Mark V. Reedy

Neural crest development: the interplay between morphogenesis and cell differentiation.

The final pattern of tissues established during embryogenesis reflects the outcome of two developmental processes: differentiation and morphogenesis. Avian neural crest cells are an excellent system in which to study this interaction. In the first phase of neural crest cell migration, neural crest cells separate from the neural epithelium via an epithelial-mesenchymal transformation. We present three models to account for this process: (1) separation by asymmetric mitosis, (2) separation by generating tractional force in order to rupture cell adhesions and (3) loss of expression or function of cell-cell adhesion molecules that keep the presumptive neural crest cells tethered to the neural epithelium. Evidence is presented that the segregation of the neural crest lineage apart from the neural epithelium is caused by the epithelial-mesenchymal transformation. Once they have detached from the neural tube, neural crest cells take two pathways in the trunk of the chick embryo: (1) the ventral path between the neural tube and somite, where neural crest cells give rise to neurons and glial cells of the peripheral nervous systems, and (2) the dorsolateral path between the ectoderm and dermamyotome of the somite, where they differentiate into pigment cells of the skin. We present data to suggest that the migration and differentiation along the ventral path is controlled primarily by environmental cues, which we refer to as the environment-directed model of neural crest morphogenesis. Conversely, only melanoblasts can migrate into the dorsolateral space, and the ability to invade that path is dependent upon their early specification as melanoblasts. We call this the phenotype-directed model for neural crest cell migration and suggest that this latter model for the positioning of neural crest derivatives in the embryo may be more common than previously suspected. These observations invite a re-examination of patterning of other crest derivates, which previously were believed to be controlled by environmental cues.

Mutant myocilin nonsecretion in vivo is not sufficient to cause glaucoma.

ABSTRACT Glaucoma is a leading cause of blindness, affecting over 70 million people worldwide. Vision loss is the result of death of the retinal ganglion cells. The best-known risk factor for glaucoma is an elevated intraocular pressure (IOP); however, factors leading to IOP elevation are poorly understood. Mutations in the MYOC gene are an important cause of open-angle glaucoma. Over 70 MYOC mutations have been identified, and they lead to approximately 5% of all primary open-angle glaucoma cases. Nevertheless, the pathogenic mechanisms by which these mutations elevate IOP are presently unclear. Data suggest that a dominant interfering effect of misfolded mutant MYOC molecules may be pathogenic. To test this hypothesis, we have generated mice carrying a mutant allele of Myoc that is analogous to a human mutation that leads to aggressive glaucoma in patients. We show that mutant MYOC is not secreted into the aqueous humor. Instead of being secreted, mutant MYOC accumulates within the iridocorneal angle of the eye, consistent with the behavior of abnormally folded protein. Surprisingly, the accumulated mutant protein does not activate the unfolded protein response and lead to elevated intraocular pressure or glaucoma in aged mice of different strains. These data suggest that production, apparent misfolding, and nonsecretion of mutant MYOC are not, by themselves, sufficient to cause glaucoma in vivo.

Specification and migration of melanoblasts at the vagal level and in hyperpigmented silkie chickens

The final pattern of neural crest derivatives used to be believed to be the result of unspecified neural crest cells haphazardly entering migratory paths and then receiving cues unique to that path that direct their differentiation. An alternative model, which we have coined the phenotype‐directed model, is that neural crest cells are fate‐specified first and then select a migratory pathway based on their developmental specification. Support for this model comes from recent studies demonstrating that, at the thoracic level, neural crest cells are specified as melanocyte precursors (melanoblasts) prior to entering the dorsolateral path, and that only melanoblasts have the ability to migrate dorsolaterally. Here we examine two examples of melanocyte patterning in birds that apparently contradict this model.The first is neural crest at the vagal level, where early crest cells migrate dorsolaterally and enter the branchial arches. Despite the fact that these cells migrate dorsolaterally (suggesting that they are melanoblasts), branchial arch‐derived neural crest cells fail to differentiate as melanocytes in vitro. These observations suggest that the branchial arch environment may not support the survival or differentiation of melanogenic neural crest cells. The second example is the hyperpigmented Silkie chickens, which exhibit extensive internal pigmentation. The Silkie defect has been linked to a difference in the neural crest migratory environment that potentially causes (or allows) unspecified neural crest cells to undergo melanogenesis in the ventral path. In both of these situations, it appears that the final distribution of pigment cells is controlled by environmental factors, which would contradict the phenotype‐directed model. Here we show that the final pattern of melanocytes at the vagal level and in Silkie chickens reflects the migratory behavior of lineage‐specified melanoblasts, as predicted by the phenotype‐directed model. At the vagal level, the early, dorsolaterally migrating crest cells that colonize the branchial arches are not melanoblasts and are biased against melanogenesis in vitro. Melanoblasts are not specified until later, just prior to a second wave of dorsolateral migration, and although these cells migrate dorsolaterally they do not invade the branchial arches. In Silkie embryos, melanoblasts are specified late and only invade the dorsolateral path after they have been specified. Unlike quail and White leghorn melanoblasts, however, Silkie melanoblasts also migrate ventrally, but again only after they are specified. Dev. Dyn. 1998;213:476–485.

Homocysteine Inhibits Cardiac Neural Crest Cell Formation and Morphogenesis In Vivo

Elevated homocysteine increases the risk of neurocristopathies. Here, we determined whether elevating homocysteine altered the proliferation or number of chick neural crest cells that form between the midotic and third somite in vivo. Homocysteine increased the number of neural tube cells but decreased neural crest cell number. However, the sum total of cells was not different from controls. In controls, the 5‐bromo‐2′‐deoxyuridine‐labeling index was higher in newly formed neural crest cells than in their progenitors, paralleling reports showing these progenitors must pass the restriction point before undergoing epithelial–mesenchymal transition. Homocysteine decreased the labeling index of newly formed neural crest cells, suggesting that it inhibited cell cycle progression of neural crest progenitors or the S‐phase entry of newly formed neural crest cells. Homocysteine also inhibited neural crest dispersal and decreased the distance they migrated from the neural tube. These results show neural crest morphogenesis is directly altered by elevated homocysteine in vivo. Developmental Dynamics 229:63–73, 2004.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) expression during cardiac neural crest cell migration and its role in proMMP-2 activation

Matrix metalloproteinases (MMPs) are important mediators of neural crest (NC) cell migration. Here, we examine the distribution of tissue inhibitor of metalloproteinase (TIMP) ‐2 and TIMP‐3 and test whether manipulating TIMP levels alters chicken cardiac NC cell migration. TIMP‐2 mRNA is expressed at stage 11 in the neural epithelium and only in migrating cardiac NC cells. TIMP‐3 mRNA is expressed only in the notochord at stage 8 and later in the outflow tract myocardium. Exogenous TIMP‐2 increases NC motility in vitro at low concentrations but has no effect when concentrations are increased. In vitro, NC cells express membrane type‐1 matrix metalloproteinase (MT1‐MMP) and TIMP‐2 and they secrete and activate proMMP‐2. Antisense TIMP‐2 oligonucleotides block proMMP‐2 activation, decrease NC cell migration from explants, and perturb NC morphogenesis in ovo. Because TIMP‐2 is required for activation of proMMP‐2 by MT1‐MMP, this finding suggests TIMP‐2 expression by cardiac NC cells initiates proMMP‐2 activation important for their migration. Developmental Dynamics 231:709–719, 2004.

The expression patterns of c-kit and Sl in chicken embryos suggest unexpected roles for these genes in somite and limb development

We have used whole-mount in situ hybridization to investigate the patterns of c-kit and Sl expression in stage 11-22 chicken embryos. Our analysis shows that c-kit and Sl are expressed quite differently in chicken embryos compared to the reported expression patterns of these genes in embryos of other taxa. Most notably, chicken c-kit is expressed in primordial germ cells as well as in the developing somite, the apical ectodermal ridge, and in the early foregut endoderm. Sl is expressed in the lateral and intermediate mesoderm and in extraembryonic membranes. These data suggest that chicken c-kit and Sl may play novel and unexpected roles in somitogenesis, limb development, and foregut development in avian embryos.

MT2-MMP Expression During Early Avian Morphogenesis

Membrane‐type 2 matrix metalloproteinase (MT2‐MMP; also called MMP15) is a membrane‐bound protease that degrades extracellular matrix and activates proMMPs such as proMMP‐2. MMP‐2 expression in avian embryos is well documented, but it is not clear how proMMP‐2 is activated during avian embryogenesis. Herein, we report that MT2‐MMP mRNA is expressed in several tissues including the neural folds and epidermal ectoderm, intermediate mesoderm, pharyngeal arches, limb buds, and dermis. Several, but not all, of these tissues are known to express MMP‐2. These observations suggest MT2‐MMP may play a role during embryonic development not only through its own proteolytic activity but also by activating proMMP‐2. Anat Rec, 2013.

Algal Morphogenesis: How Volvox Turns Itself Inside-Out

Abstract During its development, the multicellular green alga Volvox undergoes inversion, in which spherical embryos turn their multicellular sheet completely inside out. A mutant analysis has revealed that a novel kinesin motor protein is essential for completing this process.