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Dispatch R783

Axis development: The mouse becomes a dachshund Jacqueline M. Gad and Patrick P.L. Tam

Targeted deletion of the for GDF11, a novel growth [7]. Other members of the GDF family have dif- member of the TGFβ family, has been found to cause an ferent functions during development (Table 1). For increase in the number of thoracic and lumbar vertebrae example, GDF5 has a critical role in chondrogenesis, in the mouse. This is the first hint that a secreted factor whereas GDF9 is required for the formation of ovarian may influence the specification of segment identity. follicles. Like other GDFs, GDF11 has seven conserved cysteine residues, and it shows 90% sequence identity Address: Embryology Unit, Children’s Medical Research Institute, University of Sydney, Australia. across this region with GDF8 [5,6]. During normal E-mail: [email protected]; [email protected] embryonic development, Gdf11 is expressed in the somitic precursors; its expression is also seen in the maxillary Current Biology 1999, 9:R783–R786 process, mandibular and hyoid arches, the limbs and 0960-9822/99/$ – see front matter parts of the brain. © 1999 Elsevier Science Ltd. All rights reserved. Consistent with these observations, mice homozygous for a Vertebrates are segmented creatures and some of the targeted deletion of Gdf11 were found to show palatal mal- most interesting questions about their development formations and defects of vertebral morphogenesis [4]. Most concern the mechanisms by which their body axis is strikingly, the mice were found to have an elongated trunk divided up and the resulting units specified with distinct and a much reduced or absent tail. This elongation was pri- positional identities. The segmentation is perhaps most marily the result of an increase in the number of thoracic evident in the vertebrae themselves, which develop along the anterior–posterior body axis of the mouse embryo Table 1 from paraxial mesodermal segments derived from tissues originating from the primitive streak and tail bud. Our Expression patterns and functions of GDFs. understanding of the mechanisms of axial development is Expression Function steadily increasing, though we are still far from under- standing how the precision and consistency of the GDF1 Cortex and hippocampus Unknown number and morphology of vertebrae are maintained in GDF3 Adult bone marrow, spleen, Unknown different species. thymus and GDF5 Neurons and bone Chondrogenesis, joint The Hox have been shown to have important roles morphogenesis, in specifying segmental characteristics along the ante- induces tendon formation, rior–posterior axis; some signalling systems have also extends the life span of neurons, induces been implicated in axial development, apparently by angiogenesis modulating Hox gene expression [1–3]. This field now has a new player [4] — growth/differentiation factor 11 GDF6 Muscle and bone Induces tendon formation (GDF11), a member of the transforming growth factor β GDF7 Neural tissues Survival and differentiation (TGFβ) family of secreted signalling molecules. Mice of neurons with targeted deletions of the Gdf11 gene have been GDF8 Muscle Negative regulation of found to display extensive morphological changes skeletal muscle growth throughout the axial skeleton, resulting in the formation GDF9 Ovary Ovarian follicular of additional thoracic and lumbar vertebrae at the development expense of caudal segments [4]. GDF11 may be the first GDF10 Embryo: skeletal tissues, Unknown secreted signalling molecule shown to play a role in the cerebellum. Adult: craniofacial specification of the segmental characteristics of vertebrae, tissues, vertebral column, and thus offers a functional link between cell–cell inter- brain and actions and axial patterning. GDF11 Primitive streak, tail bud Specification of mesenchyme vertebral identity Gdf11 was isolated as a gene coding for a novel member GDF15 (MIC1) Epithelial cells Unknown of the bone morphogenetic (BMP) subgroup of and macrophages the TGFβ family [5,6]; later, it was found to be more References: GDF1 [14]; GDF3 [15]; GDF5 [16–21]; GDF6 [19]; related to Gdf8, an essential gene that encodes a protein GDF7 [22]; GDF8 [23]; GDF9 [24,25]; GDF10 [26,27]; GDF11 also known as , a negative regulator of muscle [4–6]; GDF15 [28]. R784 Current Biology Vol 9 No 20

Figure 1 As a consequence of the extended thoracic and lumbar regions of the mutant mice, their hindlimbs are located Wild type Gdf11-/- more posteriorly along the body axis than normal — they seem superficially to be the mouse equivalent of the dachshund breed of dog, which has a proportionally longer trunk than other canines. In heterozygous Gdf11+/– mice, the skeletal defects are much milder, suggesting Cervical 7 that Gdf11 acts in a dosage-dependent manner. It is 7 * * important to note that deformities were not observed in * the cervical vertebrae. This is consistent with the notion that the first six to eight somites — the ones that form the occipital bone and upper cervical vertebrae — are already specified before Gdf11 expression initiates in the somitic precursors of the primitive streak at late gastrulation.

Thoracic 13 The mutant phenotype strongly suggests that, when the 18 vertebral column develops in the absence of GDF11, a posteriorising signal is missing, allowing some somites to acquire a more anterior morphology than usual. Does GDF11 act as a negative regulator of anterior vertebral morphogenesis, in a similar manner to the suppression of muscle growth by the closely related GDF8? In the homozygous Gdf11 mutant, an apparently normal number Lumbar of somites is formed by late organogenesis. This suggests 6 that, although Gdf11 is expressed in the tissue compart- ments — presomitic mesoderm, primitive streak and tail bud — where the recruitment and segmentation of the paraxial mesoderm take place throughout the whole 7–9 period of axis formation, the loss of its function may have Sacral and caudal little impact on the initial phases of somitogenesis. The ~34 homeotic changes of the vertebrae point, rather, to GDF11 having an essential role in the specification of segmental identity by regulating the differentiation of the sclerotomal tissues. This raises the possibility that the }Fused loss of GDF11 function has an effect on the differentia- tion of the prevertebral tissues at a stage later than the Current Biology onset of transcriptional activity, that is, when cells are Homeotic changes of the axial skeleton in Gdf11 mutant mice. The about to undergo vertebral morphogenesis. normal seven cervical vertebrae are formed, but both the sixth and seventh segments are decorated with the anterior tubercle McPherron et al. [4] suggest that GDF11 is a secreted (asterisks), characteristic of the normal sixth cervical vertebra. There are additional rib-bearing thoracic vertebrae, with an extra four or five molecule that affects cells that are more anterior to ribs fused to the posterior aspect of the sternum. The vertebrae where its mRNA is produced. So, if the protein is generally display more anterior characteristics. For example, the indeed secreted, what are the target cells of GDF11 and transitional articular and spinous processes (arrows) normally found what is the immediate biological effect on the respond- on the tenth thoracic vertebra are now situated on the thirteenth thoracic vertebra. One to three extra lumbar vertebrae are formed, ing cells? The answers to these questions hinge on the but there are fewer sacral and caudal vertebrae which are often localization of the GDF11 protein and its receptors in fused and malformed. The overall effect is the lengthening of the the somitic tissues of the embryo. Interestingly, muta- thoracic and lumbar part of the backbone at the expense of the tion of one of the activin receptors, the type IIB recep- pelvis and the tail. tor [8], results in mutant mice with skeletal defects similar to those of the Gdf11 mutant. Furthermore, type IIA and type IIB activin receptors are known to bind segments, from the usual 13 to 17 or 18; furthermore, the TGFβ molecules, such as BMP7, and therefore may also vertebrae appeared to have acquired more anterior charac- act as GDF11 receptors — but if they do, they may not teristics. This mutant phenotype was interpreted as being be the sole GDF11 receptors, given the more severe due to a global homeotic transformation of the vertebrae to skeletal defects of Gdf11 mutants, compared to the more anterior developmental fates (Figure 1) [4]. mutants. Dispatch R785

How is GDF11 integrated into the molecular mechanisms important to establish the relative timing and cellular of anterior-posterior patterning of the axial skeleton? In specificity of their actions. It will also be important to the Gdf11 mutant, the expression domain of four Hox determine the intracellular pathway that presumably links genes is altered: the Hoxc6 and Hoxc8 expression domains GDF11 to transcription factors inside the cell. are extended to include more posterior prevertebrae, whereas those of Hoxc10 and Hoxc11 are shifted posteri- Acknowledgements orly relative to the position of the limb buds. These We thank Peter Rowe, Timothy Cox and Bruce Davidson for helpful discus- sions, and Meredith O’Rourke for reading the manuscript. changes are consistent with the adoption of thoracic and lumbar characteristics by the trunk vertebrae. A recent References study of python embryos [9] has shown that the persistent 1. Pownall ME, Tucker AS, Slack JM, Isaacs HV: eFGF, Xcad3 and Hox expression of Hoxc6, Hoxc8 and Hoxb5 is associated with genes form a molecular pathway that establishes the anteroposterior axis in Xenopus. Development 1996, the formation of a vertebral column almost exclusively of 122:3881-3892. thoracic identity in the trunk of the snake. It would be 2. Partanen J, Schwartz L, Rossant J: Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a intriguing to determine the activity of Gdf11 in the snake function for Fgfr1 in anteroposterior patterning of mouse paraxial mesoderm. If indeed Gdf11 is required for the embryos. Genes Dev 1998, 12:2332-2344. acquisition of a posterior segment identity, one would 3. Oh SP, Li E: The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry expect that the Gdf11 equivalent is functionally inert or in the mouse. Genes Dev 1997, 11:1812-1826. even absent in the snake. 4. McPherron AC, Lawler AM, Lee S-J: Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11. Nat Genet 1999, 22:260-264. From comparative studies, it appears that the mammalian 5. Gamer LW, Wolfman NM, Celeste AJ, Hattersley G, Hewick R, Rosen Hox genes have evolved from a single ancestral cluster to V: A novel BMP expressed in developing mouse limb, , four gene clusters [10]. These genes encode transcription and tail bud is a potent mesoderm inducer in Xenopus embryos. Dev Biol 1999, 208:222-232. factors with significantly overlapping expression domains 6. Nakashima M, Toyono T, Akamine A, Joyner A: Expression of and functions, providing a system with considerable func- growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis. Mech tional redundancy. Hox genes are recognised to be impor- Dev 1999, 80:185-189. tant in developmental processes, particularly in the 7. McPherron AC, Lawler AM, Lee SJ: Regulation of skeletal muscle patterning of embryonic organs/structures including, axial mass in mice by a new TGF-beta superfamily member. Nature 1997, 387:83-90. skeleton and limbs [10,11]. But the functional role of Hox 8. Oh SP, Li E: The signaling pathway mediated by the type IIB genes during vertebral development is still not clearly activin receptor controls axial patterning and lateral asymmetry understood. In the mouse, gain-of-function perturbations in the mouse. Genes Dev 1997, 11:1812-1826. 9. Cohn MJ, Tickle C: Developmental basis of limblessness and of Hoxc6 and Hoxc8 cause anterior transformation of the axial pattering in snakes. Nature 1999, 399:474-479. axial skeleton, such that the segmentally posterior verte- 10. Krumlauf R: Hox genes in vertebrate development. Cell 1994, 78:191-201. brae acquire a more anterior phenotype, similar to the 11. Mark M, Rijli FM, Chambon P: Homeobox genes in embryogenesis Gdf11 mutant. But loss-of-function mutation of Hoxc8 and pathogenesis. Pediatric Res 1997, 42:421-429. causes a similar phenotype, emphasizing the complexity 12. Ault KT, Xu RH, Kung HF, Jamrich M: The homeobox gene PV.1 mediates specification of the prospective neural ectoderm in of the system. Xenopus embryos. Dev Biol 1997, 192:162-171. 13. Xu X, Yin Z, Hudson JB, Ferguson EL, Frasch M: Smad act Analysis of the Gdf11 mutant phenotype has provided in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm. Genes Dev strong genetic evidence that the four Hoxc genes are 1998, 12:2354-2370. functionally downstream of Gdf11 activity. The obvious 14. Lee SJ: Expression of growth/differentiation factor 1 in the questions to ask are whether a GDF11-activated cellular nervous system: conservation of a bicistronic structure. Proc Natl Acad Sci USA 1991, 88:4250-4254. pathway interacts directly with these Hox genes, which 15. McPherron AC, Lee SJ: GDF-3 and GDF-9: two new members of then are responsible for the specification of segment iden- the transforming growth factor-beta superfamily containing a novel pattern of cysteines. J Biol Chem 1993, 268:3444-3449. tity, or whether the expression patterns of these Hox 16. Storm EE, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM, Lee genes are just an indirect reflection of the change in SJ: Limb alterations in brachypodism mice due to mutations in a segment identity. It is not clear from the Gdf11 study [4] new member of the TGF beta-superfamily. Nature 1994, 368:639-643. which is more likely. But there are examples of functional 17. Thomas JT, Lin K, Nandedkar M, Camargo M, Cervenka J, Luyten FP: interactions between cellular pathways activated by A human chondroplasia due to a mutation in a TGF-beta TGFβ molecules and genes coding for homeobox-contain- superfamily member. Nat Genet 1996, 12:315-317. 18. Francis-West PH, Richardson MK, Bell E, Chen P, Luyten F, ing proteins, such as those between BMP-4 and PV.1 [12] Adelfattah A, Barlow AJ, Brickell PM, Wolpert L, Archer CW: The and Dpp and tinman [13]. The confounding issue in the effect of overexpression of BMPs and GDF-5 on the development Gdf11 mutant mice is that the four homeobox genes — of chick limb skeletal elements. Ann NY Acad Sci 1996, 785:254-255. Hoxc6, Hoxc8, Hoxc10 and Hoxc11 — are expressed inap- 19. Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, propriately in somites before any overt sign of vertebral Dube JL, DiBalsio-Smith E, Nove J, Song JJ, et al.: Ectopic induction of tendon and ligament in rats by growth and differentiation morphogenesis has occurred. To resolve the functional factors 5, 6 and 7, members of the TGF-beta gene family. J Clin relationship between GDF11 and the Hox genes, it will be Invest 1997, 100:321-330. R786 Current Biology Vol 9 No 20

20. Yamashita H, Shimizu A, Kato M, Nishitoh H, Ichijo H, Hanyu A, Morita J, Kimura M, Makishima F, Miyazono K: Growth/differentiation factor-5 induces angiogenesis in vivo. 235: Exp Cell Res 1997, 218-226. If you found this dispatch interesting, you might also want 21. Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R, Allen S, MacPherson S, Luyten FP, Archer CW: Mechanisms of to read the October 1999 issue of GDF-5 action during skeletal development. Development 1999, 126:1305-1315. Current Opinion in 22. Lee KJ, Mendelsohn M, Jessell TM: Neuronal patterning by BMPs: a requirement for GDF7 in the generation of a discrete class of commissural interneurons in the mouse spinal cord. Genes Dev Genetics & Development 1998, 12:3394-3407. 23. McPherron AC, Lawler AM, Lee SJ: Regulation of skeletal muscle which included the following reviews, edited mass in mice by a new TGF-beta superfamily member. Nature 1997, 387:83-90. by Michael Levine and Peter Rigby, on 24. McGrath SA, Esquela AF, Lee SJ: Oocyte-specific expression of Differentiation and gene regulation: growth/differentiation factor-9. Mol Endocrinol 1995, 9:131-136. 25. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Makzuk MM: Nuclear receptor cofactors as chromatin remodelers Growth differentiation factor-9 is required during early ovarian Bryan D Lemon and Leonard P Freedman folliculogenesis. Nature 1996, 383:531-535. 26. Cunningham NS, Jenkins NA, Gilbert DJ, Copeland NG, Reddi AH, Lee SJ: Growth/differentiation factor-10: a new member of the Distant liaisons: long-range enhancer–promoter transforming growth factor-beta superfamily related to bone interactions in Drosophila morphogenetic protein-3. Growth Factors 1995, 12:99-109. Dale Dorsett 27. Zhao R, Lawler AM, Lee SJ: Characterization of GDF-10 expression patterns and null mice. Dev Biol 1999, 212:68-79. From factors to mechanisms: translation and 28. Bottner M, Suter-Crazzolara C, Schober A, Unsicker K: Expression of a translational control in eukaryotes novel member of the TGF-beta superfamily growth/differentiation Thomas Preiss and Matthias W Hentze factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult tissues. Cell Tissue Res 1999, 297:103-110. Controls in patterning and diversification of somatic muscles during Drosophila embryogenesis Manfred Frasch A view from the genome: spatial control of transcription in sea urchin development Eric H Davidson Developmental gene activities in ascidian embryos Yutaka Satou and Nori Satoh Transcriptional regulation during zebrafish embryogenesis Sharon L Amacher Transcriptional regulation in Xenopus: a bright and froggy future David Kimelman Notch around the clock Olivier Pourquié Combinatorial codes in signaling and synergy: lessons from pituitary development Jeremy S Dasen and Michael G Rosenfeld Transcription factors in hematopoiesis Isaac Engel and Cornelis Murre Remembrance of things PAS: regulation of development by bHLH-PAS proteins Stephen T Crews and Chen-Ming Fan The ins and outs of circadian timekeeping Steven A Brown and Ueli Schibler APC: the plot thickens Mariann Bienz The full text of Current Opinion in Genetics & Development is in the BioMedNet library at http://BioMedNet.com/cbiology/gen