Apical expansion of calvarial osteoblasts and suture patency is dependent on fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Surgery for internal rotation contracture in infancy may obviate the need for brachial plexus nerve reconstruction: early experience.

BACKGROUND: Shoulder internal rotation contracture and subluxation in the first year of life has long been recognized in some patients with brachial plexus birth injury (BPBI). Surgical management of shoulder pathology has traditionally been undertaken following nerve reconstruction as necessary. In some patients; however, shoulder pathology may impair or obscure functional neuromuscular recovery of the upper extremity. As a proof of concept, we report a highly selected subset of patients with BPBI in whom shoulder surgery undertaken before one year of age obviated the need for neuroma resection and nerve grafting. METHODS: A retrospective review was performed of all patients with upper trunk BPBI who underwent shoulder surgery before one year of age from 2015 to 2018. Upper extremity motor function was evaluated with preoperative and postoperative Active Movement Scale scores, Cookie tests, and the requirement for subsequent neuroma resection and nerve grafting. RESULTS: Fifteen patients with BPBI meeting the inclusion criteria underwent shoulder surgery (including a subscapularis slide and tendon transfers of the teres major and latissimus dorsi muscles) before 1 year of age. Preoperatively, no patients of the appropriate age passed the Cookie test for elbow flexion. Thirteen patients either passed the Cookie test or scored Active Movement Scale score 7 for elbow flexion at or before the last available follow-up undertaken at a median age of 3.4 [1.4, 5.2] years. One of those 13 patients underwent single fascicular distal nerve transfer to improve elbow flexion before subsequently passing the Cookie test. Two patients did not have sufficient follow-up to assess elbow flexion. CONCLUSION: Although the exact role of shoulder surgery in infancy for BPBI remains to be defined, the findings from this study provide proof of concept that early, targeted surgical treatment of the shoulder may obviate the need for brachial plexus nerve reconstruction in a highly selected group of infants with BPBI.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral for calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. During apical expansion, we found that mouse calvarial primordia have consistent cellular proliferation, density, and survival with complex tissue scale deformations, raising the possibility that morphogenetic movements underlie expansion. Time lapse light sheet imaging of mouse embryos revealed that calvarial progenitors intercalate in 3D to converge supraorbital arch mesenchyme mediolaterally and extend it apically. In contrast, progenitors located further apically exhibited protrusive and crawling activity. CM cells express non-canonical Wnt/Planar Cell Polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand, Wnt5a-/- mutants have less dynamic cell rearrangements, protrusive activity, and a flattened head shape. Loss of cranial mesenchyme-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of OSX+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin cytoskeleton protein along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis and provide tissue level cues for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

Shape-driven confluent rigidity transition in curved biological tissues.

Collective cell motions underlie structure formation during embryonic development. Tissues exhibit emergent multicellular characteristics such as jamming, rigidity transitions, and glassy dynamics, but there remain questions about how those tissue-scale dynamics derive from local cell-level properties. Specifically, there has been little consideration of the interplay between local tissue geometry and cellular properties influencing larger-scale tissue behaviors. Here, we consider a simple two-dimensional computational vertex model for confluent tissue monolayers, which exhibits a rigidity phase transition controlled by the shape index (ratio of perimeter to square root area) of cells, on surfaces of constant curvature. We show that the critical point for the rigidity transition is a function of curvature such that positively curved systems are likely to be in a less rigid, more fluid, phase. Likewise, negatively curved systems (saddles) are likely to be in a more rigid, less fluid, phase. A phase diagram we generate for the curvature and shape index constitutes a testable prediction from the model. The curvature dependence is interesting because it suggests a natural explanation for more dynamic tissue remodeling and facile growth in regions of higher surface curvature. Conversely, we would predict stability at the base of saddle-shaped budding structures without invoking the need for biochemical or other physical differences. This concept has potential ramifications for our understanding of morphogenesis of budding and branching structures.

Indentation induces instantaneous nuclear stiffening and unfolding of nuclear envelope wrinkles.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm and regulates transport between the cytosol and the nucleus in eukaryotes. Nuclear stiffening enables the cell nucleus to protect itself from extensive deformation, loss of NE integrity, and genome instability. It is known that the reorganization of actin, lamin, and chromatin can contribute to nuclear stiffening. In this work, we show that structural alteration of NE also contributes to instantaneous nuclear stiffening under indentation. In situ mechanical characterization of cell nuclei in intact cells shows that nuclear stiffening and unfolding of NE wrinkles occur simultaneously at the indentation site. A positive correlation between the initial state of NE wrinkles, the unfolding of NE wrinkles, and the stiffening ratio (stiffness fold-change) is found. Additionally, NE wrinkles unfold throughout the nucleus outside the indentation site. Finite element simulation, which involves the purely passive process of structural unfolding, shows that unfolding of NE wrinkles alone can lead to an increase in nuclear stiffness and a reduction in stress and strain levels. Together, these results provide a perspective on how cell nucleus adapts to mechanical stimuli through structural alteration of the NE.

Primitive Myxoid Mesenchymal Tumor of Infancy With Brain Metastasis Case Report and Literature Review.

Primitive myxoid mesenchymal tumor of infancy (PMMTI), a rare soft tissue tumor with distinct characteristics. PMMTI tends to have an aggressive local course, with multiple relapses and poor response to treatment. Rare cases of distant metastases have been described before. We described the second case of PMMTI with brain metastasis.

Management of Brachial Plexus Birth Injury: The SickKids Experience.

This article describes the approach utilized by the multidisciplinary team at Sick Kids Hospital to evaluate and treat patients with brachial plexus birth injury (BPBI). This approach has been informed by more than 30 years of experience treating over 1,800 patients with BPBI and continues to evolve over time. The objective of this article is to provide readers with a practical overview of the Sick Kids approach to the management of infants with BPBI.

Recent Advances in the Treatment of Brachial Plexus Birth Injury.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe methods of clinical evaluation for neurologic recovery in brachial plexus birth injury. 2. Understand the role of different diagnostic imaging modalities to evaluate the upper limb. 3. List nonsurgical strategies and surgical procedures to manage shoulder abnormality. 4. Explain the advantages and disadvantages of microsurgical nerve reconstruction and distal nerve transfers in brachial plexus birth injury. 5. Recognize the prevalence of pain in this population and the need for greater sensory outcomes evaluation. SUMMARY: Brachial plexus birth injury (BPBI) results from closed traction injury to the brachial plexus in the neck during an infant's vertex passage through the birth canal. Although spontaneous upper limb recovery occurs in most instances of BPBI, some infants do not demonstrate adequate motor recovery within an acceptable timeline and require surgical intervention to restore upper limb function. This article reviews major advances in the management of BPBI in the past decade that include improved understanding of shoulder pathology and its impact on observed motor recovery, novel surgical techniques, new insights in sensory function and pain, and global efforts to develop standardized outcomes assessment scales.

Rotationplasty with Tibial Nerve Coaptation: A Case Report.

CASE: We present the case of a 14-year-old adolescent boy with a distal femoral osteosarcoma partially encasing the tibial nerve. He underwent rotationplasty with resection and coaptation (end-to-end repair) of the tibial nerve. By 1 year postoperatively, he had recovered sensation on the plantar aspect of his foot and Medical Research Council scale 4+/5 gastro-soleus contraction that powered extension of the new knee. CONCLUSION: Tibial nerve resection is not an absolute contraindication for rotationplasty, even in an adolescent. Nerve coaptation allows for well-functioning rotationplasty as an alternative to endoprosthetic reconstruction or above-knee amputation.

Apical expansion of calvarial osteoblasts and suture patency is dependent on graded fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bone. Premature suture fusion (craniosynostosis, CS) or persistent fontanelles are common defects in calvarial development. Although some of the genetic causes of these disorders are known, we lack an understanding of the instructions directing the growth and migration of progenitors of these bones, which may affect the suture patency. Here, we identify graded expression of Fibronectin (FN1) protein in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvarial osteoblasts. Syndromic forms of CS exhibit dysregulated FN1 expression, and we find FN1 expression is altered in a mouse CS model as well. Conditional deletion of Fn1 in CM causes diminished frontal bone expansion by altering cell polarity and shape. To address how osteoprogenitors interact with the observed FN1 prepattern, we conditionally ablate Wasl/N-Wasp to disrupt F-actin junctions in migrating cells, impacting lamellipodia and cell-matrix interaction. Neural crest-targeted deletion of Wasl results in a diminished actin network and reduced expansion of frontal bone primordia similar to conditional Fn1 mutants. Interestingly, defective calvaria formation in both the Fn1 and Wasl mutants occurs without a significant change in proliferation, survival, or osteogenesis. Finally, we find that CM-restricted Fn1 deletion leads to premature fusion of coronal sutures. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Transgenic force sensors and software to measure force transmission across the mammalian nuclear envelope in vivo.

Nuclear mechanotransduction is a growing field with exciting implications for the regulation of gene expression and cellular function. Mechanical signals may be transduced to the nuclear interior biochemically or physically through connections between the cell surface and chromatin. To define mechanical stresses upon the nucleus in physiological settings, we generated transgenic mouse strains that harbour FRET-based tension sensors or control constructs in the outer and inner aspects of the nuclear envelope. We knocked-in a published esprin-2G sensor to measure tensions across the LINC complex and generated a new sensor that links the inner nuclear membrane to chromatin. To mitigate challenges inherent to fluorescence lifetime analysis in vivo, we developed software (FLIMvivo) that markedly improves the fitting of fluorescence decay curves. In the mouse embryo, the sensors responded to cytoskeletal relaxation and stretch applied by micro-aspiration. They reported organ-specific differences and a spatiotemporal tension gradient along the proximodistal axis of the limb bud, raising the possibility that mechanical mechanisms coregulate pattern formation. These mouse strains and software are potentially valuable tools for testing and refining mechanotransduction hypotheses in vivo.

Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFß to rotate the gut.

The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-ß (TGFß), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.

Patterning the embryonic pulmonary mesenchyme.

Smooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

Bone Health in Osteosarcoma at Presentation and Its Impact on Cancer Treatment: A Case Series of 3 Pediatric Patients.

Osteosarcoma is the most common pediatric malignant bone tumor. Concomitant osteoporosis has typically been attributed to oncologic therapy. The present case series is aimed to describe 3 patients who presented with osteoporosis or osteopenia before, or early in, their oncology treatment. In our patients, bone health and its complications had significant impacts including pain, reduced mobility, prolonged admission, and delays in recovery. Our patients experienced improvement with resection of their primary tumor and with bisphosphonate infusion. Future studies are required to determine the prevalence osteoporosis at presentation of osteosarcoma and the role of bisphosphonates.

Live imaging YAP signalling in mouse embryo development.

YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATS kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behaviour of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signalling in vivo in many different situations.

Reconstruction for bone tumours of the shoulder and humerus in children and adolescents.

Reconstructions for paediatric bone tumours of the shoulder girdle and humerus are intended to optimize placement of the hand in space. Given the longevity of paediatric survivors of sarcoma, durability is an important planning consideration. Here, I review a subset of approaches based on anatomical site with an emphasis on function and longevity. Often, biological reconstructions that combine living bone with tendon repairs and transfers best address those goals.

Non-rhabdomyosarcoma soft tissue sarcomas diagnosed in patients at a young age. An overview of clinical, pathological, and molecular findings.

OBJECTIVE: Disease spectrum in pediatric sarcoma differs substantially from adults. We report a cohort of very young children with non-rhabdomyosarcoma soft tissue sarcoma (NRSTS) detailing their molecular features, treatment, and outcome. METHODS: We report features of consecutive children (age <2 years) with NRSTS (2000-2017). Archival pathological material was re-reviewed, with additional molecular techniques applied where indicated. RESULTS: Twenty-nine patients (16 females, 55%) were identified (median age 6 months; range 0-23). Most common diagnoses included infantile fibrosarcoma (IFS, n = 14, 48%), malignant rhabdoid tumor (MRT, n = 4, 14%), and undifferentiated sarcoma (n = 4, 14%). Twenty-seven of 29 (93%) had tumor molecular characterization to confirm diagnosis. Clinical presentation included a swelling/mass (n = 23, 79%). Disease extent was localized (n = 20, 69%), locoregional (n = 6, 21%), or metastatic (n = 3, 10%). Seventeen of 29 (59%) who underwent surgery achieved complete resection (R0). Other treatments included conventional chemotherapy (n = 26, 90%), molecularly targeted therapies (n = 3, 10%), and radiation (n = 5, 17%). At last follow-up (median 3 years; range 0.3-16.4), 23 (79%) were alive, disease-free and six (21%) had died of disease. All patients with IFS were alive and all those with MRT died. A cancer predisposition syndrome (CPS) was confirmed in three of 10 (30%) genetically tested patients. CONCLUSION: We recommend tumor molecular characterization in all young patients including evaluation for CPS to optimize treatment options and prognostication.

Radiological Assessment and Outcome of Local Disease Progression after Neoadjuvant Chemotherapy in Children and Adolescents with Localized Osteosarcoma.

OBJECTIVE: We examined the interobserver reliability of local progressive disease (L-PD) determination using two major radiological response evaluation criteria systems (Response evaluation Criteria in Solid Tumors (RECIST) and the European and American Osteosarcoma Study (EURAMOS)) in patients diagnosed with localized osteosarcoma (OS). Additionally, we describe the outcomes of patients determined to experience L-PD. MATERIALS AND METHODS: Forty-seven patients diagnosed with localized OS between 2000 and 2012 at our institution were identified. Paired magnetic resonance imaging of the primary tumor from diagnosis and post-neoadjuvant chemotherapy were blindly assessed by two experienced radiologists and determined L-PD as per RECIST and EURAMOS radiological criteria. Interobserver reliability was measured using the kappa statistic (κ). The Kaplan Meier method and log-rank test was used to assess differences between groups. RESULTS: Of 47 patients (median age at diagnosis 12.9 years), 16 (34%) had L-PD (by RECIST or EURAMOS radiological definition). There was less agreement between the radiologists using EURAMOS radiological criteria for L-PD (80.9%, κ = 0.48) than with RECIST criteria (97.9%, κ = 0.87). Patients with radiologically defined L-PD had a 5-year progression-free survival (PFS) of 55.6%, compared to a 5 year-PFS of 82.7% in the group of patients without L-PD (n = 31) (Log rank p = 0.0185). CONCLUSIONS: The interobserver reliability of L-PD determination is higher using RECIST than EURAMOS. RECIST can be considered for response assessment in OS clinical trials. The presence of L-PD was associated with worse outcomes.

IRX3/5 regulate mitotic chromatid segregation and limb bud shape.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.

Magnetic Micromanipulation for In Vivo Measurement of Stiffness Heterogeneity and Anisotropy in the Mouse Mandibular Arch.

The mechanical properties of tissues are pivotal for morphogenesis and disease progression. Recent approaches have enabled measurements of the spatial distributions of viscoelastic properties among embryonic and pathological model systems and facilitated the generation of important hypotheses such as durotaxis and tissue-scale phase transition. There likely are many unexpected aspects of embryo biomechanics we have yet to discover which will change our views of mechanisms that govern development and disease. One area in the blind spot of even the most recent approaches to measuring tissue stiffness is the potentially anisotropic nature of that parameter. Here, we report a magnetic micromanipulation device that generates a uniform magnetic field gradient within a large workspace and permits measurement of the variation of tissue stiffness along three orthogonal axes. By applying the device to the organ-stage mouse embryo, we identify spatially heterogenous and directionally anisotropic stiffness within the mandibular arch. Those properties correspond to the domain of expression and the angular distribution of fibronectin and have potential implications for mechanisms that orient collective cell movements and shape tissues during development. Assessment of anisotropic properties extends the repertoire of current methods and will enable the generation and testing of hypotheses.