Robert Maxson

A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis

Craniosynostosis, the premature fusion of calvarial sutures, is a common developmental anomaly that causes abnormal skull shape. The locus for one autosomal dominant form of craniosynostosis has been mapped to chromosome 5qter. The human MSX2 gene localizes to chromosome 5, and a polymorphic marker in the MSX2 intron segregates in a kindred with the disorder with no recombination. Moreover, a histidine substitutes for a highly conserved proline at position 7 of the MSX2 homeodomain exclusively in affected members. In the mouse, transcripts of the Msx2 gene are localized to calvarial sutures. These results provide compelling evidence that the mutation causes this craniosynostosis syndrome.

Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

Recent advances in craniofacial morphogenesis

Craniofacial malformations are involved in three fourths of all congenital birth defects in humans, affecting the development of head, face, or neck. Tremendous progress in the study of craniofacial development has been made that places this field at the forefront of biomedical research. A concerted effort among evolutionary and developmental biologists, human geneticists, and tissue engineers has revealed important information on the molecular mechanisms that are crucial for the patterning and formation of craniofacial structures. Here, we highlight recent advances in our understanding of evo–devo as it relates to craniofacial morphogenesis, fate determination of cranial neural crest cells, and specific signaling pathways in regulating tissue–tissue interactions during patterning of craniofacial apparatus and the morphogenesis of tooth, mandible, and palate. Together, these findings will be beneficial for the understanding, treatment, and prevention of human congenital malformations and establish the foundation for craniofacial tissue regeneration. Developmental Dynamics 235:2353–2375, 2006.

Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development

Development of the vertebrate limb bud depends on reciprocal interactions between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Sonic hedgehog (SHH) and fibroblast growth factors (FGFs) are key signalling molecules produced in the ZPA and AER, respectively. Experiments in chicks suggested that SHH expression in the ZPA is maintained by FGF4 expression in the AER, and vice versa, providing a molecular mechanism for coordinating the activities of these two signalling centres. This SHH/FGF4 feedback loop model is supported by genetic evidence showing that Fgf4 expression is not maintained in Shh−/− mouse limbs. We report here that Shh expression is maintained and limb formation is normal when Fgf4 is inactivated in mouse limbs, thus contradicting the model. We also found that maintenance of Fgf9 and Fgf17 expression is dependent on Shh, whereas Fgf8 expression is not. We discuss a model in which no individual Fgf expressed in the AER (AER–Fgf) is solely necessary to maintain Shh expression, but, instead, the combined activities of two or more AER–Fgfs function in a positive feedback loop with Shh to control limb development.

Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation

To facilitate the elucidation of the genetic events that may play an important role in the development or tumorigenesis of the prostate gland, we have generated a transgenic mouse line with prostate-specific expression of Cre recombinase. This line, named PB-Cre4, carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter. Based on RT-PCR detection of Cre mRNA in PB-Cre4 mice or Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice, it is conclusively demonstrated that Cre expression is post-natal and prostatic epithelium-specific. Although the Cre recombination is detected in all lobes of the mouse prostate, there is a significant difference in expression levels between the lobes, being highest in the lateral lobe, followed by the ventral, and then the dorsal and anterior lobes. Besides the prostate gland, no other tissues of the adult PB-Cre4 mice demonstrate significant Cre expression, except for a few scattered areas in the gonads and the stroma of the seminal vesicle. By crossing the PB-Cre4 animals with floxed RXRalpha allelic mice, we demonstrate that mice, whose conventional knockout of this gene is lethal in embryogenesis, could be propagated with selective inactivation of RXRalpha in the prostate. Taken together, the results show that the PB-Cre4 mice have high levels of Cre expression and a high penetrance in the prostatic epithelium. The PB-Cre4 mice will be a useful resource for genetic-based studies on prostate development and prostatic disease.

Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification.

The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non-syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34–q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation.

Targeted Disruption in Murine Cells Reveals Variable Requirement for Smad4 in Transforming Growth Factor β-related Signaling

The tumor suppressor gene Smad4 has been proposed to be a common mediator of transforming growth factor β (TGFβ)-related signaling pathways. We investigated the role ofSmad4 in TGFβ-related pathways by targeted disruption of its locus in murine cell lines. TGFβ responses, including growth arrest, induction of the endogenous PAI-1 gene, and other extracellular matrix components, were normal in Smad4-deficient fibroblasts. Assembly of a TGFβ-induced DNA-binding complex on one of two regulatory regions in the human plasminogen activator inhibitor (PAI)-1 promoter did not require Smad4 but was, instead, dependent on a TFE-3 binding site. In contrast, Smad4 was required for activation of the Xenopus Mix.2 promoter in response to TGFβ/activin. Smad4 was also involved in the regulation of the Msx homeobox protein family members in response to bone morphogenetic protein (BMP). Interestingly, the expression of the endogenous Msx-2 was reduced, whereas that of Msx-3 was activated in differentiating Smad4 −/− ES cells relative to wild-type cells. Moreover, reporter assays of the Msx-2promoter revealed an absolute requirement for Smad4 in fibroblasts and ES cells for activation. Our results indicate that Smad4 is dispensable for critical TGFβ-induced responses but is required for others in murine fibroblasts. We have identified transcriptional targets for Smad4 in the BMP signaling pathway, which may contribute to the genetic defect observed in theSmad4-deficient embryos.

Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development†

The knowledge concerning fetal hepatic stellate cells (HSCs) is scarce, and their cell lineage and functions are largely unknown. The current study isolated fetal liver mesenchymal cells from a mouse expressing β‐galactosidase under the control of Msx2 promoter by fluorescence‐activated cell sorting (FACS) and surveyed marker genes by microarray analysis. Based on the location and immunostaining with conventional and newly disclosed markers, we have identified three distinct populations of fetal liver mesenchymal cells expressing both desmin and p75 neurotrophin receptor (p75NTR): HSCs in the liver parenchyma; perivascular mesenchymal cells expressing α‐smooth muscle actin (α‐SMA); and submesothelial cells associated with the basal lamina beneath mesothelial cells and expressing activated leukocyte cell adhesion molecule (ALCAM) and platelet‐derived growth factor receptor α. A transitional cell type from the submesothelial cell phenotype to fetal HSCs was also identified near the liver surface. Mesothelial cells expressed podoplanin and ALCAM. Ki‐67 staining showed that proliferative activity of the submesothelial cells is higher than that of mesothelial cells and transitional cells. Using anti‐ALCAM antibodies, submesothelial and mesothelial cells were isolated by FACS. The ALCAM+ cells expressed hepatocyte growth factor and pleiotrophin. In culture, the ALCAM+ cells rapidly acquired myofibroblastic morphology and α‐SMA expression. The ALCAM+ cells formed intracellular lipid droplets when embedded in collagen gel and treated with retinol, suggesting the potential for ALCAM+ cells to differentiate to HSCs. Finally, we demonstrated that fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are all derived from mesoderm by using MesP1‐Cre and ROSA26 reporter mice. Conclusion: Fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are mesodermal in origin, and ALCAM+ submesothelial cells may be a precursor for HSCs in developing liver. (HEPATOLOGY 2009.)

Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault.

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest.

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2α, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.