Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels.

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. Here, we show that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, we provide evidence that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill.

Smaug1 membrane-less organelles respond to AMPK and mTOR and affect mitochondrial function.

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused.

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Chest imaging findings of chronic respiratory disease in HIV-infected adolescents on combined anti retro viral therapy.

Early treatment with combination antiretroviral therapy (cART) has improved survival of children perinatally infected with HIV into adolescence. This population is at risk of long term complications related to HIV infection, particularly chronic respiratory disease. Limited data on chest imaging findings in HIV-infected adolescents, suggest that the predominant disease is of small and large airways: predominantly bronchiolitis obliterans or bronchiectasis. Single cases of emphysema have been reported. Lung fibrosis, lymphocytic interstitial pneumonitis, post tuberculous apical fibrocystic changes and malignancies do not feature in this population. Chest radiograph (CXR) is easily accessible and widely used, especially in resource limited settings, such as sub Saharan Africa, where the greatest burden of HIV disease occurs. Lung ultrasound has been described for the diagnosis of pneumonia in children, pulmonary oedema and interstitial lung disease [1-3]. The use of this modality in chronic respiratory disease in adolescents where the predominant finding is small airway disease and bronchiectasis has however not been described. CXR is useful to evaluate structural/post infective changes, parenchymal opacification and nodules, hyperinflation or extensive bronchiectasis. CXR however, is inadequate for diagnosing small airway disease, for which high resolution computed tomography (HRCT) is the modality of choice. Where available, low dose HRCT should be used early in the course of symptomatic disease in adolescents and for follow up in children who are non responsive to treatment or clinically deteriorating. This article provides a pictorial review of the spectrum of CXR and HRCT imaging findings of chronic pulmonary disease in perinatally HIV-infected adolescents on cART and guidelines for imaging.

Yield of CT angiography in penetrating lower extremity trauma.

PURPOSE: CT angiography (CTA) has become a valuable tool in the assessment of suspected arterial injury in patients with penetrating lower extremity trauma. However, expensive imaging such as CTA should be judiciously utilized to ensure value-based care. We therefore assessed the yield of CTA in this setting at a level-1 trauma unit and correlated it with the clinical history provided. METHODS: A retrospective descriptive study from 1 July 2013 to 31 June 2018 at a 1386-bed, tertiary-level, public-sector teaching hospital in Cape Town, South Africa.. All patients undergoing CTA for suspected arterial injury following penetrating lower extremity trauma were included. The imaging yield of clinically significant arterial injury and the predictive value of specific clinical signs were determined. RESULTS: A total of 983 patients (median age 27 years, 91% male) were included; 90% (886/983) had gunshots, 9% (89/983) stabs, and 1% (8/983) other injuries. Despite an average 13% year-on-year increase in CTA performed, there was no change in the proportion demonstrating arterial injury. Thirty-four percent (23/68) of patients with strong (hard) signs of arterial injury (active pulsatile bleeding, rapidly expanding hematoma, absent pulse, palpable thrill, or audible bruit), 11% (49/459) with moderate (soft) signs (history of an arterial bleed, excessive non-pulsatile bleeding, large non-expanding hematoma, major neurological deficit, diminished but appreciable pulse, and arterial proximity), and 5% (24/456) with no indication for imaging had clinically significant arterial injuries. Significant positive correlations were rapidly expanding hematoma (p = 0.009), an absent pulse (p < 0.001), and a diminished pulse (p < 0.001). Significant negative correlations were proximity to a major artery (p = 0.005) and no clinical indication provided (p < 0.001). CONCLUSION: There is poor correlation between clinical details provided and the presence of arterial injury at our institution. In this context, CTA serves a pivotal role in the definitive identification of arterial injury.

The bZIP transcription factor SPA Heterodimerizing Protein represses glutenin synthesis in Triticum aestivum.

The quality of wheat grain is mainly determined by the quantity and composition of its grain storage proteins (GSPs). Grain storage proteins consist of low- and high-molecular-weight glutenins (LMW-GS and HMW-GS, respectively) and gliadins. The synthesis of these proteins is essentially regulated at the transcriptional level and by the availability of nitrogen and sulfur. The regulation network has been extensively studied in barley where BLZ1 and BLZ2, members of the basic leucine zipper (bZIP) family, activate the synthesis of hordeins. To date, in wheat, only the ortholog of BLZ2, Storage Protein Activator (SPA), has been identified as playing a major role in the regulation of GSP synthesis. Here, the ortholog of BLZ1, named SPA Heterodimerizing Protein (SHP), was identified and its involvement in the transcriptional regulation of the genes coding for GSPs was analyzed. In gel mobility shift assays, SHP binds cis-motifs known to bind to bZIP family transcription factors in HMW-GS and LMW-GS promoters. Moreover, we showed by transient expression assays in wheat endosperm that SHP acts as a repressor of the activity of these gene promoters. This result was confirmed in transgenic lines overexpressing SHP, which were grown with low and high nitrogen supply. The phenotype of SHP-overexpressing lines showed a lower quantity of both LMW-GS and HMW-GS, while the quantity of gliadin was unchanged, whatever the nitrogen availability. Thus, the gliadin/glutenin ratio was increased, which suggests that gliadin and glutenin genes may be differently regulated.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused.

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

Quantitative CT analysis for bronchiolitis obliterans in perinatally HIV-infected adolescents-comparison with controls and lung function data.

OBJECTIVE: To compare quantitative chest CT parameters in perinatally HIV-infected adolescents with and without bronchiolitis obliterans compared with HIV-uninfected controls and their association with lung function measurements. MATERIALS AND METHODS: Seventy-eight (41 girls) HIV-infected adolescents with a mean age of 13.8 ± 1.65 years and abnormal pulmonary function tests in the prospective Cape Town Adolescent Antiretroviral Cohort underwent contrast-enhanced chest CT on inspiration and expiration. Sixteen age-, sex-, and height-matched non-infected controls were identified retrospectively. Fifty-one HIV-infected adolescents (28 girls) displayed mosaic attenuation on expiration suggesting bronchiolitis obliterans. Pulmonary function tests were collected. The following parameters were obtained: low- and high-attenuation areas, mean lung density, kurtosis, skewness, ventilation heterogeneity, lung mass, and volume. RESULTS: HIV-infected adolescents showed a significantly higher mean lung density, ventilation heterogeneity, mass, and high- and low-attenuation areas compared with non-infected individuals. Kurtosis and skewness were significantly lower as well. HIV-infected adolescents with bronchiolitis obliterans had a significantly lower kurtosis and skewness compared with those without bronchiolitis obliterans. Lung mass and volume showed the strongest correlations with forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and alveolar volume. Low-attenuation areas below - 950 HU and ventilation heterogeneity showed the strongest correlation with FEV1/FVC (range, - 0.51 to - 0.34) and forced expiratory flow between 25 and 75% of FVC (range, - 0.50 to - 0.35). CONCLUSION: Quantitative chest CT on inspiration is a feasible technique to differentiate perinatally HIV-infected adolescents with and without bronchiolitis obliterans. Quantitative CT parameters correlate with spirometric measurements of small-airway disease. KEY POINTS: • Perinatally HIV-infected adolescents showed a more heterogeneous attenuation of the lung parenchyma with a higher percentage of low- and high-attenuation areas compared with non-infected patients. • Kurtosis and skewness are able to differentiate between HIV-infected adolescents with and without bronchiolitis obliterans using an inspiratory chest CT. • Quantitative CT parameters of the chest correlate significantly with pulmonary function test. Low-attenuation areas and ventilation heterogeneity are particularly associated with spirometric parameters related to airway obstruction.

Risk-Stratification of Children Presenting to Ambulatory Paediatrics with First-Onset Seizures: Should We Order an Urgent CT Brain?

INTRODUCTION: A computed tomography (CT) brain scan is an often-utilised emergency department imaging modality to detect emergent intra-cranial pathology in a child with a first seizure. Identifying children at low risk of having a clinically significant intra-cranial abnormality could prevent unnecessary radiation exposure and contrast/sedation-related risks. OBJECTIVES: To identify clinical variables which could predict clinically significant CT brain abnormalities and use recursive partitioning analysis to define a low-risk group of children in whom emergent CT brain can be deferred. METHODS: Retrospective cross-sectional review of 468 children who underwent emergent CT brain after presenting to a low- and middle-income paediatric emergency department following first seizure. RESULTS: In total 133/468 (28.4%) of CT brain scans had clinically significant abnormalities. Failure to return to neurological baseline and focal neurological deficit persisting >36 h had statistical significance in a multiple regression analysis. Recursive partitioning analysis, applied to a subgroup without suspected tuberculous meningitis (n = 414), classified 153 children aged between 6 months and 5 years, who had a normal neurological baseline, had returned to baseline post-seizure, and were not in status epilepticus, as non-clinically significant scans and 98% were correctly classified. CONCLUSION: Our study re-inforces the American Academy of Neurology recommendation that children with persistent post-ictal abnormal neurological status and/or post-ictal focal deficit be prioritised for emergent CT brain. Having excluded children with suspected tuberculous meningitis, the remaining subgroup aged 6 months to 5 years presenting with a non-status first seizure, normal neurological baseline and return to baseline post-seizure, are at very low risk of having a clinically significant CT brain abnormality.

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments.

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference.

"Barbell Sign": A Diagnostic Imaging Finding in Progressive Multifocal Leukoencephalopathy.

The recently proposed diagnostic algorithm for progressive multifocal leukoencephalopathy underscores the importance of imaging and emphasizes the role of the radiologist in the diagnostic workup. We describe a case series of patients with visual symptoms and a typical pattern of brain involvement in definite progressive multifocal leukoencephalopathy, for which we have coined the term barbell sign.

Transcriptional and metabolic alternations rebalance wheat grain storage protein accumulation under variable nitrogen and sulfur supply.

Wheat (Triticum aestivum L.) grain storage proteins (GSPs) are major determinants of flour end-use value. Biological and molecular mechanisms underlying the developmental and nutritional determination of GSP accumulation in cereals are as yet poorly understood. Here we timed the accumulation of GSPs during wheat grain maturation relative to changes in metabolite and transcript pools in different conditions of nitrogen (N) and sulfur (S) availability. We found that the N/S supply ratio modulated the duration of accumulation of S-rich GSPs and the rate of accumulation of S-poor GSPs. These changes are likely to be the result of distinct relationships between N and S allocation, depending on the S content of the GSP. Most developmental and nutritional modifications in GSP synthesis correlated with the abundance of structural gene transcripts. Changes in the expression of transport and metabolism genes altered the concentrations of several free amino acids under variable conditions of N and S supply, and these amino acids seem to be essential in determining GSP expression. The comprehensive data set generated and analyzed here provides insights that will be useful in adapting fertilizer use to variable N and S supply, or for breeding new cultivars with balanced and robust GSP composition.

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis.

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO-induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO-dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development.

Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development.

CUP-SHAPED COTYLEDON2 (CUC2) and the interacting microRNA miR164 regulate leaf margin dissection. Here, we further investigate the evolution and the specific roles of the CUC1 to CUC3 genes during Arabidopsis thaliana leaf serration. We show that CUC2 is essential for dissecting the leaves of a wide range of lobed/serrated Arabidopsis lines. Inactivation of CUC3 leads to a partial suppression of the serrations, indicating a role for this gene in leaf shaping. Morphometric analysis of leaf development and genetic analysis provide evidence for different temporal contributions of CUC2 and CUC3. Chimeric constructs mixing CUC regulatory sequences with different coding sequences reveal both redundant and specific roles for the three CUC genes that could be traced back to changes in their expression pattern or protein activity. In particular, we show that CUC1 triggers the formation of leaflets when ectopically expressed instead of CUC2 in the developing leaves. These divergent fates of the CUC1 and CUC2 genes after their formation by the duplication of a common ancestor is consistent with the signature of positive selection detected on the ancestral branch to CUC1. Combining experimental observations with the retraced origin of the CUC genes in the Brassicales, we propose an evolutionary scenario for the CUC genes.

Association study of wheat grain protein composition reveals that gliadin and glutenin composition are trans-regulated by different chromosome regions.

Wheat grain storage protein (GSP) content and composition are the main determinants of the end-use value of bread wheat (Triticum aestivum L.) grain. The accumulation of glutenins and gliadins, the two main classes of GSP in wheat, is believed to be mainly controlled at the transcriptional level through a network of transcription factors. This regulation network could lead to stable cross-environment allometric scaling relationships between the quantity of GSP classes/subunits and the total quantity of nitrogen per grain. This work conducted a genetic mapping study of GSP content and composition and allometric scaling parameters of grain N allocation using a bread wheat worldwide core collection grown in three environments. The core collection was genotyped with 873 markers for genome-wide association and 167 single nucleotide polymorphism markers in 51 candidate genes for candidate association. The candidate genes included 35 transcription factors (TFs) expressed in grain. This work identified 74 loci associated with 38 variables, of which 19 were candidate genes or were tightly linked with candidate genes. Besides structural GSP genes, several loci putatively trans-regulating GSP accumulation were identified. Seven candidate TFs, including four wheat orthologues of barley TFs that control hordein gene expression, were associated or in strong linkage disequilibrium with markers associated with the composition or quantity of glutenin or gliadin, or allometric grain N allocation parameters, confirming the importance of the transcriptional control of GSP accumulation. Genome-wide association results suggest that the genes regulating glutenin and gliadin compositions are mostly distinct from each other and operate differently.

Storage protein activator controls grain protein accumulation in bread wheat in a nitrogen dependent manner.

The expression of cereal grain storage protein (GSP) genes is controlled by a complex network of transcription factors (TFs). Storage protein activator (SPA) is a major TF acting in this network but its specific function in wheat (Triticum aestivum L.) remains to be determined. Here we generated an RNAi line in which expression of the three SPA homoeologs was reduced. In this line and its null segregant we analyzed GSP accumulation and expression of GSP and regulatory TF genes under two regimes of nitrogen availability. We show that down regulation of SPA decreases grain protein concentration at maturity under low but not high nitrogen supply. Under low nitrogen supply, the decrease in SPA expression also caused a reduction in the total quantity of GSP per grain and in the ratio of GSP to albumin-globulins, without significantly affecting GSP composition. The slight reduction in GSP gene expression measured in the SPA RNAi line under low nitrogen supply did not entirely account for the more significant decrease in GSP accumulation, suggesting that SPA regulates additional levels of GSP synthesis. Our results demonstrate a clear role of SPA in the regulation of grain nitrogen metabolism when nitrogen is a limiting resource.

High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened.

The oncogenic G-protein-coupled receptor (GPCR) Smoothened (SMO) is a key transducer of the hedgehog (HH) morphogen, which plays an essential role in the patterning of epithelial structures. Here, we examine how HH controls SMO subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that HH promotes the stabilization of SMO by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of protein kinase A, casein kinase I, and the Fused (FU) kinase. Moreover, in the presence of very high levels of HH, the second effect of FU leads to the local enrichment of SMO in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of HH to the apico-basal distribution of SMO and provide a novel mechanism for the regulation of a GPCR.

Costal2 functions as a kinesin-like protein in the hedgehog signal transduction pathway.

The Hedgehog (Hh) signaling pathway initiates an evolutionarily conserved developmental program required for the proper patterning of many tissues [1]. Although Costal2 (Cos2) is a requisite component of the Hh pathway, its mechanistic role is not well understood. Because of its primary sequence, Cos2 was initially predicted to function as a kinesin-like protein [2]. However, evidence showing that Cos2 function might require kinesin-like properties has been lacking [2-6]. Thus, the prevailing dogma in the field is that Cos2 functions solely as a scaffolding protein [7, 8]. Here, we show that Cos2 motility is required for its biological function and that this motility may be Hh regulated. We show that Cos2 motility requires an active motor domain, ATP, and microtubules. Additionally, Cos2 recruits and transports other components of the Hh signaling pathway, including the transcription factor Cubitus interruptus (Ci). Drosophila expressing cos2 mutations that encode proteins that lack motility are attenuated in their ability to regulate Ci activity and exhibit phenotypes consistent with attenuated Cos2 function [9]. Combined, these results demonstrate that Cos2 motility plays an important role in its function, regulating the amounts and activity of Ci that ultimately interpret the level of Hh to which cells are exposed.