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The Hedgehog Family of Paracrine Factors

The hedgehog family of paracrine factors is one of the most important group of signaling molecules. In Drosophila, there is only one type of Hedgehog (the hh gene), and it functions in specifying the segmental polarity of the blastoderm (see p. 282-285 in the textbook) and the specification of cell fate in imaginal discs (such as those of the leg, wing, and eye) during metamorphosis (p. 560).

Sonic Hedgehog

In vertebrates, there are several hedeghog genes. Sonic hedgehog plays numerous important developmental roles including:

In fish, there are two "extra" Hedgehog genes, echidna hedgehog and tiggywinkle hedgehog. Both of these genes are expressed along the midline of the fish embryo and may act with Sonic hedgehog in establishing the fates of the midline cells.

Indian Hedgehog

Endodermal differentiation

Indian hedgehog (Ihh) seems to play at least two roles in vertebrate development. The first role of Ihh appears to be in endoderm development. In the earliest stages of endoderm formation, Ihh appears to be critical for the differentiation of the visceral endoderm (Maye et al., 2000). Later, Ihh appears to regulate the proliferation and differentiation of the gut epithelial stem cell (Ramalho-Santos, et al., 2000). It appears that in the development of the gut, both Sonic hedgehog and Indian hedgehog are needed.

Postnatal bone growth

The bones of human infants grow at an astonishing rate. The long bones of the average infant lengthen by 50% their original length during the first year after birth. By age 3 this has slowed to only 7%. (Otherwise, our kindergartens would be full of seven-footers.) New research (Lanske et al., 1996; Vortkamp et al., 1996) has implicated two proteins—Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP)—in putting on the brakes to chondrocyte differentiation. This research was the result of a collaboration between a group of developmental biologists (who figured out that Ihh was important but couldn't work out its mechanism) and two groups of endocrinologists (who discovered PTHrP and its receptor but who did not understand how they were regulated) (Roush, 1996).

These investigators found that prehypertrophic chondrocytes, those cells making the transition from proliferating chondrocytes to hypertrophic chondrocytes, have two distinctive properties. First, they secrete Ihh. Second, they have the receptor for the PTHrP protein. They showed that these cells begin to secrete Ihh as they enter into the hypertrophic phase of growth, and that addition of extra Ihh slowed the rate of differentiation. Ihh acts by inducing Gli and patched proteins in the periosteum adjacent to these prehypertrophic chondrocytes. In some way, this signal is communicated to the periarticular periosteum at the end of the cartilagenous model. Here, PTHrP is made, and it prevents other prehypertrophic chondrocytes from entering into this pathway of differentiation. If mice are engineered to lack either PTHrP or its receptor, ossification occurs prematurely. So there is a negative feedback loop: Ihh induces patched and Gli proteins. These proteins induce the synthesis of PTHrP, and PTHrP blocks the recruitment of more chondrocytes into this pathway.

Recent research shows that Indian Hedgehog (Ihh) coordinates endochondral bone growth and morphogenesis by at least two mechanisms, and only one of them is dependent on PTHrP. Targeted deletion of Ihh in mice results in short-limbed dwarfism, with decreased chondrocyte proliferation and extensive hypertrophy. This syndrome is also characteristic of loss-of-function mutations of PTHrP and its receptor. Activation of Ihh signaling upregulates PTHrP at the articular surface and prevents chondrocyte hypertrophy in wild-type but not PTHrP null explants. This implies that Ihh acts through PTHrP. Karp and his colleagues (2000) constructed mice with various combinations of an Ihh null mutation (Ihh-/-), a PTHrP null mutation (PTHrP-/-), and a constitutively active PTHrP/Parathyroid hormone Receptor expressed under the control of the Collagen II promoter (PTHrPR*). PTHrPR* rescues PTHrP-/- embryos, demonstrating that an active PTHrP receptor can completely compensate for lack of PTHrP signaling. At embryonic day 18.5, limb skeletons of Ihh, PTHrP compound mutants were identical to Ihh single mutants, suggesting Ihh is necessary for PTHrP function. Expression of PTHrPR* in chondrocytes of Ihh knockout mice prevented premature chondrocyte hypertrophy, showing that PtHrP signaling could bypass this genetic block. However, the expression of activated PTHrPR did not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation. These experiments demonstrate that the molecular mechanism that prevents chondrocyte hypertrophy is distinct from that which drives chondrocyte proliferation. Ihh positively regulates PTHrP, which is sufficient to prevent chondrocyte hypertrophy and maintain a normal domain of cells competent to undergo proliferation. In contrast, Ihh is necessary for normal chondrocyte proliferation in a pathway that use signaling. Thus, Indian Hedgehog is a major coordinator of skeletal growth and morphogenesis, and it acts in several ways.

Ihh may also play a role in the healing of fractures in the long bones (Muramaki and Noda, 2000).

Desert Hedgehog

Desert Hedgehog (Dhh) appears to also have two major roles in development. One is in the male germ line. The other involves the differentiation of glial cells.

Dhh in the male germ line

Sertoli cells are thought to play a central role in male-specific cell interactions. The male primordial germ cells reside in the Sertoli cells, and they remain there as they differentiate. The molecular bases of Sertoli cell function on spermatogenesis is not known, but one clue is that Dhh encodes a signaling molecule expressed in the testis, but not in the ovary. Therefore, it may play a role in the regulation of spermatogenesis. Bitgood et al. (1996) demonstrated that Dhh expression is initiated in Sertoli cell precursors shortly after they express Sry, and that this expression persists in the testis into the adult. Male mice homozygous for a knock-out allele of Dhh are sterile, owing to the complete absence of mature sperm, while female mice homozygous for this loss-of-function Dhh mutation show no obvious morphological changes or infertility. Dhh appears to regulates both early and late stages of spermatogenesis.

Dhh in the nervous system

In the nervous system, Schwann cell-derived Dhh signals the connective tissue surrounding the peripheral neurons to form a collagenous sheath around the peripheral nerves. The mRNAs for Dhh are expressed in the Schwann cells, and the receptor for hedgehogs, the patched (ptc) protein, is expressed in the fibroblasts surrounding these nerves. In Dhh-/- mice, epineurial collagen is reduced, while the basal lamina surrounding the nerve is thin and disorganized.

Literature Cited

Bitgood, M. J., Shen, L. and McMahon, A. P. 1996. Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr. Biol. 6 :298-304.

Currie, P.D. and Ingham, P.W. Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature. 382: 452-455.

Karp, S. J. , Schipani, E., St-Jacques, B., Hunzelman, J., Kronenberg, H. and McMahon, A. P. 2000. Indian Hedgehog coordinates endochondral bone growth and morphogenesis via Parathyroid Hormone related-Protein-dependent-and-independent pathways. Development 127: 543-548.

Lanske, B. and several others. 1996. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273: 663-666.

Maye P., Becker S., Kasameyer, E., Byrd, N. and Grabel, L. 2000. Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies. Mech Dev. 2000 94:117-132.

Murakami, S. and Noda, M. 2000. Expression of Indian hedgehog during fracture healing in adult rat femora. Calcif Tissue Int. 66: 272-276.

Parmantier, E., Lynn, B., Lawson, D., Turmaine, M., Namini, S. S., Chakrabarti, L., McMahon, A. P., Jessen, K. R., and Mirsky, R. 1999. Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths. Neuron 23: 713- 724.

Ramalho-Santos, M., Melton, D. A., and McMahon, A. P. 2000. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127: 2763-2772.

Roush, W. 1996. Putting the brakes on bone growth. Science 273: 579.

Vortkamp, A., Lee, K., Lanske, B., Segre, G. V., Kronenberg, H. M., and Tabin, C. J. 1996. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273: 613-622.

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