Sampling rate and heart rate variability: On metrics and health outcomes.

Sampling rate impacts the quality of HRV estimates. In the context of the recent research article by Burma et al published in JBI which systematically examined this matter, I discuss this notion more deeply with practical implications to biomedical informatics. Not all HRV metrics are created equal regarding their sensitivity to sampling rate errors when their health predictive performance is concerned. A combination of several, especially nonlinear HRV metrics can remedy these sampling rate constraints. I present methodology for comprehensive validation of the effect of sampling rate on HRV.

Fetal heart rate variability responsiveness to maternal stress, non-invasively detected from maternal transabdominal ECG.

PURPOSE: Prenatal stress (PS) during pregnancy affects in utero- and postnatal child brain-development. Key systems affected are the hypothalamic-pituitary-adrenal axis and the autonomic nervous system (ANS). Maternal- and fetal ANS activity can be gauged non-invasively from transabdominal electrocardiogram (taECG). We propose a novel approach to assess couplings between maternal (mHR) and fetal heart rate (fHR) as a new biomarker for PS based on bivariate phase-rectified signal averaging (BPRSA). We hypothesized that PS exerts lasting impact on fHR. METHODS: Prospective case-control study matched for maternal age, parity, and gestational age during the third trimester using the Cohen Perceived Stress Scale (PSS-10) questionnaire with PSS-10 over or equal 19 classified as stress group (SG). Women with PSS-10 < 19 served as control group (CG). Fetal electrocardiograms were recorded by a taECG. Coupling between mHR and fHR was analyzed by BPRSA resulting in fetal stress index (FSI). Maternal hair cortisol, a memory of chronic stress exposure for 2-3 months, was measured at birth. RESULTS: 538/1500 pregnant women returned the questionnaire, 55/538 (10.2%) mother-child pairs formed SG and were matched with 55/449 (12.2%) consecutive patients as CG. Maternal hair cortisol was 86.6 (48.0-169.2) versus 53.0 (34.4-105.9) pg/mg (p = 0.029). At 36 + 5 weeks, FSI was significantly higher in fetuses of stressed mothers when compared to controls [0.43 (0.18-0.85) versus 0.00 (- 0.49-0.18), p < 0.001]. CONCLUSION: Prenatal maternal stress affects the coupling between maternal and fetal heart rate detectable non-invasively a month prior to birth. Lasting effects on neurodevelopment of affected offspring should be studied. TRIAL REGISTRATION: Clinical trial registration: NCT03389178.

Non-invasive acquisition of fetal ECG from the maternal xyphoid process: a feasibility study in pregnant sheep and a call for open data sets.

OBJECTIVE: The utility of fetal heart rate (FHR) monitoring can only be achieved with an acquisition sampling rate that preserves the underlying physiological information on the millisecond time scale (1000 Hz rather than 4 Hz). For such acquisition, fetal ECG (fECG) is required, rather than the ultrasound to derive FHR. We tested one recently developed algorithm, SAVER, and two widely applied algorithms to extract fECG from a single-channel maternal ECG signal recorded over the xyphoid process rather than the routine abdominal signal. APPROACH: At 126dG, ECG was attached to near-term ewe and fetal shoulders, manubrium and xyphoid processes (n = 12). fECG served as the ground-truth to which the fetal ECG signal extracted from the simultaneously-acquired maternal ECG was compared. All fetuses were in good health during surgery (pH 7.29 ± 0.03, pO2 33.2 ± 8.4, pCO2 56.0 ± 7.8, O2Sat 78.3 ± 7.6, lactate 2.8 ± 0.6, BE -0.3 ± 2.4). MAIN RESULT: In all animals, single lead fECG extraction algorithm could not extract fECG from the maternal ECG signal over the xyphoid process with the F1 less than 50%. SIGNIFICANCE: The applied fECG extraction algorithms might be unsuitable for the maternal ECG signal over the xyphoid process, or the latter does not contain strong enough fECG signal, although the lead is near the mother's abdomen. Fetal sheep model is widely used to mimic various fetal conditions, yet ECG recordings in a public data set form are not available to test the predictive ability of fECG and FHR. We are making this data set openly available to other researchers to foster non-invasive fECG acquisition in this animal model.

α7 nicotinic acetylcholine receptor signaling modulates the inflammatory phenotype of fetal brain microglia: first evidence of interference by iron homeostasis.

Neuroinflammation in utero may result in life-long neurological disabilities. Microglia play a pivotal role, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of microglia. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal microglia will augment their neuroprotective transcriptome profile, while the antagonistic stimulation of α7nAChR will achieve the opposite. Using an in vivo - in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep microglia cultures re-exposed to LPS in presence of a selective α7nAChR agonist or antagonist. Our RNAseq and protein level findings show that a pro-inflammatory microglial phenotype acquired in vitro by LPS stimulation is reversed with α7nAChR agonistic stimulation. Conversely, antagonistic α7nAChR stimulation potentiates the pro-inflammatory microglial phenotype. Surprisingly, under conditions of LPS double-hit an interference of a postulated α7nAChR - ferroportin signaling pathway may impede this mechanism. These results suggest a therapeutic potential of α7nAChR agonists in early re-programming of microglia in neonates exposed to in utero inflammation via an endogenous cerebral cholinergic anti-inflammatory pathway. Future studies will assess the role of interactions between inflammation-triggered microglial iron sequestering and α7nAChR signaling in neurodevelopment.

T-Box Genes in Drosophila Mesoderm Development.

In Drosophila there are eight genes encoding transcription factors of the T-box family, which are known to exert a variety of crucial developmental functions during ectodermal patterning processes, neuronal cell specification, mesodermal tissue development, and the development of extraembryonic tissues. In this review, we focus on the prominent roles of Drosophila T-box genes in mesodermal tissues. First, we describe the contributions of brachyenteron (byn) and optomotor-blind-related-gene-1 (org-1) to the development of the visceral mesoderm. Second, we provide an overview on the functions of the three Dorsocross paralogs (Doc1-3) and the two Tbx20-related paralogs (midline and H15) during Drosophila heart development. Third, we portray the roles of org-1 and midline/H15 in the specification of individual body wall and organ-attached muscles, including the function of org-1 in the transdifferentiation of certain heart-attached muscles during metamorphosis. The functional analysis of these evolutionarily conserved T-box genes, along with their interactions with other types of transcription factors and various signaling pathways, has provided key insights into the regulation of Drosophila visceral mesoderm, muscle, and heart development.

Development of somatosensory-evoked potentials in foetal sheep: effects of betamethasone.

AIM: Antenatal glucocorticoids are used to accelerate foetal lung maturation in babies threatened with premature labour. We examined the influence of glucocorticoids on functional and structural maturation of the central somatosensory pathway in foetal sheep. Somatosensory-evoked potentials (SEP) reflect processing of somatosensory stimuli. SEP latencies are determined by afferent stimuli transmission while SEP amplitudes reveal cerebral processing. METHODS: After chronic instrumentation of foetal sheep, mothers received saline (n = 9) or three courses of betamethasone (human equivalent dose of 2 × 110 µg kg-1 betamethasone i.m. 24 h apart, n = 12) at 0.7, 0.75 and 0.8 of gestational age. Trigeminal SEP were evoked prior to, 4 and 24 h after each injection and at 0.8 of gestational age before brains were histologically processed. RESULTS: Somatosensory-evoked potentials were already detectable at 0.7 of gestation age. The early and late responses N20 and N200 were the only reproducible peaks over the entire study period. With advancing gestational age, SEP latencies decreased but amplitudes remained unchanged. Acutely, betamethasone did not affect SEP latencies and amplitudes 4 and 24 h following administration. Chronically, betamethasone delayed developmental decrease in the N200 but not N20 latency by 2 weeks without affecting amplitudes. In parallel, betamethasone decreased subcortical white matter myelination but did not affect network formation and synaptic density in the somatosensory cortex. CONCLUSION: Somatosensory stimuli are already processed by the foetal cerebral cortex at the beginning of the third trimester. Subsequent developmental decrease in SEP latencies suggests ongoing maturation of afferent sensory transmission. Antenatal glucocorticoids affect structural and functional development of the somatosensory system with specific effects at subcortical level.

RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain.

BACKGROUND: The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. NEW METHOD: Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. RESULTS: We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1ß) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. COMPARISON WITH EXISTING METHODS: This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. CONCLUSIONS: The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.

Decreased neuroinflammation correlates to higher vagus nerve activity fluctuations in near-term ovine fetuses: a case for the afferent cholinergic anti-inflammatory pathway?

BACKGROUND: Neuroinflammation in utero may contribute to brain injury resulting in life-long neurological disabilities. The pivotal role of the efferent cholinergic anti-inflammatory pathway (CAP) in controlling inflammation, e.g., by inhibiting the HMGB1 release, via the macrophages' α7 nicotinic acetylcholine receptor (α7nAChR) has been described in adults, but its importance in the fetus is unknown. Moreover, it is unknown whether CAP may also exert anti-inflammatory effects on the brain via the anatomically predominant afferent component of the vagus nerve. METHODS: We measured microglial activation in the ovine fetal brain near term 24 h after the umbilical cord occlusions mimicking human labor versus controls (no occlusions) by quantifying HMGB1 nucleus-to-cytosol translocation in the Iba1+ and α7nAChR+ microglia. Based on multiple clinical studies in adults and our own work in fetal autonomic nervous system, we gauged the degree of CAP activity in vivo using heart rate variability measure RMSSD that reflects fluctuations in vagus nerve activity. RESULTS: RMSSD correlated to corresponding plasma IL-1ß levels at R = 0.57 (p = 0.02, n = 17) and to white matter microglia cell counts at R = -0.89 (p = 0.03). The insult increased the HMGB1 translocation in α7nAChR+ microglia in a brain region-dependent manner (p < 0.001). In parallel, RMSSD at 1 h post insult correlated with cytosolic HMGB1 of thalamic microglia (R = -0.94, p = 0.005), and RMSSD at pH nadir correlated with microglial α7nAChR in the white matter (R = 0.83, p = 0.04). Overall, higher RMSSD values correlated with lower HMGB1 translocation and higher α7nAChR intensity per area in a brain region-specific manner. CONCLUSIONS: Afferent fetal CAP may translate increased vagal cholinergic signaling into suppression of cerebral inflammation in response to near-term hypoxic acidemia as might occur during labor. Our findings suggest a new control mechanism of fetal neuroinflammation via the vagus nerve, providing novel possibilities for its non-invasive monitoring in utero and for targeted treatment.

Fetal cholinergic anti-inflammatory pathway and necrotizing enterocolitis: the brain-gut connection begins in utero.

Necrotizing enterocolitis (NEC) is an acute neonatal inflammatory disease that affects the intestine and may result in necrosis, systemic sepsis and multisystem organ failure. NEC affects 5-10% of all infants with birth weight ≤ 1500 g or gestational age less than 30 weeks. Chorioamnionitis (CA) is the main manifestation of pathological inflammation in the fetus and is strong associated with NEC. CA affects 20% of full-term pregnancies and upto 60% of preterm pregnancies and, notably, is often an occult finding. Intrauterine exposure to inflammatory stimuli may switch innate immunity cells such as macrophages to a reactive phenotype ("priming"). Confronted with renewed inflammatory stimuli during labour or postnatally, such sensitized cells can sustain a chronic or exaggerated production of proinflammatory cytokines associated with NEC (two-hit hypothesis). Via the cholinergic anti-inflammatory pathway, a neurally mediated innate anti-inflammatory mechanism, higher levels of vagal activity are associated with lower systemic levels of proinflammatory cytokines. This effect is mediated by the α7 subunit nicotinic acetylcholine receptor (α7nAChR) on macrophages. The gut is the most extensive organ innervated by the vagus nerve; it is also the primary site of innate immunity in the newborn. Here we review the mechanisms of possible neuroimmunological brain-gut interactions involved in the induction and control of antenatal intestinal inflammatory response and priming. We propose a neuroimmunological framework to (1) study the long-term effects of perinatal intestinal response to infection and (2) to uncover new targets for preventive and therapeutic intervention.

Time scales of autonomic information flow in near-term fetal sheep.

Autonomic information flow (AIF) characterizes fetal heart rate (FHR) variability (fHRV) in the time scale dependent complexity domain and discriminates sleep states [high voltage/low frequency (HV/LF) and low voltage/high frequency (LV/HF) electrocortical activity (ECoG)]. However, the physiologic relationship of AIF time scales to the underlying sympathetic and vagal rhythms is not known. Understanding this relationship will enhance the benefits derived from using fHRV to monitor fetal health non-invasively. We analyzed AIF measured as Kullback-Leibler entropy (KLE) in fetal sheep in late gestation as function of vagal and sympathetic modulation of fHRV, using atropine and propranolol, respectively (n = 6), and also analyzed changes in fHRV during sleep states (n = 12). Atropine blockade resulted in complexity decrease at 2.5 Hz compared to baseline HV/LF and LV/HF states and at 1.6 Hz compared to LV/HF. Propranolol blockade resulted in complexity increase in the 0.8-1 Hz range compared to LV/HF and in no changes when compared to HV/LF. During LV/HF state activity, fHRV complexity was lower at 2.5 Hz and higher at 0.15-0.19 Hz than during HV/LF. Our findings show that in mature fetuses near term vagal activity contributes to fHRV complexity on a wider range of time scales than sympathetic activity. Related to sleep, during LV/HF we found lower complexity at short-term time scale where complexity is also decreased due to vagal blockade. We conclude that vagal and sympathetic modulations of fHRV show sleep state-dependent and time scale-dependent complexity patterns captured by AIF analysis of fHRV. Specifically, we observed a vagally mediated and sleep state-dependent change in these patterns at a time scale around 2.5 Hz (0.2 s). A paradigm of state-dependent non-linear sympathovagal modulation of fHRV is discussed.

Fetal body weight and the development of the control of the cardiovascular system in fetal sheep.

Reduced birth weight predisposes to cardiovascular diseases in later life. We examined in fetal sheep at 0.76 (n = 18) and 0.87 (n = 17) gestation whether spontaneously occurring variations in fetal weight affect maturation of autonomic control of cardiovascular function. Fetal weights at both gestational ages were grouped statistically in low (LW) and normal weights (NW) (P < 0.01). LW fetuses were within the normal weight span showing minor growth dysproportionality at 0.76 gestation favouring heart and brain, with a primary growth of carcass between 0.76 and 0.87 gestation (P < 0.05). While twins largely contributed to LW fetuses, weight differences between singletons and twins were absent at 0.76 and modest at 0.87 gestation, underscoring the fact that twins belong to normality in fetal sheep not constituting a major malnutritive condition. Mean fetal blood pressure (FBP) of all fetuses was negatively correlated to fetal weight at 0.76 but not 0.87 gestation (P < 0.05). At this age, FBP and baroreceptor reflex sensitivity were increased in LW fetuses (P < 0.05), suggesting increased sympathetic activity and immaturity of circulatory control. Development of vagal modulation of fetal heart rate depended on fetal weight (P < 0.01). These functional associations were largely independent of twin pregnancies. We conclude, low fetal weight within the normal weight span is accompanied by a different trajectory of development of sympathetic blood pressure and vagal heart rate control. This may contribute to the development of elevated blood pressure in later life. Examination of the underlying mechanisms and consequences may contribute to the understanding of programming of cardiovascular diseases.

Autonomic organization of respirocardial function in healthy human neonates in quiet and active sleep.

BACKGROUND AND AIM: It is not known on which time scales the nonlinear respirocardial interactions occur. This work's aim is to quantitatively assess functional respirocardial organization during quiet and active sleep of healthy full-term neonates by autonomic information flow (AIF) without limitation on specific time scales. Representing respirocardial interactions on a global time scale AIF carries information on a wider scope of interdependencies than known linear and nonlinear measures described. It assesses the complexity of heart rate fluctuations (HRF) and respiratory movements (RM) and their interaction comprising both linear and nonlinear properties. Thus, we hypothesized AIF to characterize novel aspects of sleep state-dependent respirocardial interaction. METHODS: RM and ECG-derived HRF of six healthy full-term neonates were studied. We analyzed their power spectra, coherence, auto- and cross-correlation and complexity estimated on local ("next sample" prediction) and global time scales (an integral over AIF predicting for all time lags in HRF and RM). RESULTS: We found the global AIF of HRF and RM to differ significantly between active and quiet sleep in all neonates, whereas on a local time scale this applied to the HRF AIF only. HRF complexity was larger in quiet than in active sleep. Respirocardial interaction was less complex in quiet versus active sleep in the high frequency band only. CONCLUSION: Complex sleep state-related changes of respirocardial interdependencies cannot be identified completely on the local time scale. Considering the global time scale of respirocardial interactions allows a more complete physiological interpretation with regard to the underlying autonomic dynamics.

Cardiogenesis in the Drosophila model: control mechanisms during early induction and diversification of cardiac progenitors.

The dorsal vessel of Drosophila displays developmental, functional, and morphological similarities to the primitive linear heart tube of early vertebrate embryos. Because these similarities extend to the genetic and molecular level, Drosophila has become a fruitful model to study control mechanisms of early heart development. Herein we summarize recently obtained insights into control mechanisms during early induction and diversification of cardiac progenitors in Drosophila. We also show that induction of tinman, a key cardiogenic gene, in the dorsal mesoderm by Dpp (Drosophila BMP) involves protein/protein interactions between Tinman and the Smad proteins Mad and Medea, in addition to their DNA-binding activities to specific tinman enhancer sequences. Furthermore, we present evidence that binding of a high-mobility-group protein, HMG-D, to the Dpp-responsive enhancer of tinman as well as to the Tinman protein may be involved in the formation of a fully active enhancer complex.

biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila.

The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut musculature versus body wall musculature. Here we report that biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature. biniou expression is activated in the trunk visceral mesoderm primordia downstream of dpp, tinman, and bagpipe and is maintained in all types of developing gut muscles. We show that biniou activity is essential for maintaining the distinction between splanchnic and somatic mesoderm and for differentiation of the splanchnic mesoderm into midgut musculature. biniou is required not only for the activation of differentiation genes that are expressed ubiquitously in the trunk visceral mesoderm but also for the expression of dpp in parasegment 7, which governs proper midgut morphogenesis. Activation of dpp is mediated by specific Biniou binding sites in a dpp enhancer element, which suggests that Biniou serves as a tissue-specific cofactor of homeotic gene products in visceral mesoderm patterning. Based upon these and other data, we propose that the splanchnic mesoderm layers in Drosophila and vertebrate embryos are homologous structures whose development into gut musculature and other visceral organs is critically dependent on FoxF genes.

A role for the COUP-TF-related gene seven-up in the diversification of cardioblast identities in the dorsal vessel of Drosophila.

The Drosophila gene tinman is essential for dorsal vessel (heart) formation and is structurally and functionally conserved in vertebrates. In the mature embryonic dorsal vessel, tinman is expressed in four of the six pairs of cardioblasts in each segment. We provide evidence that seven-up, which is homologous to the vertebrate COUP-TF transcription factor and is expressed in the non-Tinman-expressing cardioblasts, represses tinman in these cells. Loss of function seven-up mutations derepress tinman expression in these cardioblasts while ectopic expression of seven-up represses tinman in the cardioblasts that normally express it. These changes are correlated with alterations in the expression of additional molecular markers for each of these two types of cardioblasts, such as the novel T-box-containing gene Tb66F2 and the potassium channel-encoding gene sur. These observations suggest that seven-up has a role in diversifying cardioblast identities within each segment. We also describe the tinman cis sequences that mediate tinman repression by seven-up and examine whether Seven-up can bind these sequences to directly inhibit tinman.

Testosterone alters membrane binding of progesterone in male rat brains / La testosterona altera la fijación de membrana de la progesterona en el cerebro de ratas machos

The steroid progesterone (P) is important in control of male sexual behaviors. In this study binding sites for P conjugated to bovine serum albumin (P-BSA) were analyzed in the brains of gonadectomized male rats that were implanted with silastic capsules either filled with testosterone or empty. Frozen brains were sliced, thaw mounted on microscope slides, and incubated with radiolabeled P-BSA (P-[125I-BSA]) alone or with 1000 fold P-BSA competitor. Microscope slides were then dried, dipped in photographic emulsion and placed in light-tight boxes for four days before being developed and counterstained. Grain densities over cells were then analyzed with an image analysis system. Testosterone treatment significantly increased specific binding of P-[125I-BSA] in the caudal medial basal hypothalamus, the paraventricular nucleus-anterior hypothalamus, and the medial preoptic area. Testosterone treatment significantly decreased P-[125I-BSA] binding in the amygdala. There was no significant effect of testosterone in the rostral medial basal hypothalamus. Testosterone treatment changes P-[125I-BSA] binding in several areas that are important for male sexual behavior suggesting a function for P-[125I-BSA] binding sites in endocrine systems and behavior (AU)

El esteroide progesterona (P) es importante en el control del comportamiento sexual del macho. En este estudio se analizaron los lugares de fijación de la P conjugada con albúmina sérica bovina (P-BSA) en el cerebro de ratas machos gonadectomizadas que fueron implantadas con cápsulas silásticas rellenas con testosterona o bien vacías. Se hicieron secciones de los cerebros congelados que fueron montadas en portas e incubadas con P-BSA radiomarcada (P-[125I-BSA]) sola o con un competidor 1000 veces mayor de P-BSA. Posteriormente, los cortes microscópicos fueron secados, sumergidos en emulsión fotográfica y colocados en cajas en oscuridad durante cuatro días antes de ser revelados y teñidos con tinción de contraste. Se analizaron después las densidades de los granos situados sobre las células con un sistema de análisis de imagen. El tratamiento con testosterona elevó significativamente la fijación específica de (P-[125I-BSA]) en el hipotálamo basal caudal y medial, el núcleo paraventricular del hipotálamo anterior y el área preóptica medial. El tratamiento con testosterona disminuyó de forma significativa la fijación de (P-[125I-BSA]) en la amígdala. La testosterona no tuvo efecto significativo en el hipotálamo basal rostral y medial. El tratamiento con testosterona cambia la fijación de (P-[125I-BSA]) en diversas áreas que son importantes para el comportamiento sexual del macho, sugiriendo una función para los lugares de fijación de (P-[125I-BSA]) en los sistemas endocrinos y en el comportamiento (AU)

A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs.

Although genes involved in common developmental programs are usually scattered throughout the metazoan genome, there are some important examples of functionally interconnected regulatory genes that display close physical linkage. In particular the homeotic genes, which determine the identities of body parts, are clustered in the Hox complexes and clustering is thought to be crucial for the proper execution of their developmental programs. Here we describe the organization and functional properties of a more recently identified cluster of six homeobox genes at 93DE on the third chromosome of Drosophila. These genes, which include tinman, bagpipe, ladybird early, ladybird late, C15, and slouch, all participate in mesodermal patterning and differentiation programs and show multiple regulatory interactions among each other. We propose that their clustering, through unknown mechanisms, is functionally significant and discuss the similarities and differences between the 93DE homeobox gene cluster and the Hox complexes.

Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors.

Individual somatic muscles and heart progenitors are specified at defined positions within the mesodermal layer of Drosophila. The expression of the homeobox gene even-skipped (eve) identifies one specific subset of cells in the dorsal mesoderm, which give rise to particular pericardial cells and dorsal body wall muscles. Genetic analysis has shown that the induction of eve in these cells involves the combined activities of genes encoding mesoderm-intrinsic factors, such as Tinman (Tin), and spatially restricted signaling activities that are largely derived from the ectoderm, particularly those encoded by wingless (wg) and decapentaplegic (dpp). Here we show that a Dpp-activated Smad protein, phosphorylated Mad, is colocalized in eve-expressing cells during an extended developmental period. We demonstrate further that a mesodermally active enhancer of eve contains several Smad and Tin binding sites that are essential for enhancer activity in vivo. This enhancer also contains a number of binding sites for the Wg-effector Pangolin (Pan/Lef-1), which are required for full levels of enhancer activity. However, we find that their main function is to prevent ectopic enhancer activity in the dorsal mesoderm. This suggests that, in the absence of Wg signaling, Pan binding serves to abrogate the synergistic activities of Smads and Tin in eve activation while, in cells that receive Wg signals, Pan is converted into a coactivator that promotes eve induction. Together, these data show that the eve enhancer integrates several regulatory pathways via the combinatorial binding of the mesoderm-intrinsic regulator Tin and the effectors of the Dpp and Wg signals.