Faculty Bibliography

Dlx transcription factors are implicated in patterning the mammalian jaw, based on their nested expression patterns in the first branchial arch (primordium for jaw) and mutant phenotypes; inactivation of Dlx1 and Dlx2 (Dlx1/2-/-) causes defects in the upper jaw, whereas Dlx5/6(-/-) results in homeotic transformation of the lower jaw into upper jaw. Therefore, the 'Dlx codes' appear to regionalize the jaw primordium such that Dlx1/2 regulate upper jaw development, while Dlx5/6 confer the lower jaw fate. Towards identifying the genetic pathways downstream of Dlx5/6, we compared the gene expression profiles of the wild-type and Dlx5/6(-/-) mouse mandibular arch (prospective lower jaw). We identified 20 previously unrecognized Dlx5/6-downstream genes, of which 12 were downregulated and 8 upregulated in the mutant. We found a Dlx-regulated transcriptional enhancer in close proximity to Gbx2, one of the Dlx5/6-downstream genes, strongly suggesting that Gbx2 is a direct target of Dlx5/6. We also showed that Pou3f3 is normally expressed in the maxillary (prospective upper jaw) but not mandibular arch, is upregulated in the mandibular arch of Dlx5/6(-/-), and is essential for formation of some of the maxillary arch-derived skeleton. A comparative analysis of the morphological and molecular phenotypes of various Dlx single and double mutants revealed that Dlx1, 2, 5 and 6 act both partially redundantly and antagonistically to direct differential expression of downstream genes in each domain of the first branchial arch. We propose a new model for Dlx-mediated mammalian jaw patterning.

Sonic hedgehog (Shh) ligand secreted by the notochord induces distinct ventral cell identities in the adjacent neural tube by a concentration-dependent mechanism. To study this process, we genetically engineered mice that produce bioactive, fluorescently labeled Shh from the endogenous locus. We show that Shh ligand concentrates in close association with the apically positioned basal body of neural target cells, forming a dynamic, punctate gradient in the ventral neural tube. Both ligand lipidation and target field response influence the gradient profile, but not the ability of Shh to concentrate around the basal body. Further, subcellular analysis suggests that Shh from the notochord might traffic into the neural target field by means of an apical-to-basal-oriented microtubule scaffold. This study, in which we directly observe, measure, localize and modify notochord-derived Shh ligand in the context of neural patterning, provides several new insights into mechanisms of Shh morphogen action.

Upregulation of Patched (Ptc), the Drosophila Hedgehog (Hh) receptor in response to Hh signaling limits the range of signaling within a target field by sequestering Hh. In vertebrates, Ptch1 also exhibits ligand-dependent transcriptional activation, but mutants lacking this response show surprisingly normal early development. The identification of Hh-interacting protein 1 (Hhip1), a vertebrate-specific feedback antagonist of Hh signaling, raises the possibility of overlapping feedback controls. We addressed the significance of feedback systems in sonic hedgehog (Shh)-dependent spinal cord patterning. Mouse embryos lacking both Ptch1 and Hhip1 feedback activities exhibit severe patterning defects consistent with an increased magnitude and range of Hh signaling, and disrupted growth control. Thus, Ptc/Ptch1-dependent feedback control of Hh morphogens is conserved between flies and mice, but this role is shared in vertebrates with Hhip1. Furthermore, this feedback mechanism is crucial in generating a neural tube that contains appropriate numbers of all ventral and intermediate neuronal cell types.

Previous studies have demonstrated that Disp1 function is essential for Shh and Ihh signaling in the mouse, and Disp1 gene dose regulates the level of Shh signaling activity in vivo. To determine whether Disp1 activity is required in Shh-producing cells for paracrine signaling in Shh target fields, we used a ShhGFP-Cre (here shortened to ShhCre) knock-in allele and a Disp1 conditional allele to knock down Disp1 activity specifically within Shh-producing cells. The resulting facial and neural tube phenotypes support the conclusion that the primary and probably exclusive role for Disp1 is within hedgehog protein-producing cells. Furthermore, using an allele that produces N-Shh (a noncholesterol modified form of the Shh protein), we demonstrate that N-Shh is sufficient to rescue most of the early embryonic lethal defects in a Disp1-null mutant background. Thus, Disp1 activity is only required for paracrine hedgehog protein signaling by the cholesterol modified form of Shh (N-Shhp), the normal product generated by auto-processing of a Shh precursor protein. In both respects, Disp function is conserved from Drosophila to mice.

Facial abnormalities in human SHH mutants have implicated the Hedgehog (Hh) pathway in craniofacial development, but early defects in mouse Shh mutants have precluded the experimental analysis of this phenotype. Here, we removed Hh-responsiveness specifically in neural crest cells (NCCs), the multipotent cell type that gives rise to much of the skeleton and connective tissue of the head. In these mutants, many of the NCC-derived skeletal and nonskeletal components are missing, but the NCC-derived neuronal cell types are unaffected. Although the initial formation of branchial arches (BAs) is normal, expression of several Fox genes, specific targets of Hh signaling in cranial NCCs, is lost in the mutant. The spatially restricted expression of Fox genes suggests that they may play an important role in BA patterning. Removing Hh signaling in NCCs also leads to increased apoptosis and decreased cell proliferation in the BAs, which results in facial truncation that is evident by embryonic day 11.5 (E11.5). Together, our results demonstrate that Hh signaling in NCCs is essential for normal patterning and growth of the face. Further, our analysis of Shh-Fox gene regulatory interactions leads us to propose that Fox genes mediate the action of Shh in facial development.

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. We show that the morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicate that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and show that a morphogen can also act as an axonal chemoattractant.