Virginia M. Diewert

TARGETED DISRUPTION OF THE HUNTINGTON'S DISEASE GENE RESULTS IN EMBRYONIC LETHALITY AND BEHAVIORAL AND MORPHOLOGICAL CHANGES IN HETEROZYGOTES

Huntingtons disease (HD) is an incurable neuropsychiatric disease associated with CAG repeat expansion within a widely expressed gene that causes selective neuronal death. To understand its normal function, we have created a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homolog of the HD gene. Homozygotes die before embryonic day 8.5, initiate gastrulation, but do not proceed to the formation of somites or to organogenesis. Mice heterozygous for the Hdhex5 mutation display increased motor activity and cognitive deficits. Neuropathological assessment of two heterozygous mice shows significant neuronal loss in the subthalamic nucleus. These studies show that the HD gene is essential for postimplantation development and that it may play an important role in normal functioning of the basal ganglia.

Initiation and patterning of the snake dentition are dependent on Sonic Hedgehog signaling

Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation.

Recent Advances in Primary Palate and Midface Morphogenesis Research

During the sixth week of human development, the primary palate develops as facial prominences enlarge around the nasal pits to form the premaxillary region. Growth of craniofacial components changes facial morphology and affects the extent of contact between the facial prominences. Our recent studies have focused on developing methods to analyze growth of the primary palate and the craniofacial complex to define morphological phases of normal development and to determine alterations leading to cleft lip malformation. Analysis of human embryos in the Carnegie Embryology Collection and mouse embryos of cleft lip and noncleft strains showed that human and mouse embryos have similar phases of primary palate development: first, an epithelial seam, the nasal fin, forms; then a mesenchymal bridge develops through the nasal fin and enlarges rapidly. A robust mesenchymal bridge must form between the facial prominences before advancing midfacial growth patterns tend to separate the facial components as the medial nasal region narrows and elongates, the nasal pits narrow, and the primary choanae (posterior nares) open posterior to the primary palate. In mouse strains with cleft lip gene, maxillary growth, nasal fin formation, and mesenchymal replacement of the nasal fin were all delayed compared with noncleft strains of mice. Successful primary palate formation involves a sequence of local cellular events that are closely timed with spatial changes associated with craniofacial growth that must occur within a critical developmental period.

Phenotypic variability and craniofacial dysmorphology: increased shape variance in a mouse model for cleft lip

Cleft lip and palate (CL/P), as is true of many craniofacial malformations in humans, is etiologically complex and highly variable in expression. A/WySn mice are an intriguing model for human CL/P because they develop this dysmorphology with a variable expression pattern, incomplete penetrance and frequent unilateral expression on a homogeneous genetic background. The developmental basis for this variation in expression is unknown, but of great significance for understanding such expression patterns in humans. As a step towards this goal, this study used three‐dimensional geometric morphometric and novel high throughput morphometric techniques based on three‐dimensional computed microtomography of mouse embryos to analyze craniofacial shape variation during primary palate formation. Our analysis confirmed previous findings based on two‐dimensional analyses that the midface in A/WySn embryos, and the maxillary prominence in particular, is relatively reduced in size and appears to be developmentally delayed. In addition, we find that shape variance is increased in A/WySn embryos during primary palate formation compared to both C57BL/6J mice and the F1 crosses between these strains. If the reduction in midfacial growth caused by the Wnt9b hypomorphic mutation pushes A/WySn mice closer on average to the threshold for cleft lip formation, the elevated shape variance may explain why some, but not all, embryos develop the dysmorphology in a genetically homogeneous inbred line of mice.

Short‐faced mice and developmental interactions between the brain and the face

The length of the face represents an important axis of variation in mammals and especially in primates. Mice with mutations that produce variation along this axis present an opportunity to study the developmental factors that may underlie evolutionary change in facial length. The Crf4 mutant, obtained from the C57BL/6J (wt/wt) background by chemical mutagenesis by the Baylor Mouse Mutagenesis Resource, is reported to have a short‐faced phenotype. As an initial step towards developing this model, we performed 3D geometric morphometric comparisons of Crf4 mice to C57BL/6J wild‐type mice focusing on three stages of face development and morphology – embryonic (GD 9.5–12), neonatal, and adult. Morphometric analysis of adult Crf4 mutants revealed that in addition to a shortened face, these mice exhibit a significant reduction in brain size and basicranial length. These same features also differ at the neonatal stage. During embryonic face formation, only dimensions related to brain growth were smaller, whereas the Crf4 face actually appeared advanced relative to the wild‐type at the same somite stage. These results show that aspects of the Crf4 phenotype are evident as early as embryonic face formation. Based on our anatomical findings we hypothesize that the reduction in facial growth in Crf4 mice is a secondary consequence of reduction in the growth of the brain. If correct, the Crf4 mutant will be a useful model for studying the role of epigenetic interactions between the brain and face in the evolutionary developmental biology of the mammalian craniofacial complex as well as human craniofacial dysmorphology.

A cephalometric study of orofacial structures during secondary palate closure in the rat

Abstract The relationships between orofacial structures prior to and subsequent to secondary palate closure were evaluated in midsagittal sections of 30 foetal rats. Efforts were made to differentiate between the effects of shelf elevation and the effects of growth of individual orofacial components. In the day before palatal closure, the growth rates (percentage increase per hour) of the tongue and Meckels cartilage were greater than those of the mandible and the maxilla. The tongue, Meckels cartilage and the mandible grew in a downward and forward direction similar to the angulation of the incline formed by the nasal septum and the medial palatal process of the primary palate. Space for growth of the lower face was provided by maxillary growth, opening of the cranial base, and by extension of the head during the day before palatal closure. Palatal closure took place when the length of Meckels cartilage exceeded the length of the oronasal cavity measured to the medial palatal process. During palatal closure, the medial palatal process increased in infero-posterior prominence, the cross-section of Meckels cartilage and the anterior portion of the mandible rotated forward, counter-clockwise, head facing right, and the vertical distance of Meckels cartilage from the primary palate increased. Anterior tongue protrusion was present during the day of palatal closure but the degree of protrusion relative to the mandible did not change when the shelves elevated. In the day after palatal closure, mandibular growth became more horizontally directed and the rotation of Meckels cartilage and the anterior portion of the mandible continued. The rate of growth of the mandible was similar to its rate before palatal closure. Growth of the tongue, Meckels cartilage, the mandible and the medial palatal process appeared to be important factors in creating the environment conducive to secondary palatal closure. Rotation of the anterior portion of the mandible and Meckels cartilage beginning at the time of palatal closure may produce a change in position of the mid-portion of the mandible, allowing for tongue and palatine shelf exchange.

The fate of Meckel's cartilage chondrocytes in ocular culture.

Modulation of the chondrocyte phenotype was observed in an organ culture system using Meckels cartilage. First branchial arch cartilage was dissected from fetal rats of 16- and 17-day gestation. Perichondrium was mechanically removed, cartilage was split at the rostral process, and each half was grafted into the anterior chamber of an adult rat eye. The observed pattern of development in nonirradiated specimens was the following: hypertrophy of the rostral process and endochondral-type ossification, fibrous atrophy in the midsection, and mineralization of the malleus and incus. A change in matrix composition of the implanted cartilage was demonstrated with immunofluorescence staining for cartilage-specific proteoglycan (CSPG). After 15 days of culture, CSPG was found in the auricular process but not in the midsection or rostral process. In order to mark the implanted cells and follow their fate, cartilage was labeled in vitro with [3H]thymidine [3H]TdR). Immediately after labeling 20% of the chondrocytes contained [3H]TdR. After culturing for 5 days, 20% of the chondrocytes were still labeled and 10% of the osteogenic cells also contained radioactive label. The labeling index decreased in both cell types with increased duration of culture. Multinucleated clast-type cells did not contain label. Additional cartilages not labeled with [3H]TdR were exposed to between 20000 and 6000 rad of gamma irradiation before ocular implantation. Irradiated cartilage did not hypertrophy or form bone but a fibrous region developed in the midsection. Cells of the host animal were not induced to form bone around the irradiated cartilage. Our studies suggest that fully differentiated chondrocytes of Meckels cartilage have the capacity to become osteocytes, osteoblasts, and fibroblasts.

Comparative morphometrics of embryonic facial morphogenesis: Implications for cleft‐lip etiology

Cleft lip (CL) with or without cleft palate (CL[P]) has a complex etiology but is thought to be due to either genetic or environmentally induced disruptions of developmental processes affecting the shape and size of the facial prominences (medial nasal, lateral nasal, and maxilla). Recent advances in landmark‐based morphometrics enable a rigorous reanalysis of phenotypic shape variation associated with facial clefting. Here we use geometric morphometric (GM) tools to characterize embryonic shape variation in the midface and head of six strains of mice that are both cleft‐liable (A, A/WySn, CL/Fr) and normal (BALB/cBy, C57BL, CD1). Data were comprised of two‐dimensional landmarks taken from frontal and lateral photographs of embryos spanning the time period in which the facial prominences fuse (GD10‐12). Results indicate that A/‐ strain mice, and particularly A/WySn, have overall smaller midfaces compared to other strains. The A/WySn strain also has significant differences in facial shape related to retarded development. Overall, CL/Fr strain mice are normal‐sized, but tend to have undersized maxillary prominences that do not project anteriorly and have a small nasal contact area. These results suggest that the etiology of clefting differs in A/WySn and CL/Fr strains, with the former strain suffering disruptions to developmental processes affecting overall size (e.g., neural crest migration deficiencies and lower mitotic activity), while the latter strain has defects restricted to the shape and size of the maxilla. A combination of molecular experimentation and phenotypic analysis of shape is required to test these hypotheses further. Anat Rec, 290:123–139, 2007.

A quantitative coronal plane evaluation of craniofacial growth and spatial relations during secondary palate development in the rat.

Abstract Movement of the lateral palatine processes from the vertical to the horizontal plane occurs in a complex three-dimensional environment. Selected coronal sections of foetal Sprague-Dawley rat heads were magnified and measured. Mean group outlines were mapped and graphic composite illustrations made to demonstrate relative changes. During the two-day period about the time of palatal closure, although the height of the oro-nasal cavity doubled, there was little change in the width. As the height increased, the position of the tongue became lower relative to the palato-maxillary processes and a space developed above the tongue in the middle-palate region. During shelf elevation, the width of the oral cavity and the maxilla decreased, but the vertical dimension was unchanged.

Cell Proliferation and Expression of EGF, TGF-α, and EGF Receptor in the Developing Primary Palate

Growth factors such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-a) play an important role in cell proliferation during embryogenesis. The purposes of the study were to characterize the expression patterns of EGF and TGF-a and their receptor, EGF receptor (EGF-R), and to analyze regional patterns of cell proliferation during primary palate morphogenesis when facial primordia outgrow and fuse to form the premaxillary and upper lip regions. The expression of all molecules was studied with indirect immunohistochemistry with conventional and/or confocal microscopes in normal days 10 and 11 CD1 mice. 5-Bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA) were used as markers of cell proliferation. EGF, TGF-a, and EGF-R were found to have similar distribution patterns at all stages examined. In the anterior region of the face, the molecules were intensely localized at the tips and peripheral regions of the medial and lateral nasal prominences. Upon fusion of the facial prominences, all three molecules were present mainly at the fusion area and the tips and peripheral areas of the maxillary and nasal prominences. BrdU and PCNA were found to have distribution patterns similar to those of EGF, TGF-a, and EGF-R, with intense staining at the tips and peripheral regions of the facial prominences. These results show that EGF, TGF-a, and their receptor were expressed more intensely in regions of the developing primary palate where cell proliferation was most pronounced, and suggest that EGF, TGF-a, and EGF-R may play a role in cell proliferation during morphogenesis of the primary palate.