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Biomedical subjects

Jill A Helms

Publications and source records attributed to Jill A Helms.

At least 19 recordsLinked to original sources
PubMed2006

Effect of mechanical stimuli on skeletal regeneration around implants.

Due to the aging population and the increasing need for total joint replacements, osseointegration is of a great interest for various clinical disciplines. Our objective was to investigate the molecular and cellular foundation that underlies this process. Here, we used an in vivo mouse model to study the cellular and molecular response in three distinct areas of unloaded implants: the periosteum, the gap between implant and cortical bone, and the marrow space. Our analyses began with the early phases of healing, and continued until the implants were completely osseointegrated. We investigated aspects of osseointegration ranging from vascularization, cell proliferation, differentiation, and bone remodeling. In doing so, we gained an understanding of the healing mechanisms of different skeletal tissues during unloaded implant osseointegration. To continue our analysis, we used a micromotion device to apply a defined physical stimulus to the implants, and in doing so, we dramatically enhanced bone formation in the peri-implant tissue. By comparing strain measurements with cellular and molecular analyses, we developed an understanding of the correlation between strain magnitudes and fate decisions of cells shaping the skeletal regenerate.

Adult Stem Cells↗
PubMed2006

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm.

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.

Animals↗
PubMed2006

The molecular origins of species-specific facial pattern.

The prevailing approach within the field of craniofacial development is focused on finding a balance between tissues (e.g., facial epithelia, neuroectoderm, and neural crest) and molecules (e.g., bone morphogenetic proteins, fibroblast growth factors, Wnts) that play a role in sculpting the face. We are rapidly learning that neither these tissues nor molecular signals are able to act in isolation; in fact, molecular cues are constantly reciprocating signals between the epithelia and the neural crest in order to pattern and mold facial structures. More recently, it has been proposed that this crosstalk is often mediated and organized by discrete organizing centers within the tissues that are able to act as a self-contained unit of developmental potential (e.g., the rhombomere and perhaps the ectomere). Whatever the molecules are and however they are interpreted by these tissues, it appears that there is a remarkably conserved mechanism for setting up the initial organization of the facial prominences between species. Regardless of species, all vertebrates appear to have the same basic bauplan. However, sometime during mid-gestation, the vertebrate face begins to exhibit species-specific variations, in large part due to differences in the rates of growth and differentiation of cells comprising the facial prominences. How do these differences arise? Are they due to late changes in molecular signaling within the facial prominences themselves? Or are these late changes a reflection of earlier, more subtle alterations in boundaries and fields that are established at the earliest stages of head formation? We do not have clear answers to these questions yet, but in this chapter we present new studies that shed light on this age-old question. This chapter aims to present the known signals, both on a molecular and cellular level, responsible for craniofacial development while bringing to light the events that may serve to create difference in facial morphology seen from species to species.

Animals↗
PubMed2005

Molecular interactions coordinating the development of the forebrain and face.

From an architectural point of view, the forebrain acts as a framework upon which the middle and upper face develops and grows. In addition to serving a structural role, we present evidence that the forebrain is a source of signals that shape the facial skeleton. In this study, we inhibited Sonic hedgehog (Shh) signaling from the neuroectoderm then examined the molecular changes and the skeletal alterations resulting from the treatment. One of the first changes we noted was that the dorsoventral polarity of the forebrain was disturbed, which manifested as a loss of Shh in the ventral telencephalon, a reduction in expression of the ventral markers Nkx2.1 and Dlx2, and a concomitant expansion of the dorsal marker Pax6. In addition to changes in the forebrain neuroectoderm, we observed altered gene expression patterns in the facial ectoderm. For example, Shh was not induced in the frontonasal ectoderm, and Ptc and Gli1 were reduced in both the ectoderm and adjacent mesenchyme. As a consequence, a signaling center in the frontonasal prominence was disrupted and the prominence failed to undergo proximodistal and mediolateral expansion. After 15 days of development, the upper beaks of the treated embryos were truncated, and the skeletal elements were located in more medial and proximal locations in relation to the skeletal elements of the lower jaw elements. These data indicate that a role of Shh in the forebrain is to regulate Shh expression in the face, and that together, these Shh domains mediate patterning within the frontonasal prominence and proximodistal outgrowth of the middle and upper face.

Animals↗
PubMed2005

Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development.

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh(-/-) chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh(-/-) skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh(-/-) cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh(-/-) cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh(-/-) cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage -- comprising osteoblast precursors and endothelial cells -- as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh(-/-) embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

Alleles↗
PubMed2005

Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects.

Demineralized bone matrix commonly is used to enhance and to facilitate bone grafting after skeletal injury or disease; however, the biologic bases for its bone-inducing abilities remain obscure. We have taken advantage of a mouse model of cortical bone defect healing to elucidate its mechanisms of action in vivo. Demineralized bone matrix combined with hyaluronan improved skeletal healing by inducing early deposition of an osteoid matrix. Demineralized bone matrix combined with hyaluronan might accelerate bone formation because it serves as a scaffold on which osteoprogenitor cells attach. We tested this possibility by comparing demineralized bone matrix combined with hyaluronan with heat-inactivated demineralized bone matrix combined with hyaluronan and found that the intact material was superior in terms of its ability to stimulate new bone formation. We also compared the bone inducing capacity of demineralized bone matrix combined with hyaluronan with a synthetic collagen sponge and found that not only the synthetic collagen scaffold delayed bone healing but also impaired bony bridging at later stages of repair. Another important property of demineralized bone matrix combined with hyaluronan was its ability to become actively degraded by osteoclasts during healing. Therefore, demineralized bone matrix combined with hyaluronan may not only attract osteoblasts and stimulate their differentiation, but also induce bone matrix resorption, which is a critically important regulator of bone formation and mineralization.

Animals↗
PubMed2005

The fickle finger of fate.

In this issue of the JCI, Niedermaier and colleagues demonstrate that a chromosomal inversion in mice results in dysregulation of Sonic hedgehog (Shh), such that Shh is ectopically expressed in a skeletogenic domain typically occupied by Indian hedgehog (Ihh). This molecular reversal eliminates phalangeal joint spaces, and consequently, Short digits (Dsh) heterozygotes (Dsh/+) have brachydactyly (shortened digits). Ihh is normally downregulated in regions that will become the joint space, but in Dsh/+ mice, Shh bypasses this regulatory control and persists; accordingly, cells maintain their chondrogenic fate and the developed digits are shorter than normal. The significance of these data extends far beyond the field of skeletal biology: they hint at the very real possibility that the endogenous Shh regulatory region contains a repressor designed to segregate the activity of Shh from Ihh. The existence of such a repressor provides a window into the distant past, revealing that Shh and Ihh must once have shared responsibilities in establishing tissue boundaries and orchestrating vertebrate tissue morphogenesis.

Animals↗
PubMed2005

New insights into craniofacial morphogenesis.

No region of our anatomy more powerfully conveys our emotions nor elicits more profound reactions when disease or genetic disorders disfigure it than the face. Recent progress has been made towards defining the tissue interactions and molecular mechanisms that control craniofacial morphogenesis. Some insights have come from genetic manipulations and others from tissue recombinations and biochemical approaches, which have revealed the molecular underpinnings of facial morphogenesis. Changes in craniofacial architecture also lie at the heart of evolutionary adaptation, as new studies in fish and fowl attest. Together, these findings reveal much about molecular and tissue interactions behind craniofacial development.

Animals↗
PubMed2004

Hierarchy revealed in the specification of three skeletal fates by Sox9 and Runx2.

Across vertebrates, there are three principal skeletal tissues: bone, persistent cartilage, and replacement cartilage. Although each tissue has a different evolutionary history and functional morphology, they also share many features. For example, they function as structural supports, they are comprised of cells embedded in collagen-rich extracellular matrix, and they derive from a common embryonic stem cell, the osteochondroprogenitor. Occasionally, homologous skeletal elements can change tissue type through phylogeny. Together, these observations raise the possibility that skeletal tissue identity is determined by a shared set of genes. Here, we show that misexpression of either Sox9 or Runx2 can substitute bone with replacement cartilage or can convert persistent cartilage into replacement cartilage and vice versa. Our data also suggest that these transcription factors function in a molecular hierarchy in which chondrogenic factors dominate. We propose a binary molecular code that determines whether skeletal tissues form as bone, persistent cartilage, or replacement cartilage. Finally, these data provide insights into the roles that master regulatory genes play during evolutionary change of the vertebrate skeleton.

Animals↗
PubMed2004

Mepe is expressed during skeletal development and regeneration.

a bone metabolism regulator that is expressed by osteocytes in normal adult bone. Here, we used an immunohistochemical approach to study whether Mepe has a role in murine long bone development and regeneration. Our data showed that Mepe protein was produced by osteoblasts and osteocytes during skeletogenesis, as early as 2 days postnatal. During the healing of non-stabilized tibial fractures, which occurs through endochondral ossification, Mepe expression was first detected in fibroblast-like cells within the callus by 6 days postfracture. By 10 and 14 days postfracture (the hard callus phase of repair), Mepe was expressed within late hypertrophic chondrocytes and osteocytes in the regenerating tissues. Mepe became externalized in osteocyte lacunae during this period. By 28 days postfracture (the remodeling phase of repair), Mepe continued to be robustly expressed in osteocytes of the regenerating bone. We compared the Mepe expression profile with that of alkaline phosphatase, a marker of bone mineralization. We found that both Mepe and alkaline phosphatase increased during the hard callus phase of repair. In the remodeling phase of repair, Mepe expression levels remained high while alkaline phosphatase activity decreased. We also examined Mepe expression during cortical bone defect healing, which occurs through intramembranous ossification. Mepe immunostaining was found within fibroblast-like cells, osteoblasts, and osteocytes in the regenerating bone, through 5 to 21 days postsurgery. Thus, Mepe appears to play a role in both long bone regeneration and the latter stages of skeletogenesis.

Alkaline Phosphatase↗
PubMed2004

Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development.

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.

Animals↗
PubMed2004

Conserved molecular program regulating cranial and appendicular skeletogenesis.

The majority of in vivo studies on bone and cartilage differentiation are carried out using the appendicular skeleton as a model system, with the implicit assumption that skeletal formation is equivalent throughout the body. This assumption persists, despite differences in the cellular origins of the skeletogenic precursors. To test the hypothesis that a fundamental set of genes directs skeletal cell differentiation throughout the body, we analyzed cartilage and bone of the chick limb and head during mesenchymal condensation, and when the skeletal tissues had matured. First, we analyzed the expression patterns of transcription factors in early skeletogenic condensations, which revealed similarities among skeletal cell specification in the limb and head. For example, skeletogenic condensations that undergo endochondral ossification had equivalent expression patterns of skeletogenic transcription factors in both limb and head. In the head, many elements also differentiate through intramembranous ossification, or through persistent cartilage formation. Our analyses of these skeletogenic condensations revealed that a unique expression pattern of transcription factors distinguishes among three skeletal tissue fates. The vasculature was excluded from all three skeletogenic condensations, demonstrating that this is not a characteristic unique to endochondral ossification. Second, we compared three different types of more mature cartilage and bone tissue in both the limb and the head, by analyzing a variety of skeletal collagens and signaling molecules. Histological and molecular markers of cartilage and bone generally were conserved between the limb and head skeletons, although we uncovered subtle differences in signaling pathways that might influence cranial and appendicular skeletogenesis.

Animals↗
PubMed2004

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.

Animals↗
PubMed2003

Molecular ontogeny of the skeleton.

From a traditional viewpoint, skeletal elements form by two distinct processes: endochondral ossification, during which a cartilage template is replaced by bone, and intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts. There are inherent difficulties with this historical classification scheme, not the least of which is that bones typically described as endochondral actually form bone through an intramembranous process, and that some membranous bones may have a transient chondrogenic phase. These innate contradictions can be circumvented if molecular and cellular, rather than histogenic, criteria are used to describe the process of skeletal tissue formation. Within the past decade, clinical examinations of human skeletal syndromes have led to the identification and subsequent characterization of regulatory molecules that direct chondrogenesis and osteogenesis in every skeletal element of the body. In this review, we survey these molecules and the tissue interactions that may regulate their expression. What emerges is a new paradigm, by which we can explain and understand the process of normal- and abnormal-skeletal development.

Animals↗
PubMed2003

Markers of osteoblast differentiation in fusing and nonfusing cranial sutures.

Accumulating clinical genetic data support the hypothesis that alterations in osteoblast differentiation are closely associated with craniosynostoses. Gain-of-function mutations in FGFR1, FGFR2, FGFR3, and Msx2 and loss-of-function mutations in Twist are examples of such alterations. Several studies have examined how these mutations alter the expression patterns for transcription factors such as Runx2 and noncollagenous extracellular matrix molecules such as osteopontin and osteocalcin. One limitation of such studies is that they examine samples derived from craniosynostotic patients with sutures that have already fused, thus missing the dynamic osteogenic process of suture fusion. In this study, in situ hybridization was used to localize Runx2, osteopontin, and osteocalcin expression in the sagittal and posterior frontal sutures in mice (n = 20), before (day 13), during (days 23, 33, and 43), and after (day 53) the period of physiological posterior frontal suture fusion. The data demonstrated similar patterns of expression in fusing (posterior frontal) and nonfusing (sagittal) sutures. The expression of all three genes was primarily concentrated in the osteogenic fronts of both sutures and decreased with time. Notably, none of the three genes was expressed in the mesenchyme of either fusing or nonfusing sutures. The data suggest that the molecular signals leading to bone formation along the osteogenic fronts in fusing and nonfusing sutures are similar, raising the possibility that other factors, such as antagonists of osteogenesis, might have a role in maintaining suture patency.

Animals↗