Lee Niswander

Hedgehog signalling in the mouse requires intraflagellar transport proteins

Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, here we identify two mouse mutants, wimple (wim) and flexo (fxo), that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homologue of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homologue of IFT88. Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of Hedgehog signalling. Our data show that IFT machinery has an essential and vertebrate-specific role in Hedgehog signal transduction.

FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb

The apical ectodermal ridge plays a key role in limb development. We show that recombinant FGF-4 can substitute for the ridge to provide all the signals necessary for virtually complete outgrowth and patterning of the chick limb. FGF-4 stimulates proliferation of cells in the distal mesenchyme and maintains a signal from the posterior to the distal mesenchyme that appears to be required for elaboration of skeletal elements in the normal proximodistal sequence. Moreover, retinoic acid, which is capable of providing polarizing activity, can supply this signal. This suggests that polarizing activity plays a role in patterning along the proximodistal axis, in addition to its well-established role in anteroposterior patterning. Taken together, the data suggest a simple mechanism whereby FGF-4 links growth and pattern formation during limb development.

Requirement for BMP signaling in interdigital apoptosis and scale formation

Interdigital cell death leads to regression of soft tissue between embryonic digits in many vertebrates. Although the signals that regulate interdigital apoptosis are not known, BMPs—signaling molecules of the transforming growth factor-β superfamily—are expressed interdigitally. A dominant negative type I BMP receptor (dnBMPR-IB) was used here to block BMP signaling. Expression of dnBMPR in chicken embryonic hind limbs greatly reduced interdigital apoptosis and resulted in webbed feet. In addition, scales were transformed into feathers. The similarity of the webbing to webbed duck feet led to studies that indicate that BMPs are not expressed in the duck interdigit. These results indicate BMP signaling actively mediates cell death in the embryonic limb.

Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors

Intraflagellar transport (IFT) is an active event in which cargo is transported along microtubules by motor proteins such as kinesin and dynein. IFT proteins are required for the formation and maintenance of flagella and cilia. We have previously shown that mouse mutants for two IFT proteins, IFT88 and IFT172, as well as Kif3a, a subunit of mouse kinesin 2, exhibit ventral spinal cord patterning defects that appear to result from reduced hedgehog (Hh) signaling. Although genetic epistasis experiments place IFT proteins downstream of the Hh receptor and upstream of the Gli transcription factors, the mechanism by which IFT regulates Gli function is unknown. The developing limb provides an excellent system to study Hh signaling, in particular as it allows a biological and molecular readout of both Gli activator and repressor function. Here we report that homozygous mutants for flexo (Fxo), a hypomorphic allele of mouse IFT88 generated in our ENU mutagenesis screen, exhibit polydactyly in all four limbs. Molecular analysis indicates that expression domains of multiple posteriorly restricted genes are expanded anteriorly in the mutant limbs, similar to loss of Gli3 transcriptional repressor function. Sonic hedgehog (Shh) expression is normal, yet Ptch1 and Gli1, two known targets of Hh signaling, are greatly reduced, consistent with loss of Shh signaling. Expression of Gli3 and Hand2 in the mutant limb indicates that the limb prepattern is abnormal. In addition, we show that partial loss-of-function mutations in another mouse IFT gene, Ift52 (Ngd5), result in similar phenotypes and abnormal Hh signaling as Fxo, indicating a general requirement for IFT proteins in Hh signaling and patterning of multiple organs. Analysis of Ift88 and Shh double mutants indicates that, in mouse, IFT proteins are required for both Gli activator and repressor functions, and Gli proteins are insensitive to Hh ligand in the absence of IFT proteins. Finally, our biochemical studies demonstrate that IFT proteins are required for proteolytic processing of Gli3 in mouse embryos. In summary, our results indicate that IFT function is crucial in the control of both the positive and negative transcriptional activities of Gli proteins, and essential for Hh ligand-induced signaling cascade.

Interaction between the signaling molecules WNT7a and SHH during vertebrate limb development: Dorsal signals regulate anteroposterior patterning

Growth and patterning of the vertebrate limb are controlled by the ridge, posterior mesenchyme, and non-ridge ectoderm. Fibroblast growth factor 4 (FGF4) and Sonic hedgehog (SHH) can mediate signaling from the ridge and posterior mesenchyme, respectively. Here we show that dorsal ectoderm is required together with FGF4 to maintain Shh expression. Removal of dorsal ectoderm results in loss of posterior skeletal elements, which can be rescued by exogenous SHH. Wnt7a, which is expressed in dorsal ectoderm, provides the signal required for Shh expression and formation of posterior structures. These results provide evidence that all three axes (dorsoventral, proximodistal, and anteroposterior) are intimately linked by the respective signals WNT7a, FGF4, and SHH during limb out-growth and patterning.

Homozygous WNT3 Mutation Causes Tetra-Amelia in a Large Consanguineous Family

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.

Bone morphogenetic protein signalling and vertebrate nervous system development

Transforming growth factor-β (TGFβ) signalling, particularly signalling from the bone morphogenetic protein (BMP) members of this protein family, is crucial for the development of both the central and peripheral nervous systems in vertebrates. Experimental embryology and genetics performed in a range of organisms are providing insights into how BMPs establish the neural tissue and control the types and numbers of neurons formed. These studies also highlight the interactions between different developmental signals that are necessary to form a functional nervous system. The challenges ahead will be to uncover functions of TGFβ signalling in later stages of CNS development, as well as to determine possible associations with neurological diseases.

Plzf regulates limb and axial skeletal patterning

The promyelocytic leukaemia zinc finger (Plzf) protein (encoded by the gene Zfp145) belongs to the POZ/zinc-finger family of transcription factors. Here we generate Zfp145−/− mice and show that Plzf is essential for patterning of the limb and axial skeleton. Plzf inactivation results in patterning defects affecting all skeletal structures of the limb, including homeotic transformations of anterior skeletal elements into posterior structures. We demonstrate that Plzf acts as a growth-inhibitory and pro-apoptotic factor in the limb bud. The expression of members of the abdominal b (Abdb) Hox gene complex, as well as genes encoding bone morphogenetic proteins (Bmps), is altered in the developing limb of Zfp145−/− mice. Plzf regulates the expression of these genes in the absence of aberrant polarizing activity and independently of known patterning genes. Zfp145−/− mice also exhibit anterior-directed homeotic transformation throughout the axial skeleton with associated alterations in Hox gene expression. Plzf is therefore a mediator of anterior-to-posterior (AP) patterning in both the axial and appendicular skeleton and acts as a regulator of Hox gene expression.

Pattern formation: old models out on a limb

The vertebrate limb is an excellent model for studying fundamental aspects of embryonic development. Cell proliferation, death and movement, and the assignment and interpretation of positional information, must be coordinated if an exquisitely patterned limb is to form. Recent results from gene targeting in mice and from experimental manipulation of the chick embryonic limb have significantly altered the way in which developmental biologists have conceptualized limb patterning.

LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes.