Notochordal and foregut abnormalities correlate with elevated neural crest apoptosis in Patch embryos.

Although Patch mutants show severe abnormalities in many neural crest-derived structures including the face and the heart, there is a paucity of information characterizing the mechanisms underlying these congenital defects. Via manipulating the genetic background to circumvent early embryonic lethality, our results revealed that Patch phenotypes are most likely due to a significant decrease in migratory neural crest lineage due to diminished neural crest survival and elevated apoptosis. Homozygous mutant neural crest precursors can undergo typical expansion within the neural tube, epithelial-to-mesenchymal transformation, and initiate normal neural crest emigration. Moreover, in vitro explant culture demonstrated that when isolated from the surrounding mesenchyme, Patch mutant neural crest cells (NCCs) can migrate appropriately. Additionally, Patch foregut, notochord and somitic morphogenesis, and Sonic hedgehog expression profiles were all perturbed. Significantly, the timing of lethality and extent of apoptosis correlated with the degree of severity of Patch mutant foregut, notochord, and somite dysfunction. Finally, analysis of Balb/c-enriched surviving Patch mutants revealed that not all the neural crest subpopulations are affected and that Patch mutant neural crest-derived sympathetic ganglia and dorsal root ganglia were unaffected. We hypothesize that loss of normal coordinated signaling from the notochord, foregut, and somites underlies the diminished survival of the neural crest lineage within Patch mutants resulting in subsequent neural crest-deficient phenotypes.

Periostin is required for maturation and extracellular matrix stabilization of noncardiomyocyte lineages of the heart.

The secreted periostin protein, which marks mesenchymal cells in endocardial cushions following epithelial-mesenchymal transformation and in mature valves following remodeling, is a putative valvulogenesis target molecule. Indeed, periostin is expressed throughout cardiovascular morphogenesis and in all 4 adult mice valves (annulus and leaflets). Additionally, periostin is expressed throughout the fibrous cardiac skeleton and endocardial cushions in the developing heart but is absent from both normal and/or pathological mouse cardiomyocytes. Periostin (peri(lacZ)) knockout mice exhibit viable valve disease, with neonatal lethality in a minority and latent disease with leaflet abnormalities in the viable majority. Surviving peri(lacZ)-null leaflets are truncated, contain ectopic cardiomyocytes and smooth muscle, misexpress the cartilage proteoglycan aggrecan, demonstrate disorganized matrix stratification, and exhibit reduced transforming growth factor-beta signaling. Neonatal peri(lacZ) nulls that die (14%) display additional defects, including leaflet discontinuities, delamination defects, and deposition of acellular extracellular matrix. Assessment of collagen production, 3D lattice formation ability, and transforming growth factor-beta responsiveness indicate periostin-deficient fibroblasts are unable to support normal valvular remodeling and establishment of a mature cardiac skeleton. Furthermore, pediatric stenotic bicuspid aortic valves that have lost normal extracellular matrix trilaminar stratification have greatly reduced periostin. This suggests that loss of periostin results in inappropriate differentiation of mesenchymal cushion cells and valvular abnormalities via a transforming growth factor-beta-dependent pathway during establishment of the mature heart. Thus, peri(lacZ) knockouts provide a new model of viable latent valve disease.

Gene replacement strategies to test the functional redundancy of basic helix-loop-helix transcription factor.

Basic helix-loop-helix (bHLH) transcription factors control developmental decisions for a wide range of embryonic cell types. Hand1 and Hand2 are closely related bHLH proteins that control cardiac, craniofacial, and limb development. Within the developing heart, Hand1 expression becomes restricted predominantly to the left ventricle, whereas Hand2 becomes restricted predominantly to the left ventricle, for which findings have shown each Hand factor to be necessary for normal chamber formation. Forced overexpression of Hand1 throughout the early developing heart induces abnormal interventricular septal development, with resulting pathogenesis of congenital heart defects. To investigate the potential transcriptional mechanisms involved in heart morphogenesis by Hand2, this study used a replacement targeting approach to knock Hand2 into the Hand1 locus and ectopically express one copy of Hand2 within the endogenous Hand1 expression domain in the developing hearts of transgenic mice. The findings show that high-percentage Hand1 ( Hand2 ) chimeras die at birth and exhibit a range of congenital heart defects. These findings suggest that Hand factors may act via unique transcriptional mechanisms mediated by bHLH factor partner choice, supporting the notion that alterations of Hand factor stoichiometry may be as deleterious to normal heart morphogenesis as Hand factor loss of function.

Building, scaling, and sustaining a learning health system for surgical quality improvement: A toolkit.

This article describes how to start, replicate, scale, and sustain a learning health system for quality improvement, based on the experience of the Michigan Surgical Quality Collaborative (MSQC). The key components to operationalize a successful collaborative improvement infrastructure and the features of a learning health system are explained. This information is designed to guide others who desire to implement quality improvement interventions across a regional network of hospitals using a collaborative approach. A toolkit is provided (under Supporting Information) with practical information for implementation.

Lineage-specific responses to reduced embryonic Pax3 expression levels.

Pax3 is an essential paired- and homeodomain-containing transcription factor that is necessary for closure of the neural tube, and morphogenesis of the migratory neural crest and myoblast lineages. Homozygous loss-of-function mutation results in mid-gestational lethality with defects in myogenesis, neural tube closure and neural crest-derived lineages including melanocytes, Schwann cells and insufficient mesenchymal cells to septate the cardiac outflow tract. To address the function of Pax3 in later fetal stages and in specific adult tissues, we generated a floxed Pax3 allele (Pax3(flox)). An intermediate allele (Pax3(neo)) was produced via creation of the floxed allele, in which the TK-neo(R) cassette is present between exons 5 and 6. It was deduced to be a hypomorph, as Pax3 protein expression is reduced by 80% and homozygote hypomorphs die postnatally. To assess the consequences of reduced Pax3 levels on the various Pax3-expressing lineages and to determine the underlying cause of lethality, we examined Pax3 spatiotemporal expression and the resultant defects. Defective limb and tongue musculature were observed and lethality was due to an inability to suckle. However, the heart, diaphragm, trunk musculature, as well as the various neural crest-derived lineages and neural tube were all unaffected by reduced Pax3 levels. Significantly, elevated levels of the related Pax7 protein were present in unaffected neural tube and epaxial somatic component. The limb and tongue myogenic defects were found to be due to a significant increase in apoptosis within the somites that leads to a paucity of migratory hypaxial myoblasts. These effects were attributed to the hypomorphic effect of the Pax3(neo) allele, as removal of the TK-neo(R) cassette completely relieves the hypomorphic effect, as 100% of the Pax3(flox/flox) mice were normal. These data demonstrate a lineage-specific response to approximately 80% loss of Pax3 protein expression, with myogenesis of limb and tongue being most sensitive to reduced Pax3 levels. Thus, we have established that there are different minimum threshold requirements for Pax3 within different Pax3-expressing lineages.

Generation and characterization of Csrp1 enhancer-driven tissue-restricted Cre-recombinase mice.

Cell type-specific genetic modification using the LoxP/Cre system is a powerful tool for genetic analysis of distinct cell lineages. Because of the unique arterial smooth muscle-restricted expression of a 5.0 kb cysteine-rich protein (Csrp1) enhancer (Lilly et al.,2001, Dev Biol 240:531-547), we hypothesized that a transgenic Cre line would prove useful for the smooth muscle lineage-specific genetic manipulation. Here we describe a transgenic mouse line, ECsrp1(Cre), where Cre is initially specifically expressed in arterial smooth muscle cells. Use of the ROSA26R reporter allele confirmed that Cre-mediated recombination in vascular smooth muscle cells began at approximately E10.0 and was highly proficient. Subsequently, Cre is expressed in restricted skeletal and nonvascular smooth muscle lineages. This lineage tracing data is important for future conditional knockout studies to understand where and when Cre-mediated deletion occurs and where Cre-expressing daughter cells finally localize. Additionally, we crossed the ECsrp1(Cre) mice to the ROSA26(-eGFP-DTA) diphtheria toxin A-expressing mice to genetically ablate ECsrp1(Cre) expressing cells. This ECsrp1(Cre) transgenic line should thus prove useful for genetic analysis of diverse aspects of cardiovascular morphogenesis and as a general smooth muscle lineage deletor line.

periostin null mice exhibit dwarfism, incisor enamel defects, and an early-onset periodontal disease-like phenotype.

Periostin was originally identified as an osteoblast-specific factor and is highly expressed in the embryonic periosteum, cardiac valves, placenta, and periodontal ligament as well as in many adult cancerous tissues. To investigate its role during development, we generated mice that lack the periostin gene and replaced the translation start site and first exon with a lacZ reporter gene. Surprisingly, although periostin is widely expressed in many developing organs, periostin-deficient (peri(lacZ)) embryos are grossly normal. Postnatally, however, approximately 14% of the nulls die before weaning and all of the remaining peri(lacZ) nulls are severely growth retarded. Skeletal analysis revealed that trabecular bone in adult homozygous skeletons was sparse, but overall bone growth was unaffected. Furthermore, by 3 months, the nulls develop an early-onset periodontal disease-like phenotype. Unexpectedly, these mice also show a severe incisor enamel defect, although there is no apparent change in ameloblast differentiation. Significantly, placing the peri(lacZ) nulls on a soft diet that alleviated mechanical strain on the periodontal ligament resulted in a partial rescue of both the enamel and periodontal disease-like phenotypes. Combined, these data suggest that a healthy periodontal ligament is required for normal amelogenesis and that periostin is critically required for maintenance of the integrity of the periodontal ligament in response to mechanical stresses.

Comparison of the four mouse fasciclin-containing genes expression patterns during valvuloseptal morphogenesis.

All four mammalian fasciclin-containing genes are expressed in the adult valves and are localized in partially overlapping and reciprocal patterns during cardiovascular development. Spatiotemporal comparison of the fasciclin-containing secreted adhesion genes, TGFbeta induced clone H3 (betaigH3) and periostin, revealed that they are co-localized within the outflow tract endocardial cushions, but that betaigH3 expression is restricted to the septal cushions within the atrioventricular canal. Conversely, the fasciclin-containing transmembrane gene, stabilin-1, is predominately expressed in the endocardial layer overlaying the cushions and lining the developing heart. However, expression of the fasciclin-containing transmembrane gene, stabilin-2 is only present in the post-natal mature valve endothelial cells. These data illustrate for the first time that the primitive endocardial cushions dynamically express multiple fasciclin-containing adhesion molecules as they undergo the key steps of seeding, proliferation, differentiation, fusion, mesenchymal condensation and remodeling during mouse heart development.

Identification and characterization of a novel Schwann and outflow tract endocardial cushion lineage-restricted periostin enhancer.

Periostin is a fasciclin-containing adhesive glycoprotein that facilitates the migration and differentiation of cells that have undergone epithelial-mesenchymal transformation during embryogenesis and in pathological conditions. Despite the importance of post-transformational differentiation as a general developmental mechanism, little is known how periostin's embryonic expression is regulated. To help resolve this deficiency, a 3.9-kb periostin proximal promoter was isolated and shown to drive tissue-specific expression in the neural crest-derived Schwann cell lineage and in a subpopulation of periostin-expressing cells in the cardiac outflow tract endocardial cushions. In order to identify the enhancer and associated DNA binding factor(s) responsible, in vitro promoter dissection was undertaken in a Schwannoma line. Ultimately a 304-bp(peri) enhancer was identified and shown to be capable of recapitulating 3.9 kb(peri-lacZ)in vivo spatiotemporal patterns. Further mutational and EMSA analysis helped identify a minimal 37-bp region that is bound by the YY1 transcription factor. The 37-bp enhancer was subsequently shown to be essential for in vivo 3.9 kb(peri-lacZ) promoter activity. Taken together, these studies identify an evolutionary-conserved YY1-binding 37-bp region within a 304-bp periostin core enhancer that is capable of regulating simultaneous novel tissue-specific periostin expression in the cardiac outflow-tract cushion mesenchyme and Schwann cell lineages.

Periostin is expressed within the developing teeth at the sites of epithelial-mesenchymal interaction.

Periostin was originally isolated as an osteoblast-specific factor that functions as a cell adhesion molecule for preosteoblasts and is thought to be involved in osteoblast recruitment, attachment, and spreading. The protein was renamed "periostin" because of its expression in the periosteum and periodontal ligament, indicating a potential role in bone and maintenance of tooth structure. Periostin has structural similarity to insect fasciclin-I and can be induced by TGF-beta and Bmp2. Because tooth and periodontium development is a well-described genetic model for organogenesis governed by a reciprocal set of epithelial-mesenchymal interactions, thought to be controlled by various TGF-beta superfamily members, we investigated whether periostin is present during tooth morphogenesis. Both periostin mRNA and protein expression were analyzed throughout normal tooth development (embryonic day [E] 9.5-newborn) and within both Bmp4- and Msx2-null embryos. Periostin mRNA is initially present within the E9.5 first branchial arch epithelium and then shifts to underlying ectomesenchyme. Both mRNA and protein are asymmetrically localized to the lingual/palatal and buccal side during the early epithelial-mesenchymal interactions. Periostin is also present in dental papilla cells and within the trans-differentiating odontoblasts during the bell and hard tissue formation stages of tooth development. We suggest that periostin plays multiple roles as a primary responder molecule during tooth development and may be linked to deposition and organization of other extracellular matrix adhesion molecules during maintenance of the adult tooth, particularly at the sites of hard-soft tissue interface.