Beate Lanske, PhD
Associate Professor
Department of Developmental Biology
Office: REB 303
Telephone: 617-432-5748
Email: beate_lanske@hsdm.harvard.edu

Click here for a list of Dr. Lanske's available publications in PubMed

Skeletogenesis takes place by two different mechanisms. Craniofacial bones are formed by intramembranous ossification, in which precursor cells directly differentiate into osteoblasts. In contrast, the axial as well as the appendicular skeleton develop through endochondral ossification, in which a cartilaginous bone template is formed first. Many signaling molecules and their receptors are known to influence bone formation, including parathyroid hormone-related peptide (PTHrP), the parathyroid hormone (PTH)/PTHrP receptor (PPR), Indian hedgehog (Ihh) and many others. Today, the advanced techniques in gene targeting allow us to examine the physiological role of a gene in a specific cell/tissue or at a certain time in vivo.

Role of Indian hedgehog in bone versus cartilage
Hedgehog proteins are crucial in vertebrate and invertebrate species where they participate in embryonic patterning events. In mammals three members have been identified and are homologues to the Drosophila hedgehog gene ( Hh ). Sonic hedgehog (Shh) is expressed in several vertebrate organizing centers and is a key signal in coordinating the regulation of cell proliferation and cell-fate determination. Desert hedgehog (Dhh) is expressed in testes and has been shown to be crucial for their development and fertility. Indian hedgehog (Ihh) , which is expressed in the developing cartilage elements, together with parathyroid hormone-related peptide (PTHrP), is responsible for regulating the rate of chondrocyte differentiation.

Extensive efforts have been undertaken to elucidate the function of the Ihh gene in mice by homologous recombination. Unfortunately, the phenotype of mice lacking both copies of the Ihh gene is early lethal and, therefore, studies were very limited. For this reason we are generating cell type- or tissue-specific knockouts for the Ihh gene to determine the role of Ihh in cartilage versus bone. We are using the cre/loxP system to specifically delete Ihh in chondrocytes. Transgenic mice carrying the Cre recombinase will be under the control of the collagen type II promoter or collagen type X promoter. Furthermore, we are interested in examining the role of Ihh in postnatal chondrocytes. For this purpose, we are using a tamoxifen-inducible collagen type II-cre transgenic line. The ablation of the Ihh gene from the growth plate after endochondral bones have already formed, will provide us with new information about the important function of Ihh in bone remodeling. Histological, molecular and biochemical measurements will be performed to evaluate the role of the Ihh gene in the deleted cell type.

Phenotype of mice, in which the Ihh gene is ablated in all chondrocytes. Full body skeletons at E18.5 were stained by Alizarin Red/Alcian Blue. Long bones of mutants were only one-third the length of those in Ihh fl /Ihh fl littermates (A, B, C, D), compared to controls. Although the failure in bone growth is the most striking feature of the c ol2 a 1-Cre; Ihh d /Ihh d phenotype, differentiation is also abnormal. Calcification was proportionally more extensive in bones such as sternum (E), vertebrae (F), and the cartilaginous synchodrosis of the base of the skull (G, H). We also observed the failure in digit segmentation (I, J). Arrows point to the anomalies in endochondral bones.

Physiological role of FGF23 in vivo
Intracellular phosphate is an essential structural component of nucleic acids and other macromolecules, and it is crucial for energy metabolism and signal transduction. Extracellular phosphate is an integral component of bone and is thus important for formation, growth and turnover of bone. However, in contrast to the regulation of calcium homeostasis which is primarily achieved through PTH-dependent mechanisms, the factors involved in the regulation of phosphate remain poorly understood. Recent studies have led to the identification of several novel molecules that clearly have important, albeit incompletely defined, roles in the regulation of phosphate homeostasis. These molecules include PHEX, an endopeptidase which is mutated in patients with X-linked hypophosphatemia (XLH) and fibroblast growth factor 23 (FGF-23), which is mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR) and was found to be highly expressed in tumors that cause oncogenic osteomalacia.

We are currently investigating the physiological role of FGF23 in vivo . The further characterization of those knockout mice will give detailed insights into the regulation of phosphate in man.

Fig 1: (A) Schematic representation of the murine Fgf-23 gene and the corresponding knock-out/in targeting vector. Exons 1 to 3 are shown in black boxes. Vertical and horizontal shaded boxes represent the 5' and 3' flanking regions of the Fgf-23 gene, respectively, which were used for homologous recombination. The lacZ gene was cloned in frame with the initiator methionine of the Fgf-23 gene. The neomycin resistance (neo) gene is driven by the phosphoglycerate kinase-1 (PGK-1) promoter and contains a Sv40 polyA adenylation site. Probe A was used as external probe to hybridize genomic Southern blots ( Bgl II digest) shown in ( B) (wild-type = +/+, heterozygous = +/-, homozygous = -/-) . (C) represents lacZ staining of a wild-type (lower left) and a heterozygous Fgf-23 embryo ( Fgf-23 +/- , lower right) at E12.5. Arrows depict lacZ positive tissues (somites, liver and heart). Upper panels demonstrate lacZ staining in a wild-type (left) and Fgf-23 -/- (right) skull at 3 weeks. Blue staining represents expression of the Fgf-23 gene.

Fig 2: (A) Graphic display of total bone mineral content (BMC) of control and Fgf-23 -/- animals at 3, 6, and 11 weeks. Each value obtained for BMC was normalized to the body weight of the corresponding animal. Fgf-23 -/- mice show a statistical significant increase in total BMC when compared to control littermates (* = p<0.05; *** = p<0.0001). A statistical significant increase in total BMC was also observed among Fgf-23 -/- mice with time (### = p<0.0001). (B) X-ray autoradiography of hindlimbs from a wild-type (WT) and an Fgf-23 -/- mouse. Brackets depict length, and arrowhead thickness of femur in Fgf-23 -/- mouse. (C) Graph represents bone mineral density of hindlimbs measured by PIXImus analysis. Fgf-23 -/- mice show a statistical significant decrease in BMD at 3, 6 and 11 weeks when compared to controls (*** = p<0.0001). (D) illustrates BMD obtained from femoral shaft (left) and femoral methaphysis (right) of wild-type (white bar) and Fgf-23 -/- animals (dark bar) by QCT at 4 weeks of age. Fgf-23 -/- mice show a statistical significant decrease in BMD (** = <0.001).

Fig 3: Three-µm-thick undecalcified sections from 4 week-old wild-type (upper panels) and Fgf-23 -/- (lower panels) bones (cortical bone, growth plate, ribs, vertebra) were stained with von Kossa/McNeal (magnification x20, x10). Black staining represents mineralization. More mineral deposition is found in the area below the growth plate (methaphysis), ribs and in vertebra. In contrast, areas of unmineralized osteoid (light blue) are found in cortical bone.