A gene regulatory network anchored by LIM homeobox 1 for embryonic head development.

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.

Context-specific function of the LIM homeobox 1 transcription factor in head formation of the mouse embryo.

Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.

Transcriptional targets of TWIST1 in the cranial mesoderm regulate cell-matrix interactions and mesenchyme maintenance.

TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.

Head formation: OTX2 regulates Dkk1 and Lhx1 activity in the anterior mesendoderm.

The Otx2 gene encodes a paired-type homeobox transcription factor that is essential for the induction and the patterning of the anterior structures in the mouse embryo. Otx2 knockout embryos fail to form a head. Whereas previous studies have shown that Otx2 is required in the anterior visceral endoderm and the anterior neuroectoderm for head formation, its role in the anterior mesendoderm (AME) has not been assessed specifically. Here, we show that tissue-specific ablation of Otx2 in the AME phenocopies the truncation of the embryonic head of the Otx2 null mutant. Expression of Dkk1 and Lhx1, two genes that are also essential for head formation, is disrupted in the AME of the conditional Otx2-deficient embryos. Consistent with the fact that Dkk1 is a direct target of OTX2, we showed that OTX2 can interact with the H1 regulatory region of Dkk1 to activate its expression. Cross-species comparative analysis, RT-qPCR, ChIP-qPCR and luciferase assays have revealed two conserved regions in the Lhx1 locus to which OTX2 can bind to activate Lhx1 expression. Abnormal development of the embryonic head in Otx2;Lhx1 and Otx2;Dkk1 compound mutant embryos highlights the functional intersection of Otx2, Dkk1 and Lhx1 in the AME for head formation.

Conditional restoration and inactivation of Rbm47 reveal its tissue-context requirement for viability and growth.

Rbm47 encodes a RNA binding protein that is necessary for Cytidine to Uridine RNA editing. Rbm47(gt/gt) mutant mice that harbor inactivated Rbm47 display poor viability. Here it was determined that the loss of Rbm47(gt/gt) offspring is due to embryonic lethality at mid-gestation. It was further showed that growth of the surviving Rbm47(gt/gt) mutants is impaired. Rbm47 is expressed in both the visceral endoderm and the definitive endoderm. Using the utility of the switchable FlEx gene-trap cassette and the activity of Cre and FLP recombinases to generate mice that conditionally inactivate and restore Rbm47 function in tissue-specific manner, it was demonstrated that Rbm47 function is required in the embryo proper, and not the visceral endoderm, for viability and growth. genesis 54:115-122, 2016. © 2016 Wiley Periodicals, Inc.

C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing.

The mesenchymal architecture of the cranial mesoderm of mouse embryos is disrupted by the loss of Twist1 function.

The basic helix-loop-helix transcription factor Twist1 is a key regulator of craniofacial development. Twist1-null mouse embryos exhibit failure of cephalic neural tube closure and abnormal head development and die at E11.0. To dissect the function of Twist1 in the cranial mesoderm beyond mid-gestation, we used Mesp1-Cre to delete Twist1 in the anterior mesoderm, which includes the progenitors of the cranial mesoderm. Deletion of Twist1 in mesoderm cells resulted in loss and malformations of the cranial mesoderm-derived skeleton. Loss of Twist1 in the mesoderm also resulted in a failure to fully segregate the mesoderm and the neural crest cells, and the malformation of some cranial neural crest-derived tissues. The development of extraocular muscles was compromised whereas the differentiation of branchial arch muscles was not affected, indicating a differential requirement for Twist1 in these two types of craniofacial muscle. A striking effect of the loss of Twist1 was the inability of the mesodermal cells to maintain their mesenchymal characteristics, and the acquisition of an epithelial-like morphology. Our findings point to a role of Twist1 in maintaining the mesenchyme architecture and the progenitor state of the mesoderm, as well as mediating mesoderm-neural crest interactions in craniofacial development.

Stringent requirement of a proper level of canonical WNT signalling activity for head formation in mouse embryo.

In mouse embryos, loss of Dickkopf-1 (DKK1) activity is associated with an ectopic activation of WNT signalling responses in the precursors of the craniofacial structures and leads to a complete truncation of the head at early organogenesis. Here, we show that ENU-induced mutations of genes coding for two WNT canonical pathway factors, the co-receptor LRP6 and the transcriptional co-activator ß-catenin, also elicit an ectopic signalling response and result in loss of the rostral tissues of the forebrain. Compound mutant embryos harbouring combinations of mutant alleles of Lrp6, Ctnnb1 and Dkk1 recapitulate the partial to complete head truncation phenotype of individual homozygous mutants. The demonstration of a synergistic interaction of Dkk1, Lrp6 and Ctnnb1 provides compelling evidence supporting the concepts that (1) stringent regulation of the level of canonical WNT signalling is necessary for head formation, (2) activity of the canonical pathway is sufficient to account for the phenotypic effects of mutations in three different components of the signal cascade and (3) rostral parts of the brain and the head are differentially more sensitive to canonical WNT signalling and their development is contingent on negative modulation of WNT signalling activity.

Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm.

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.

Regionalized Twist1 activity in the forelimb bud drives the morphogenesis of the proximal and preaxial skeleton.

Development of the mouse forelimb bud depends on normal Twist1 activity. Global loss of Twist1 function before limb bud formation stops limb development and loss of Twist1 throughout the mesenchyme after limb bud initiation leads to polydactyly, the ulnarization or loss of the radius and malformations and reductions of the shoulder girdle. Here we show that conditional deletion of Twist1 by Mesp1-Cre in the mesoderm that migrates into the anterior-proximal part of the forelimb bud results in the development of supernumerary digits and carpals, the acquisition of ulna-like characteristics by the radius and malformations of the humerus and scapula. The mirror-like duplications and posteriorization of pre-axial tissues are preceded by disruptions to anterior-posterior Shh, Bmp and Fgf signaling gradients and dysregulation of transcription factors that regulate anterior-posterior limb patterning.

Relational practice in health, education, criminal justice, and social care: a scoping review.

BACKGROUND: Establishing and maintaining relationships and ways of connecting and being with others is an important component of health and wellbeing. Harnessing the relational within caring, supportive, educational, or carceral settings as a systems response has been referred to as relational practice. Practitioners, people with lived experience, academics and policy makers, do not yet share a well-defined common understanding of relational practice. Consequently, there is potential for interdisciplinary and interagency miscommunication, as well as the risk of policy and practice being increasingly disconnected. Comprehensive reviews are needed to support the development of a coherent shared understanding of relational practice. METHOD: This study uses a scoping review design providing a scope and synthesis of extant literature relating to relational practice focussing on organisational and systemic practice. The review aimed to map how relational practice is used, defined and understood across health, criminal justice, education and social work, noting any impacts and benefits reported. Searches were conducted on 8 bibliographic databases on 27 October 2021. English language articles were included that involve/discuss practice and/or intervention/s that prioritise interpersonal relationships in service provision, in both external (organisational contexts) and internal (how this is received by workers and service users) aspects. RESULTS: A total of 8010 relevant articles were identified, of which 158 met the eligibility criteria and were included in the synthesis. Most were opinion-based or theoretical argument papers (n = 61, 38.60%), with 6 (3.80%) critical or narrative reviews. A further 27 (17.09%) were categorised as case studies, focussing on explaining relational practice being used in an organisation or a specific intervention and its components, rather than conducting an evaluation or examination of the effectiveness of the service, with only 11 including any empirical data. Of the included empirical studies, 45 were qualitative, 6 were quantitative, and 9 mixed methods studies. There were differences in the use of terminology and definitions of relational practice within and across sectors. CONCLUSION: Although there may be implicit knowledge of what relational practice is the research field lacks coherent and comprehensive models. Despite definitional ambiguities, a number of benefits are attributed to relational practices. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42021295958.

Cell-specific crosstalk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM. Highlights: Periventricular GBM is more malignant and disrupts neurogenesis in a rodent model.Cell-specific proteomics elucidates tumor-promoting crosstalk between GBM and NPCs.NPCs induce upregulated CTSB expression in GBM, promoting tumor progression.GBM stalls neurogenesis and promotes NPC senescence via CTSB.

Leveraging the Scaling Up Nutrition Movement to Operationalize Stunting Prevention Activities: Implementation Lessons From Rural Malawi.

BACKGROUND: The rural district of Ntchisi is in the central region of Malawi. Among children aged 6 to 23 months, the stunting prevalence is 40% to 50%. To address this high prevalence, the World Food Programme, with cooperating partners, supported the Government of Malawi to implement an integrated stunting prevention program entitled The Right Foods at the Right Time from 2013 to 2018. OBJECTIVE: To provide implementation lessons learned from systematic documentation of how the Scaling Up Nutrition (SUN) movement, combined with other international and national initiatives and policies, was translated into tailored programming. METHODS: During program conception, early design, and implementation, this descriptive study systematically documented the process of translating SUN principles and government policies into an operational stunting prevention program in rural Malawi. RESULTS: We identified 8 factors that contributed to successful translation of policy into program activities: (1) well-structured National SUN framework, (2) reliable coordination platforms and district ownership, (3) systematic and evidence-informed program design, (4) multiple forms of data used to inform program planning, (5) multisectoral implementation approaches to stunting prevention, (6) innovation in technology to improve overall program efficiency, (7) systematic collaboration among diverse stakeholders, and (8) strong public health nutrition capacity of program team members. CONCLUSIONS: Lessons from this nutrition program in Ntchisi, Malawi, provide one case illustrating how the SUN movement, government policies, and global evidence base can be operationalized into tailored programming for improving nutrition.

Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo.

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.

The lived experience of the wound, ostomy, and continence nurse in wound care.

PURPOSE: The purpose of this qualitative research study was to explore the meaning of being a wound, ostomy, and continence (WOC) nurse engaged in wound care practice. SUBJECTS AND SETTING: The sample comprised 6 WOC nurses, each of whom had 3 or more years of wound care experience in a variety of care settings. Respondents were female, 49 years or older, and master's prepared. Interviews were conducted at either the place of employment of each subject or a neutral location based on the participant's preference. METHODS: A Heideggerian hermeneutic phenomenologic framework was used during data collection. An unstructured interview process was used to encourage sharing of experiences through narratives. All interviews were audiotaped and transcribed verbatim. As a WOC nurse with 30 years of experience in wound care, the primary investigator logged reflections on the interview process as a valuable source of data analysis. Interpretive analysis involved immersion in the text, data transformation, and thematic analysis. RESULTS: Eight themes emerged: (1) Essence of Practice; bond of caring; (2) Holistic Approach; putting the pieces of the puzzle together; (3) The Dichotomy... them and me; (4) Art of Wound Care; (5) Growth in Practice; (6) Allure of the Challenge. (7) Acknowledging Limits... you can't always make it better; and (8) Teaching, Mentoring and Being a Role Model. CONCLUSIONS: The narratives illuminate the essence of this nursing specialty practice, whereas the outcome of this research provides a rich description of the human experience of being a WOC nurse engaged in wound care.

Germline pharmacogenomics of DPYD*9A (c.85T>C) variant in patients with gastrointestinal malignancies treated with fluoropyrimidines.

BACKGROUND: The correlation between DPYD*9A (c.85T>C) genotype and dihydropyrimidine dehydrogenase (DPD) deficiency clinical phenotype is controversial. Reference laboratories either did not perform DPYD*9A genotyping or have stopped DPYD*9A genotyping and limited genotyping to high-risk variants (DPYD*2A, DPYD*13 and DPYD*9B) only. This study explored DPYD*9A genotype and clinical phenotype correlation in patients with gastrointestinal (GI) malignancies treated with fluoropyrimidines. METHODS: Between 2011 and 2017, 67 patients with GI malignancies were genotyped for DPYD variants. Fluoropyrimidines-associated toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0). Fisher's exact test was used for statistical analysis. RESULTS: DPYD variants were identified in 17 out of 67 (25%) patients. One patient was homozygous for DPYD*9A variant and one patient was double heterozygous for DPYD*9A and DPYD*9B variants. In patients with identified DPYD variants, 13/17 (76%) patients had DPYD*9A variant, 3/17 (18%) patients had DPYD*2A variant and 2/17 (12%) patient had DPYD*9B variant. Only patients genotyped prior to 2015 were genotyped for DPYD*9A variant (N=28). Of those, 13/28 patients (46%) had DPYD*9A variant. Grade 3-4 diarrhea was associated with DPYD*9A variant in patients treated with full dose fluoropyrimidines (P=0.0055). CONCLUSIONS: In our cohort, DPYD*9A variant was the most common diagnosed variant. The correlation between DPYD*9A genotype and DPD deficiency in clinical phenotype was noticeable in patients who received full dose fluoropyrimidines as they all experienced grade 3-4 toxicities (diarrhea).

Malignant fungating wounds: an analysis of the lived experience.

AIM: This paper reports upon a study that aimed to illuminate the meaning and experience of living with a malignant fungating wound. BACKGROUND: The current understanding of living with a malignant fungating wound is derived from professionals' rather than the patient's perspective. An appreciation of the lived experience may assist in the development of more empathetic support approaches. METHOD: A Heideggerian hermeneutic phenomenological approach was used as the philosophical framework. Unstructured interviews were conducted with a purposive sample of five participants. Content hermeneutic analysis was adopted to analyse the data. FINDINGS: Four themes were identified: representing the worst part of the patient's cancer; living within a body that cannot be trusted; a changing relationship with the patient's family and friends; and a loss of identity while continuously striving to be normal, yet feeling different. CONCLUSION: Health care professionals must possess a heightened awareness of: the importance of the impact of the wound upon day-to-day living, identity and purpose; the value of adopting the phrase used by the patient to describe their wound; and to focus more upon the subjective meaning of a visibly changing wound rather than objective measurement.

Religious beliefs, practices and treatment adherence among individuals with HIV in the southern United States.

Nonadherence with medical treatment is a critical threat to the health of those living with HIV disease. Unfortunately the search for explanatory factors for nonadherence is still not fully developed, particularly in the area of religion and spirituality. Extant literature suggests that church attendance, religious practices and spiritual beliefs may improve health and generally benefit patients. However, religious beliefs may also play a negative role in treatment adherence due to the stigma attached to HIV disease, particularly in geographical areas and in population subgroups where religious practices are strong. In this exploratory study, HIV-positive individuals (n = 306) in a southern state were surveyed as to their attitudes and beliefs surrounding HIV disease and adherence with medical treatment for the disease. The results indicate that multiple factors influence adherence with treatment and that certain religious practices are positively associated with adherence, but certain religious beliefs are negatively related to adherence. The findings of this study reinforce the importance of remembering and addressing a patient's religious beliefs as a part of medical care.

Thyroid bud morphogenesis requires CDC42- and SHROOM3-dependent apical constriction.

Early development of the gut endoderm and its subsequent remodeling for the formation of organ buds are accompanied by changes to epithelial cell shape and polarity. Members of the Rho-related family of small GTPases and their interacting proteins play multiple roles in regulating epithelial morphogenesis. In this study we examined the role of Cdc42 in foregut development and organ bud formation. Ablation of Cdc42 in post-gastrulation mouse embryos resulted in a loss of apical-basal cell polarity and columnar epithelial morphology in the ventral pharyngeal endoderm, in conjunction with a loss of apical localization of the known CDC42 effector protein PARD6B. Cell viability but not proliferation in the foregut endoderm was impaired. Outgrowth of the liver, lung and thyroid buds was severely curtailed in Cdc42-deficient embryos. In particular, the thyroid bud epithelium did not display the apical constriction that normally occurs concurrently with the outgrowth of the bud into the underlying mesenchyme. SHROOM3, a protein that interacts with Rho GTPases and promotes apical constriction, was strongly expressed in the thyroid bud and its sub-cellular localization was disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutant embryos, the thyroid bud epithelium showed no apical constriction, while the bud continued to grow and protruded into the foregut lumen. Our findings indicate that Cdc42 is required for epithelial polarity and organization in the endoderm and for apical constriction in the thyroid bud. It is possible that the function of CDC42 is partly mediated by SHROOM3.

Dataset of TWIST1-regulated genes in the cranial mesoderm and a transcriptome comparison of cranial mesoderm and cranial neural crest.

This article contains data related to the research article entitled "Transcriptional targets of TWIST1 in the cranial mesoderm regulate cell-matrix interactions and mesenchyme maintenance" by Bildsoe et al. (2016) [1]. The data presented here are derived from: (1) a microarray-based comparison of sorted cranial mesoderm (CM) and cranial neural crest (CNC) cells from E9.5 mouse embryos; (2) comparisons of transcription profiles of head tissues from mouse embryos with a CM-specific loss-of-function of Twist1 and control mouse embryos collected at E8.5 and E9.5; (3) ChIP-seq using a TWIST1-specific monoclonal antibody with chromatin extracts from TWIST1-expressing MDCK cells, a model for a TWIST1-dependent mesenchymal state.