Juhee Jeong

The Morphogen Sonic Hedgehog Is an Axonal Chemoattractant that Collaborates with Netrin-1 in Midline Axon Guidance

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.

Growth and pattern of the mammalian neural tube are governed by partially overlapping feedback activities of the hedgehog antagonists patched 1 and Hhip1

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.

Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural 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.

Lhx6 and Lhx8 Coordinately Induce Neuronal Expression of Shh that Controls the Generation of Interneuron Progenitors

Lhx6 and Lhx8 transcription factor coexpression in early-born MGE neurons is required to induce neuronal Shh expression. We provide evidence that these transcription factors regulate expression of a Shh enhancer in MGE neurons. Lhx6 and Lhx8 are also required to prevent Nkx2-1 expression in a subset of pallial interneurons. Shh function in early-born MGE neurons was determined by genetically eliminating Shh expression in the MGE mantle zone (MZ). This mutant had reduced SHH signaling in the overlying progenitor zone, which led to reduced Lhx6, Lhx8, and Nkx2-1 expression in the rostrodorsal MGE and a preferential reduction of late-born somatostatin(+) and parvalbumin(+) cortical interneurons. Thus, Lhx6 and Lhx8 regulate MGE development through autonomous and nonautonomous mechanisms, the latter by promoting Shh expression in MGE neurons, which in turn feeds forward to promote the developmental program of the rostrodorsal MGE.

Mouse Disp1 is required in sonic hedgehog-expressing cells for paracrine activity of the cholesterol-modified ligand

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.

Cholesterol modification of Hedgehog family proteins

The Hedgehog (Hh) gene family encodes a group of secreted signaling molecules that are essential for growth and patterning of many different body parts of vertebrate and invertebrate embryos (1). Depending on the context, Hh signals can promote cell proliferation, prevent apoptosis, or induce specific cell fates. Hh family members can exert their effects not only on cells neighboring the source of the signal, but also over considerable distances (up to 30-cell diameters), acting in at least some cases as classic morphogens. Such morphogens are signaling molecules that diffuse from a source to form a concentration gradient over an extended area of the target field and elicit different responses from cells according to their position within the gradient, which in turn reflects the dosage of the ligand they are exposed to. In the fruit fly Drosophila, Hh patterns the embryonic ectoderm via short-range interactions with other signaling molecules (2). On the other hand, it employs two different strategies in larval wing imaginal discs: it induces a secondary signal (Decapentaplegic [Dpp]) locally, which then acts at a long range; and Hh itself diffuses over several cell diameters and acts as a morphogen (3–5). Unlike Drosophila, which has one member of the Hh family, mice have three — Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) — with Shh being the best studied (Table ​(Table1).1). Shh is expressed at the ventral end of the neural tube (floor plate) and underlying notochord, and patterns the neural tube along its dorsoventral axis (6). Several pieces of evidence support the notion that this patterning is mediated by a direct morphogen activity of Shh. In vitro assays using undifferentiated neural tube explants demonstrate that different dosages of Shh can induce different cell types, where the relative dosages required to induce certain cell fates are consistent with their positions within the concentration gradient in vivo (7, 8). More recently, it has become possible to visualize the endogenous Shh gradient covering the ventral half of the neural tube by immunofluorescence and immunohistochemistry (9, 10). In addition, the cell-autonomous activation or inactivation of the pathway using mutant forms of receptor components results in autonomous changes of cell fates, confirming the direct role of Hh signaling (11, 12). The limb is another place where Shh may function as a morphogen. Shh is expressed in the posterior distal mesenchyme of the limb bud, called the zone of polarizing activity (ZPA), and makes a long-range gradient along the anteroposterior axis (13, 14). This concentration gradient is believed to be important in specifying digit identities across the limb bud, with high dosages of Shh close to the ZPA inducing posterior digits and low dosages inducing anterior digits (13, 15, 16). In addition to its importance during development, inappropriate activation of the Hh pathway has been implicated in human tumors such as basal cell carcinoma, medulloblastoma, fibrosarcoma, and rhabdomyosarcoma (17). Table 1 Mammalian Hh genes: some of their normal roles and pathological associations Not surprisingly, significant effort has been devoted to uncovering the molecular mechanism of this pathway (1). Genetic and biochemical studies have established the current model in which Hh receptor Patched (Ptc) is a negative regulator of the pathway repressing the downstream activator Smoothened (Smo), and binding of Hh to Ptc abrogates this inhibition, leading to cellular responses via specific transcription factors known in the fly as Ci and in the mouse as Gli. During the past decade, much excitement has been generated by the discovery of the unusual posttranslational modifications that the Hh protein undergoes (18): the addition of cholesterol (19), which is unprecedented, and palmitoylation (20), which is normally found in cytoplasmic proteins. Here, we will review our current understanding of the mechanism and biological relevance of the cholesterol modification of Hh, with additional discussion of the role of palmitoylation, which has come to light more recently.

Signaling by SHH rescues facial defects following blockade in the brain.

Background: The Frontonasal Ectodermal Zone (FEZ) is a signaling center in the face that expresses Sonic hedgehog (Shh) and regulates patterned growth of the upper jaw. Blocking SHH in the forebrain blocks Shh expression in the FEZ and creates malformations resembling holoprosencephaly (HPE), while inhibition of BMP signaling in the mesenchyme blocks FEZ formation and causes similar dysmorphology. Thus, the brain could regulate FEZ formation by SHH or BMP signaling, and if so, activating one of these pathways in the face might alleviate the effects of repression of SHH in the brain. Results: We blocked SHH signaling in the brain while adding SHH or BMP between the neural and facial ectoderm of the frontonasal process. When applied early, SHH restored Shh expression in the FEZ and significantly improved shape outcomes, which contrasts with our previous experiments that showed later SHH treatments have no effect. BMP‐soaked beads introduced early and late caused apoptosis that exacerbated malformations. Finally, removal of Smoothened from neural crest cells did not inhibit Shh expression in the FEZ. Conclusions: Collectively, this work suggests that a direct, time‐sensitive SHH signal from the brain is required for the later induction of Shh in the FEZ. We propose a testable model of FEZ activation and discuss signaling mediators that may regulate these interactions. Developmental Dynamics 241:247–256, 2012.

Cleft palate defect of Dlx1/2-/- mutant mice is caused by lack of vertical outgrowth in the posterior palate.

Background: Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2−/−) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx1 and Dlx2 as related to palate development. Results: Dlx1/2−/− mutants exhibited lack of vertical growth in the posterior palate during the earliest stage of palatogenesis. We attributed this growth deficiency to reduced cell proliferation. Expression of a cell cycle regulator Ccnd1 was specifically down‐regulated in the same region. Previous studies established that the epithelial‐mesenchymal signaling loop involving Shh, Bmp4, and Fgf10 is important for cell proliferation and tissue growth during palate development. This signaling loop was disrupted in Dlx1/2−/− palate. Interestingly, however, the decreases in Ccnd1 expression and mitosis in Dlx1/2−/− mutants were independent of this signaling loop. Finally, Dlx1/2 activity was required for normal expression of several transcription factor genes whose mutation results in palate defects. Conclusions: The functions of Dlx1 and Dlx2 are crucial for the initial formation of the posterior palatal shelves, and that the Dlx genes lie upstream of multiple signaling molecules and transcription factors important for later stages of palatogenesis. Developmental Dynamics 241:1757–1769, 2012.

Identification of a Face Enhancer Reveals Direct Regulation of LIM homeobox 8 (Lhx8) by Wingless-Int (WNT)/β-catenin Signaling

Background: Lhx8 is an important gene for craniofacial development, but its regulation had been poorly understood. Results: We identified a face enhancer of Lhx8 and discovered that it was regulated by WNT/β-catenin signaling. Conclusion: WNT/β-catenin pathway directly regulates Lhx8 in the face via a distal enhancer. Significance: We uncovered a molecular mechanism for the regulation of Lhx8 in the face. Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8_enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8_enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/β-catenin signaling pathway. We demonstrated in vitro that WNT/β-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8_enh1 was a direct target of the WNT/β-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development.

Neural crest-specific deletion of Ldb1 leads to cleft secondary palate with impaired palatal shelf elevation

BackgroundLIM domain binding protein 1 (LDB1) is a transcriptional co-factor, which interacts with multiple transcription factors and other proteins containing LIM domains. Complete inactivation of Ldb1 in mice resulted in early embryonic lethality with severe patterning defects during gastrulation. Tissue-specific deletions using a conditional knockout allele revealed additional roles of Ldb1 in the development of the central nervous system, hematopoietic system, and limbs. The goal of the current study was to determine the importance of Ldb1 function during craniofacial development in mouse embryos.ResultsWe generated tissue-specific Ldb1 mutants using Wnt1-Cre, which causes deletion of a floxed allele in the neural crest; neural crest-derived cells contribute to most of the mesenchyme of the developing face. All examined Wnt1-Cre;Ldb1fl/- mutants suffered from cleft secondary palate. Therefore, we performed a series of experiments to investigate how Ldb1 regulated palate development. First, we examined the expression of Ldb1 during normal development, and found that Ldb1 was expressed broadly in the palatal mesenchyme during early stages of palate development. Second, we compared the morphology of the developing palate in control and Ldb1 mutant embryos using sections. We found that the mutant palatal shelves had abnormally blunt appearance, and failed to elevate above the tongue at the posterior domain. An in vitro head culture experiment indicated that the elevation defect was not due to interference by the tongue. Finally, in the Ldb1 mutant palatal shelves, cell proliferation was abnormal in the anterior, and the expression of Wnt5a, Pax9 and Osr2, which regulate palatal shelf elevation, was also altered.ConclusionsThe function of Ldb1 in the neural crest-derived palatal mesenchyme is essential for normal morphogenesis of the secondary palate.