Ralph S. Marcucio

Patterns of Infantile Hemangiomas: New Clues to Hemangioma Pathogenesis and Embryonic Facial Development

OBJECTIVES. Large facial infantile hemangiomas have higher rates of complications than small localized hemangiomas, more often require treatment, and can be associated with neurological, ophthalmologic, and cardiac anomalies (PHACE syndrome). The anatomic patterns of these hemangiomas are often referred to as “segmental” despite a lack of precise anatomic definitions. Our study aims to define “segmental” hemangiomas based on clinically observed patterns. Our secondary goal is to relate the observed patterns to currently accepted developmental patterns to gain insight into hemangioma pathogenesis and craniofacial development. METHODS. Photographic data were extracted from a large cohort of patients with infantile hemangiomas. We mapped 294 hemangiomas and recorded common morphologic patterns. Anatomic descriptions of the most common patterns were described and compared with accepted concepts of craniofacial development. RESULTS. Four primary segments were identified (Seg1–Seg4). Seg2 and Seg3 correspond with the previously recognized maxillary and mandibular prominences. Seg1 and Seg4 differ from standard human embryology texts. The frontotemporal segment, Seg1, encompasses the lateral forehead, anterior temporal scalp, and lateral frontal scalp. The segment Seg4, encompassing the medial frontal scalp, nasal bridge, nasal tip, ala, and philtrum, is substantially narrower on the forehead than the previously described frontonasal prominence. CONCLUSIONS. The patterns provide new clues regarding facial development. The observed patterns resemble previously described facial developmental units on the lower face but are distinctly different on the upper face. The patterns suggest that neural crest derivatives may play a role in the development of facial hemangiomas. Finally, these patterns (Seg1–Seg4) help standardize the nomenclature of facial segmental hemangiomas to analyze more effectively hemangioma risks and behavior.

Deciphering the Palimpsest: Studying the Relationship Between Morphological Integration and Phenotypic Covariation

Organisms represent a complex arrangement of anatomical structures and individuated parts that must maintain functional associations through development. This integration of variation between functionally related body parts and the modular organization of development are fundamental determinants of their evolvability. This is because integration results in the expression of coordinated variation that can create preferred directions for evolutionary change, while modularity enables variation in a group of traits or regions to accumulate without deleterious effects on other aspects of the organism. Using our own work on both model systems (e.g., lab mice, avians) and natural populations of rodents and primates, we explore in this paper the relationship between patterns of phenotypic covariation and the developmental determinants of integration that those patterns are assumed to reflect. We show that integration cannot be reliably studied through phenotypic covariance patterns alone and argue that the relationship between phenotypic covariation and integration is obscured in two ways. One is the superimposition of multiple determinants of covariance in complex systems and the other is the dependence of covariation structure on variances in covariance-generating processes. As a consequence, we argue that the direct study of the developmental determinants of integration in model systems is necessary to fully interpret patterns of covariation in natural populations, to link covariation patterns to the processes that generate them, and to understand their significance for evolutionary explanation.

A zone of frontonasal ectoderm regulates patterning and growth in the face.

A fundamental set of patterning genes may define the global organization of the craniofacial region. One of our goals has been to identify these basic patterning genes and understand how they regulate outgrowth of the frontonasal process, which gives rise to the mid and upper face. We identified a molecular boundary in the frontonasal process ectoderm, defined by the juxtaposed domains of Fibroblast growth factor 8 and Sonic hedgehog, which presaged the initial site of frontonasal process outgrowth. Fate maps confirmed that this boundary region later demarcated the dorsoventral axis of the upper beak. Ectopic transplantation of the ectodermal boundary region activated a cascade of molecular events that reprogrammed the developmental fate of neural crest-derived mesenchyme, which resulted in duplications of upper and lower beak structures. We discuss these data in the context of boundary/morphogen models of patterning, and in view of the recent controversy regarding neural crest pre-patterning versus neural crest plasticity.

Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes

One of the most perplexing questions in clinical genetics is why patients with identical gene mutations oftentimes exhibit radically different clinical features. This inconsistency between genotype and phenotype is illustrated in the malformation spectrum of holoprosencephaly (HPE). Family members carrying identical mutations in sonic hedgehog (SHH) can exhibit a variety of facial features ranging from cyclopia to subtle midline asymmetries. Such intrafamilial variability may arise from environmental factors acting in conjunction with gene mutations that collectively reduce SHH activity below a critical threshold. We undertook a series of experiments to test the hypothesis that modifying the activity of the SHH signaling pathway at discrete periods of embryonic development could account for the phenotypic spectrum of HPE. Exposing avian embryos to cyclopamine during critical periods of craniofacial development recreated a continuum of HPE-related defects. The craniofacial malformations included hypotelorism, midfacial hypoplasia, and facial clefting and were not the result of excessive crest cell apoptosis. Rather, they resulted from molecular reprogramming of an organizing center whose activity controls outgrowth and patterning of the mid and upper face. Collectively, these data reveal one mechanism by which the variable expressivity of a disorder such as HPE can be produced through temporal disruption of a single molecular pathway.

Differentiation of Avian Craniofacial Muscles: I. Patterns of Early Regulatory Gene Expression and Myosin Heavy Chain Synthesis

Myogenic populations of the avian head arise within both epithelial (somitic) and mesenchymal (unsegmented) mesodermal populations. The former, which gives rise to neck, tongue, laryngeal, and diaphragmatic muscles, show many similarities to trunk axial, body wall, and appendicular muscles. However, muscle progenitors originating within unsegmented head mesoderm exhibit several distinct features, including multiple ancestries, the absence of several somite lineage‐determining regulatory gene products, diverse locations relative to neuraxial and pharyngeal tissues, and a prolonged and necessary interaction with neural crest cells. The object of this study has been to characterize the spatial and temporal patterns of early muscle regulatory gene expression and subsequent myosin heavy chain isoform appearance in avian mesenchyme‐derived extraocular and branchial muscles, and compare these with expression patterns in myotome‐derived neck and tongue muscles. Myf5 and myoD transcripts are detected in the dorsomedial (epaxial) region of the occipital somites before stage 12, but are not evident in the ventrolateral domain until stage 14. Within unsegmented head mesoderm, myf5 expression begins at stage 13.5 in the second branchial arch, followed within a few hours in the lateral rectus and first branchial arch myoblasts, then other eye and branchial arch muscles. Expression of myoD is detected initially in the first branchial arch beginning at stage 14.5, followed quickly by its appearance in other arches and eye muscles. Multiple foci of myoblasts expressing these transcripts are evident during the early stages of myogenesis in the first and third branchial arches and the lateral rectus‐pyramidalis/quadratus complex, suggesting an early patterned segregation of muscle precursors within head mesoderm. Myf5‐positive myoblasts forming the hypoglossal cord emerge from the lateral borders of somites 4 and 5 by stage 15 and move ventrally as a cohort. Myosin heavy chain (MyHC) is first immunologically detectable in several eye and branchial arch myofibers between stages 21 and 22, although many tongue and laryngeal muscles do not initiate myosin production until stage 24 or later. Detectable synthesis of the MyHC‐S3 isoform, which characterizes myofibers as having “slow” contraction properties, occurs within 1–2 stages of the onset of MyHC synthesis in most head muscles, with tongue and laryngeal muscles being substantially delayed. Such a prolonged, 2‐ to 3‐day period of regulatory gene expression preceding the onset of myosin production contrasts with the interval seen in muscles developing in axial (approximately 18 hr) and wing (approximately 1–1.5 days) locations, and is unique to head muscles. This finding suggests that ongoing interactions between head myoblasts and their surroundings, most likely neural crest cells, delay myoblast withdrawal from the mitotic pool. These descriptions define a spatiotemporal pattern of muscle regulatory gene and myosin heavy chain expression unique to head muscles. This pattern is independent of origin (somitic vs. unsegmented paraxial vs. prechordal mesoderm), position (extraocular vs. branchial vs. subpharyngeal), and fiber type (fast vs. slow) and is shared among all muscles whose precursors interact with cephalic neural crest populations. Dev Dyn 1999;216:96–112. ©1999 Wiley‐Liss, Inc.

A SHH-responsive signaling center in the forebrain regulates craniofacial morphogenesis via the facial ectoderm

Interactions among the forebrain, neural crest and facial ectoderm regulate development of the upper jaw. To examine these interactions, we activated the Sonic hedgehog (SHH) pathway in the brain. Beginning 72 hours after activation of the SHH pathway, growth within the avian frontonasal process (FNP) was exaggerated in lateral regions and impaired in medial regions. This growth pattern is similar to that in mice and superimposed a mammalian-like morphology on the upper jaw. Jaw growth is controlled by signals from the frontonasal ectodermal zone (FEZ), and the divergent morphologies that characterize birds and mammals are accompanied by changes in the FEZ. In chicks there is a single FEZ spanning the FNP, but in mice both median nasal processes have a FEZ. In treated chicks, the FEZ was split into right and left domains that resembled the pattern present in mice. Additionally, we observed that, in the brain, fibroblast growth factor 8 (Fgf8) was downregulated, and signals in or near the nasal pit were altered. Raldh2 expression was expanded, whereas Fgf8, Wnt4, Wnt6 and Zfhx1b were downregulated. However, Wnt9b, and activation of the canonical WNT pathway, were unaltered in treated embryos. At later time points the upper beak was shortened owing to hypoplasia of the skeleton, and this phenotype was reproduced when we blocked the FGF pathway. Thus, the brain establishes multiple signaling centers within the developing upper jaw. Changes in organization of the brain that occur during evolution or as a result of disease can alter these centers and thereby generate morphological variation.

Role of Matrix Metalloproteinase 13 in Both Endochondral and Intramembranous Ossification during Skeletal Regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13−/− mice is intrinsic to cartilage and bone. Mmp13−/− mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.

Quantitative analyses link modulation of sonic hedgehog signaling to continuous variation in facial growth and shape

Variation is an intrinsic feature of biological systems, yet developmental biology does not frequently address population-level phenomena. Sonic hedgehog (SHH) signaling activity in the vertebrate forebrain and face is thought to contribute to continuous variation in the morphology of the upper jaw, but despite its potential explanatory power, this idea has never been quantitatively assessed. Here, we test this hypothesis with an experimental design that is explicitly focused on the generation and measurement of variation in multivariate shape, tissue growth, cellular behavior and gene expression. We show that the majority of upper jaw shape variation can be explained by progressive changes in the spatial organization and mitotic activity of midfacial growth zones controlled by SHH signaling. In addition, nonlinearity between our treatment doses and phenotypic outcomes suggests that threshold effects in SHH signaling may play a role in variability in midfacial malformations such as holoprosencephaly (HPE). Together, these results provide novel insight into the generation of facial morphology, and demonstrate the value of quantifying variation for our understanding of development and disease.

Mechanisms that underlie co-variation of the brain and face.

The effect of the brain on the morphology of the face has long been recognized in both evolutionary biology and clinical medicine. In this work, we describe factors that are active between the development of the brain and face and how these might impact craniofacial variation. First, there is the physical influence of the brain, which contributes to overall growth and morphology of the face through direct structural interactions. Second, there is the molecular influence of the brain, which signals to facial tissues to establish signaling centers that regulate patterned growth. Importantly, subtle alterations to these physical or molecular interactions may contribute to both normal and abnormal variation. These interactions are therefore critical to our understanding of how a diversity of facial morphologies can be generated both within species and across evolutionary time. genesis 49:177–189, 2011.

Multiple roles for CCR2 during fracture healing

SUMMARY Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory cells to sites of injury occurs in response to specific signaling pathways. The CC chemokine receptor type 2 (CCR2) is crucial for recruiting macrophages, as well as regulating osteoclast function. In this study, we examined fracture healing in Ccr2−/− mice. We first demonstrated that the expression of Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2−/− mice compared with wild-type controls at 3 days after injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after injury in mutant mice. At day 14, Ccr2−/− mice had less bone in their calluses. At day 21, Ccr2−/− mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2−/− osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2−/− mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.