Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Patients suffering from dystrophic epidermolysis bullosa are prone to developing autoantibodies against skin proteins: A longitudinal confirmational study.

Epidermolysis bullosa (EB) is a heritable skin blistering disease caused by variants in genes coding for proteins that secure cell-cell adhesion and attachment of the epidermis to the dermis. Interestingly, several proteins involved in inherited EB are also associated with autoimmune blistering diseases (AIBD). In this study, we present a long-term follow-up of 15 patients suffering from recessive dystrophic or junctional EB. From these patients, 62 sera were analysed for the presence of autoantibodies associated with AIBD. We show that patients suffering from recessive dystrophic EB (RDEB) are more susceptible to developing autoantibodies against skin proteins than patients suffering from junctional EB (70% vs. 20%, respectively). Interestingly, no correlation with age was observed. Most patients showed reactivity to Type XVII collagen/linear IgA bullous dermatosis autoantigen (n = 5; 33%), followed by BP230 (n = 4; 27%), Type VII collagen (n = 4; 27%) and laminin-332 (n = 1; 7%). The pathogenicity of these autoantibodies remains a subject for future experiments.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

Exposed CendR Domain in Homing Peptide Yields Skin-Targeted Therapeutic in Epidermolysis Bullosa.

Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-ß (TGF-ß)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-ß inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-ß signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB.

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function.

Variably reduced expression of the basement membrane component laminin-332 (α3aß3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a ß3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

Prevalence and pathogenesis of osteopenia and osteoporosis in epidermolysis bullosa: An evidence-based review.

BACKGROUND: Osteopenia or osteoporosis is one of the many comorbidities in patients with Epidermolysis Bullosa (EB). Current literature on the prevalence of osteoporosis in EB is scarce. OBJECTIVE: This review will analyse the current literature in the field of patients with compromised bone health in EB and any articles on the prevalence of such diseases in EB groups. METHODS: A systematic search for articles related to bone health and epidermolysis bullosa (EB) (1946-2017) was performed on seven databases: MEDLINE, EMBASE and EBM, PubMed, ProQuest, Scopus and Web of Science. Search terms: epidermolysis bullosa, osteop*, bone mass, bone mineral*, fracture, dual X-ray absorptiometry, vitamin D, calcium, nutrition, exercise and physical activity. Abstracts from all search results were screened, and reference scanning of the search results was performed. Eighty-three articles met the selection criteria and were considered for review. Letters to the editor and abstract-only articles were excluded. Articles were favoured based on citation count, impact factor of their journal and study sample size. The search included all languages. RESULTS: The searches yielded a total of 1309 articles including 717 duplicates. The remaining 592 articles were screened by title and abstracts. Eighty-three full-text articles were analysed. Twenty-one articles directly relating to bone health in EB were included. Three descriptive studies and one case-control study were found, indicating a need for research of larger scale. CONCLUSION: Further investigations into osteoporosis in EB, especially the milder forms of EB, are valuable in providing evidence to support guideline developments for EB bone health management.

Epidermolysis Bullosa-Associated Squamous Cell Carcinoma: From Pathogenesis to Therapeutic Perspectives.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited skin disorders determined by mutations in genes encoding for structural components of the cutaneous basement membrane zone. Disease hallmarks are skin fragility and unremitting blistering. The most disabling EB (sub)types show defective wound healing, fibrosis and inflammation at lesional skin. These features expose patients to serious disease complications, including the development of cutaneous squamous cell carcinomas (SCCs). Almost all subjects affected with the severe recessive dystrophic EB (RDEB) subtype suffer from early and extremely aggressive SCCs (RDEB-SCC), which represent the first cause of death in these patients. The genetic determinants of RDEB-SCC do not exhaustively explain its unique behavior as compared to low-risk, ultraviolet-induced SCCs in the general population. On the other hand, a growing body of evidence points to the key role of tumor microenvironment in initiation, progression and spreading of RDEB-SCC, as well as of other, less-investigated, EB-related SCCs (EB-SCCs). Here, we discuss the recent advances in understanding the complex series of molecular events (i.e., fibrotic, inflammatory, and immune processes) contributing to SCC development in EB patients, cross-compare tumor features in the different EB subtypes and report the most promising therapeutic approaches to counteract or delay EB-SCCs.

Diagnosis of Inherited Epidermolysis Bullosa in Resource-Limited Settings: Immunohistochemistry Revisited.

BACKGROUND: Immunofluorescence (IFM) antigen mapping is the most commonly used technique to diagnose and differentiate epidermolysis bullosa (EB). In India, IFM is limited to few research laboratories and is not readily available, making the diagnosis largely clinical and often inaccurate. Ob jective of the Study: To examine the diagnostic usefulness of immunohistochemistry (IHC) as compared to IFM in resource-limited settings. METHODS: Forty-four consecutive EB patients were included in this study. IHC and IFM were performed on 7-µm frozen tissue sections using standard laboratory protocols with a limited panel of antibodies. The kappa coefficient of agreement was calculated with genetic analysis as the gold standard. RESULTS: IFM and IHC accurately identified the subtype of EB in 80.9% (p < 0.001) of the cases, when a clear blister cavity was evident on biopsy. The sensitivities and specificities of IHC and IFM for diagnosing EB simplex, junctional EB, and dystrophic EB were 100, 100, and 60% and 82.4, 100, and 100%, respectively. IHC was equally effective (p < 0.001) in establishing the type of EB as IFM. CONCLUSIONS: IHC staining and its interpretation were simple and comparable to IFM. IHC had an advantage of showing subtle changes in the epidermal architecture that could not be appreciated on IFM and hence can be considered useful in resource-limited settings.

Autoimmunity and Cytokine Imbalance in Inherited Epidermolysis Bullosa.

In order to evaluate the serum anti-skin autoantibodies and cytokine concentrations in patients with different epidermolysis bullosa (EB) types and severity, 42 EB patients and 38 controls were enrolled. Serum anti-skin antibodies were significantly higher in the patients than in the controls (p = 0.008, p < 0.001, p < 0.001, p < 0.001 and p < 0.001 for desmoglein 1 (DSG1) desmoglein 3 (DSG3), bullous pemphigoid 180 (BP180), BP230 and type VII collagen (COL7), respectively). The same trend was observed for interleukin (IL)-1ß, IL-2, IL-6, IL-10, tumor necrosis factor-ß, and interferon-γ (p < 0.001, p < 0.001, p < 0.001, p = 0.008, p < 0.001 and p = 0.002, respectively). Increases in anti-skin antibodies and cytokine concentrations were higher in patients with recessive dystrophic EB than in those with different types of EB, in generalized cases than in localized ones, and in patients with higher Birmingham Epidermolysis Bullosa Severity (BEBS) scores than in those with a lower score. The BEBS score was directly correlated with BP180, BP230, COL7 (p = 0.015, p = 0.008 and p < 0.001, respectively) and IL-6 (p = 0.03), whereas IL-6 appeared significantly associated with DSG1, DSG3, BP180, BP230 and COL7 (p = 0.015, p = 0.023, p = 0.023, p = 0.015 and p = 0.005, respectively). This study showed that autoimmunity and inflammatory responses are frequently activated in EB, mainly in severe forms, suggesting the use of immunosuppressive drugs or biologicals that are active against pro-inflammatory cytokines to reduce clinical signs and symptoms of disease.

Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair.

RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3' and 5' RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.

[Current approaches to the morphologic diagnosis of different types of congenital epidermolysis bullosa]. / Sovremennye podkhody k morfologicheskoi diagnostike razlichnykh tipov vrozhdennogo bulleznogo epidermoliza.

Congenital epidermolysis bullosa (CEB) is an extensive group of hereditary skin diseases, the differential diagnosis of which is a challenge due to the rarity of this pathology and the diversity of its clinical manifestations. The determination of the type of CEB makes it possible to estimate its prognosis and to facilitate a prenatal diagnosis. AIM: to optimize the morphological diagnosis of different types of CEB. MATERIAL AND METHODS: 28 skin biopsies from 14 patients with different types of CEB were investigated. The investigators performed routine histological examination of skin fragments taken from a bullous area and immunofluorescence antigen mapping using the indirect immunofluorescence test (IIFT) with antibodies against structural proteins of the dermal-epidermal junction (laminin α3, ß3, and γ2 chains, keratins 5 and 14, types VII and XVII collagen, α6 and ß4 integrin subunits, desmoplakin, plectin, kindlin-1, and plakophillin) of the apparently unaffected skin. The intact skin of healthy individuals, which had been obtained during cosmetic operations, was used as controls in IIFT. RESULTS: Immunofluorescence antigen mapping could determine the type of CEB in all cases and in 86% of cases identify the protein, the impaired production of which was responsible for the development of the disease. CONCLUSION: Immunofluorescence antigen mapping is an integral part of the comprehensive morphological diagnosis of CEB, acting as an intermediate between the morphological verification of CEB diagnosis and the targeted search for mutations by a molecular genetic method.

Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering.

Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.

Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes.

Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders.

Lack of plakoglobin leads to lethal congenital epidermolysis bullosa: a novel clinico-genetic entity.

Epidermal integrity is essential for skin functions. It is maintained by adhesive structures between keratinocytes, mainly the desmosomes and adherens junctions, which provide resistance against mechanical stress and regulate the formation of the skin barrier. As a constituent of both types of intercellular junctions, plakoglobin has multiple interaction partners and mutations in its gene [junction plakoglobin (JUP)] have been associated with mild cutaneous disease, palmoplantar keratoderma and arrhythmogenic heart disease. Here we report a novel lethal phenotype caused by a homozygous nonsense JUP mutation, c.1615C>T, p.Q539X, which is very different from any human or murine JUP phenotype described so far. The patient suffered from severe congenital skin fragility with generalized epidermolysis and massive transcutaneous fluid loss, but apparently no cardiac dysfunction. In contrast to previously reported JUP mutations where truncated proteins were still present, in this case there was complete loss of plakoglobin in the patient's skin, as demonstrated by immunofluorescence and immunoblot analysis. As a consequence, only very few abnormal desmosomes were formed and no adhesion structures between keratinocytes were recognizable. The expression and distribution of desmosomal components was severely affected, suggesting an essential role for plakoglobin in desmosomal assembly. Adherens junction proteins were localized to keratinocyte plasma membrane, but did not provide proper cell-cell adhesion. This lethal congenital epidermolysis bullosa highlights the fundamental role of plakoglobin in epidermal cohesion.

Structural basis of the interaction between integrin alpha6beta4 and plectin at the hemidesmosomes.

The interaction between the integrin alpha6beta4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 A resolution of the primary alpha6beta4-plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of beta4 and the actin-binding domain of plectin. Two missense mutations in beta4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 A resolution of the beta4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of beta4 on binding to plectin. This conformational switch is correlated with the way alpha6beta4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.

Partial loss of epithelial phenotype in kindlin-1-deficient keratinocytes.

Kindlin-1 is an adaptor protein that is expressed by most epithelial cells and has been implicated in integrin bidirectional signaling. Mutations in the gene encoding kindlin-1 are associated with Kindler syndrome, a recessively inherited disorder that is characterized by fragile skin. Functionally, a loss of kindlin-1 impairs the adhesion of basal keratinocytes to the extracellular matrix both in vivo and in vitro. In this study, we show that the phenotype of mutant keratinocytes deficient in kindlin-1 is characterized by the modification of the cortical actin network and increased plasticity of the plasma membrane. At the molecular level, expression of several proteins associated with an epithelial phenotype, such as α6ß4 integrin, collagen XVII, E-cadherin, and desmoglein-3, is strongly reduced, whereas, surprisingly, laminin 332 is synthesized in larger amounts than in control keratinocytes. In contrast, mesenchymal markers such as vimentin and fibronectin are increased in keratinocytes lacking kindlin-1. The switch in cell plasticity and protein expression was confirmed by siRNA-mediated down-regulation of kindlin-1 in HaCaT epithelial cells. Furthermore, there was up-regulation of matrix metalloproteinases and pro-inflammatory cytokines in kindlin-1-deficient keratinocytes. These results provide new insights into the pathogenic mechanisms that take place in Kindler syndrome. Moreover, the constellation of molecular defects associated with the loss of kindlin-1 may explain the higher incidence of skin cancer observed in patients affected with this disorder.

Kindlin-1 and -2 have overlapping functions in epithelial cells implications for phenotype modification.

Kindlins are a novel family of intracellular adaptor proteins in integrin-containing focal adhesions. Kindlin-1 and -2 are expressed in the skin, but whether and how they cooperate in adult epithelial cells have remained elusive. We uncovered the overlapping roles of kindlin-1 and -2 in maintaining epithelial integrity and show that the phenotype of kindlin-1-deficient cells can be modulated by regulating kindlin-2 gene expression and vice versa. The experimental evidence is provided by use of human keratinocyte cell lines that express both kindlins, just kindlin-1 or kindlin-2, or none of them. Double deficiency of kindlin-1 and -2 had significant negative effects on focal adhesion formation and actin cytoskeleton organization, cell adhesion, survival, directional migration, and activation of ß(1) integrin, whereas deficiency of one kindlin only showed variable perturbation of these functions. Cell motility and formation of cell-cell contacts were particularly affected by lack of kindlin-2. These results predict that kindlin-1 and -2 can functionally compensate for each other, at least in part. The high physiologic and pathologic significance of the compensation was emphasized by the discovery of environmental regulation of kindlin-2 expression. UV-B irradiation induced loss of kindlin-2 in keratinocytes. This first example of environmental regulation of kindlin expression has implications for phenotype modulation in Kindler syndrome, a skin disorder caused by kindlin-1 deficiency.

Collagen XVII (BP180) modulates keratinocyte expression of the proinflammatory chemokine, IL-8.

Collagen XVII (COL17), a transmembrane protein expressed in epidermal keratinocytes (EK), is targeted by pathogenic autoantibodies in bullous pemphigoid. Treatment of EK with anti-COL17 autoantibodies triggers the production of proinflammatory cytokines. In this study, we test the hypothesis that COL17 is involved in the regulation of the EK proinflammatory response, using IL-8 expression as the primary readout. The absence of COL17 in EK derived from a junctional epidermolysis bullosa patient or shRNA-mediated knockdown of COL17 in normal EK resulted in a dysregulation of IL-8 responses under various conditions. The COL17-deficient cells showed an abnormally high IL-8 response after treatment with lipopolysaccharide (LPS), ultraviolet-B radiation or tumor necrosis factor, but exhibited a blunted IL-8 response to phorbol 12-myristate 13-acetate exposure. Induction of COL17 expression in COL17-negative EK led to a normalization of the LPS-induced proinflammatory response. Although α6ß4 integrin was found to be up-regulated in COL17-deficient EK, siRNA-mediated knockdown of the α6 and ß4 subunits revealed that COL17's effects on the LPS IL-8 response are not dependent on this integrin. In LPS-treated cells, inhibition of NF-kappa B activity in COL17-negative EK resulted in a normalization of their IL-8 response, and expression of an NF-kappa B-driven reporter was shown to be higher in COL17-deficient, compared with normal EK. These findings support the hypothesis that COL17 plays an important regulatory role in the EK proinflammatory response, acting largely via NF-kappa B. Future investigations will focus on further defining the molecular basis of this novel control network.