Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

Shh and ROCK1 modulate the dynamic epithelial morphogenesis in circumvallate papilla development

In rodents, a circumvallate papilla (CVP) develops with dynamic changes in epithelial morphogenesis during early tongue development. Molecular and cellular studies of CVP development revealed that there would be two different mechanisms in the apex and the trench wall forming regions with specific expression patterns of Wnt11 and Shh. Molecular interactions were examined using in vitro organ culture with over-expression of Shh, important signalling molecules and various inhibitors revealed that there are two significant different mechanisms in CVP formation by Wnt11 and Shh expressions. Wnt, a well known key molecule to initiate taste papillae, would govern Rho activation and cytoskeleton formation in the apex epithelium of CVP. In contrast, Shh regulates the cell proliferation to differentiate taste buds and to invaginate the epithelium for development of von Ebner's gland (VEG). Based on these results, we suggest that these different molecular signalling cascades of Wnt11 and Shh would play crucial roles in specific morphogenesis and pattern formation of CVP during early mouse embryo development.     

Evolution of developmental roles of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages

Background

Gene duplication provides opportunities for lineage diversification and evolution of developmental novelties. Duplicated genes generally either disappear by accumulation of mutations (nonfunctionalization), or are preserved either by the origin of positively selected functions in one or both duplicates (neofunctionalization), or by the partitioning of original gene subfunctions between the duplicates (subfunctionalization). The Pax2/5/8 family of important developmental regulators has undergone parallel expansion among chordate groups. After the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the Pax2, Pax5, and Pax8 genes of most vertebrates (the sister group of the urochordates), and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. Separate from the vertebrate genome expansions, a duplication also created two Pax2/5/8 genes in the common ancestor of ascidian and larvacean urochordates.

Results

To better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate Oikopleura dioica, the embryonic gene expression patterns of Pax2/5/8 paralogs. We compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication Pax2/5/8 gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of Pax2/5/8 gene subfunctions in the common ancestor of olfactores (vertebrates + urochordates). Expression pattern differences of larvacean and ascidian Pax2/5/8 orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. Novel expression in the larvacean heart may have resulted from the neofunctionalization of a Pax2/5/8 gene in the urochordates. Expression of larvacean Pax2/5/8 in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of Pax2, Pax5 and Pax8 in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor.

Conclusion

Comparative analysis of expression patterns of chordate Pax2/5/8 duplicates, rooted on the single-copy Pax2/5/8 gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that Pax2/5/8 functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings.     

Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata)

Here we study the role of Shh signaling in tooth morphogenesis and successional tooth initiation in snakes and lizards (Squamata). By characterizing the expression of Shh pathway receptor Ptc1 in the developing dentitions of three species (Eublepharis macularius, Python regius, and Pogona vitticeps) and by performing gain- and loss-of-function experiments, we demonstrate that Shh signaling is active in the squamate tooth bud and is required for its normal morphogenesis. Shh apparently mediates tooth morphogenesis by separate paracrine- and autocrine-mediated functions. According to this model, paracrine Shh signaling induces cell proliferation in the cervical loop, outer enamel epithelium, and dental papilla. Autocrine signaling within the stellate reticulum instead appears to regulate cell survival. By treating squamate dental explants with Hh antagonist cyclopamine, we induced tooth phenotypes that closely resemble the morphological and differentiation defects of vestigial, first-generation teeth in the bearded dragon P. vitticeps. Our finding that these vestigial teeth are deficient in epithelial Shh signaling further corroborates that Shh is needed for the normal development of teeth in snakes and lizards. Finally, in this study, we definitively refute a role for Shh signaling in successional dental lamina formation and conclude that other pathways regulate tooth replacement in squamates.     

Neural and orofacial defects in Folp1 knockout mice [corrected

BACKGROUND

Folic acid is essential for the development of the nervous system and other associated structures. Mice deficient in the folic acid‐binding protein one (Folbp1) gene display multiple developmental abnormalities, including neural and craniofacial defects. To better understand potential interactions between Folbp1 gene and selected genes involved in neural and craniofacial morphogenesis, we evaluated the expression patterns of a panel of crucial differentiation markers (Pax‐3, En‐2, Hox‐a1, Shh, Bmp‐4, Wnt‐1, and Pax‐1).

METHODS

Folbp1 mice were supplemented with low dosages of folinic acid to rescue nullizygotes from dying in utero before gestational day 10. The gene marker analyses were carried out by in situ hybridization.

RESULTS

In nullizygote embryos with open cranial neural tube defects, the downregulation of Pax‐3 and En‐2 in the impaired midbrain, along with an observed upregulation of the ventralizing marker Shh in the expanded floor plate, suggested an important regulatory interaction among these three genes. Moreover, the nullizygotes also exhibit craniofacial abnormalities, such as cleft lip and palate. Pax‐3 signals in the impaired medial nasal primordia were significantly increased, whereas Pax‐1 showed no expression in the undeveloped lateral nasal processes. Although Shh was downregulated, Bmp‐4 was strongly expressed in the medial and lateral nasal processes, highlighting the antagonistic activities of these molecules.

CONCLUSIONS

Impairment of Folbp1 gene function adversely impacts the expression of several critical signaling molecules. Mis‐expression of these molecules, perhaps mediated by Shh, may potentially contribute to the observed failure of neural tube closure and the development of craniofacial defects in the mutant mice. Birth Defects Research (Part A) 67:209–218, 2003. © 2003 Wiley‐Liss, Inc.

     

Sonic Hedgehog Gene Delivery to the Rodent Heart Promotes Angiogenesis via iNOS/Netrin-1/PKC Pathway

Background

We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway.

Methods/Principal Findings

MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid (^ShhMSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in ^ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in ^ShhMSCs. Significantly improved migration and tube formation was seen in ^ShhMSCs as compared to empty vector transfected MSCs (^EmpMSCs). Significant upregulation of netrin-1 and iNOS was observed in ^ShhMSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 µl basal DMEM without cells (group-1) or containing 1×10^{6 Emp}MSCs (group-2) and ^ShhMSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of ^ShhMSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with ^ShhMSCs in group-3 animals.

Conclusions/Significance

Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.