antiserum which recognized CAP(2b) allowed the morphological characterization of a median neurosecretory system in the abdominal ventral nerve cord of M. sexta, which resembles that of cockroach embryos. Double stainings confirmed that crustacean cardioactive peptide (CCAP) becomes colocalized with CAP(2b) in median neurosecretory cells during the last larval instar. This colocalization continues in adult insects.

Homeotic regulation of segment-specific differences in neuroblast numbers and proliferation in the Drosophila central nervous system.

The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the early Drosophila embryo is identical in thoracic and abdominal segments. However, during late embryogenesis differences in the numbers of neuroblasts and in the extent of neuroblast proliferation arise between these regions. We show that the homeotic genes Ultrabithorax and abdominal-A regulate these late differences, and that misexpression of either gene in thoracic neuroblasts after segregation is sufficient to induce abdominal behaviour. However, in wild type embryos we only detect abdominal-A and Ultrabithorax proteins in early neuroblasts. Furthermore, transplantation experiments reveal that segment-specific behaviour is determined prior to neuroblast segregation. Thus, the segment-specific differences in neuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts, and executed in later programmes controlling neuroblast numbers and proliferation.

The tumorous-head-1 locus affects bristle number of the Drosophila melanogaster cuticle.

The tuh-1 maternal effect locus contains two naturally occurring isoalleles, tuh-1h and tuh-1g. Until recently there has been no possibility to distinguish between the tuh-lh and the tuh-1g maternal effects other than evaluating their effect on the Bithorax-Complex (BXC) Abdominal B (Abd-B) mutant tuh-3. However, in this report we identify a bristle phenotype associated with the tuh-1 locus that has very interesting evolutionary implications. Females homozygous for tuh-1h always produce adult offspring with more bristles than females homozygous or heterozygous for tuh-1g. The effect is global. Increased bristle number occurs in the head, the thorax, and the anterior and posterior abdomen. Females totally deficient for the tuh-1 gene produce offspring with high bristle number. Thus, the bristle phenotype results from the absence of the maternally contributed tuh-1g factor. Genetic evidence shows that the bristle phenotype is caused by the tuh-1 locus and that tuh-1h is completely recessive to tuh-1g. The tuh-1 locus is located at the euchromatin-beta-heterochromatin junction near the centromere of the X chromosome and deficiency analysis places the locus between the lethal genes extra organs (eo) and lethal B20 (lB20). The variance in bristle number attributable to the tuh-1 locus in nature is approximately 10.1%, an indication that the bristle phenotype is most likely a neutral, pleiotrophic side effect of tuh-1.

Hedgehog acts by distinct gradient and signal relay mechanisms to organise cell type and cell polarity in the Drosophila abdomen.

The epidermis of the adult Drosophila abdomen is formed by a chain of anterior (A) and posterior (P) compartments, each segment comprising one A and one P compartment. In the accompanying paper (Struhl et al., 1997), we provide evidence that Hedgehog protein (Hh), being secreted from P compartment cells, organises the pattern and polarity of A compartment cells. Here we test whether Hh acts directly or by a signal relay mechanism. We use mutations in Protein Kinase A (PKA) or smoothened (smo) to activate or to block Hh signal transduction in clones of A compartment cells. For cell type, a scalar property, both manipulations cause strictly autonomous transformations: the cells affected are exactly those and only those that are mutant. Hence, we infer that Hh acts directly on A compartment cells to specify the various types of cuticular structures that they differentiate. By contrast, these same manipulations cause non-autonomous effects on cell polarity, a vectorial property. Consequently, we surmise that Hh influences cell polarity indirectly, possibly by inducing other signalling factors. Finally, we present evidence that Hh does not polarise abdominal cells by utilising either Decapentaplegic (Dpp) or Wingless (Wg), the two morphogens through which Hh acts during limb development. We conclude that, in the abdomen, cell type and cell polarity reflect distinct outputs of Hh signalling and propose that these outputs are controlled by separable gradient and signal relay mechanisms.

Cell fates in the adult abdomen of Drosophila are determined by wingless during pupal development.

wingless activity is required for the patterning of tergites and sternites in the adult abdomen of Drosophila melanogaster. In the absence of wg, tergite and sternite cuticular differentiation is replaced by that of pleura. Temperature shift analysis of a wg temperature-sensitive allele has shown that sensory bristles and tergite/sternite histotype are determined independently and that wg is required between 15 and 20 hr after pupariation for bristle formation. The determination of sensory mother cells at this stage of development was confirmed by expression of the neuralized gene in a subset of the proliferating histoblasts. Ectopic expression of wg leads to the appearance of ectopic bristles and expanded tergite and sternite, indicating that wg expression is sufficient to promote both bristle formation and tergite/sternite differentiation. wg is expressed in the dividing and spreading histoblast population in a restricted pattern which may determine the spatial arrangement of cuticular elements.

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta.

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.