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Pitx3 plays a well understood role in directing development of lens, muscle fiber, and dopaminergic neurons; however, in Xenopus laevis, it may also play a role in early gastrulation and somitogenesis. Potential downstream targets of pitx3 possess multiple binding motifs that would not be readily accessible by conventional promoter analysis.We isolated and characterized pitx3 target genes lhx1 and xnr5 using a novel three-fluor flow cytometry tool that was designed to dissect promoters with multiple binding sites for the same transcription factor. This approach was calibrated using a known pitx3 target gene, Tyrosine hydroxylase.We demonstrate how flow cytometry can be used to detect gene regulatory changes with exquisite precision on a cell-by-cell basis, and establish that in HEK293 cells, pitx3 directly activates lhx1 and represses xnr5. Developmental Dynamics 246:657-669, 2017. © 2017 Wiley Periodicals, Inc.

The aristaless-related gene, Arx, plays a fundamental role in patterning the brain in humans and mice. Arx mutants exhibit lissencephaly among other anomalies. We have cloned a Xenopus aristaless homolog that appears to define specific regions of the developing forebrain. xArx2 is transcribed in blastula through neurula stages, and comes to be restricted to the ventra and lateral telencephalon, lateral diencephalon, neural floor plate of the anterior spinal cord, and somites. In this respect, Arx2 expresses in regions similar to Arx with the exception of the somites. Overexpression enlarges the telencephalon, and interference by means of antisense morpholino-mediated translation knockdown reduces growth of this area. Overexpression and inhibition studies demonstrate that misregulation of xArx2 imposes dire consequences upon patterns of differentiation not only in the forebrain where the gene normally expresses, but also in more caudal brain territories and derivatives as well. This suggests that evolutionary changes that expanded Arx-expression from ventral to dorsal prosencephalon might be one of the determinants that marked development and expansion of the telencephalon. genesis 47:19-31, 2009. (C) 2008 Wiley-Liss, Inc.

The polo-like kinases are a family of conserved serine/threonine kinases that play multiple roles in regulation of the cell cycle. Unlike its four other family members, the role of Plk4 in embryonic development has not been well characterized. In mice, Plk4−/−embryos arrest at E7.5, just prior to the initiation of somitogenesis. This has led to the hypothesis that Plk4 expression may be essential to somitogenesis. Recently characterized human mutations lead to Seckel Syndrome. Riboprobe in situ hybridization revealed that plk4 is ubiquitously expressed during early stages of development of Xenopus and Danio; in later stages, expression in frogs restricts to somites as well as eye, otic vesicle, and branchial arch, and brain. Expression patterns in fish remain ubiquitous. Both somite and eye development require planar cell polarity, and disruption of plk4 function in frog by means of morpholino-mediated translational knockdown yields orientational disorganization of both these structures. These results provide the first steps in defining a new role for plk4 in organogenesis and implies a role in planar cell polarity, segmentation, and in recently described PLK4 mutations in human.

Microinjection has a long and distinguished history in Xenopus and has been used to introduce a surprisingly diverse array of agents into embryos by both intra- and intercellular means. In addition to nuclei, investigators have variously injected peptides, antibodies, biologically active chemicals, lineage markers, mRNA, DNA, morpholinos, and enzymes. While enumerating many of the different microinjection approaches that can be taken, we will focus upon the mechanical operations and options available to introduce mRNA, DNA, and morpholinos intracellularly into early stage embryos for the study of neurogenesis.

Pitx3 has been identified as the causative locus in a developmental eye mutation associated with mammalian anterior segment dysgenesis, congenital cataracts, and aphakia. In recent studies of frog eye development we discovered that pitx3 expresses symmetrically in the somites and lateral plate mesoderm and asymmetrically during cardiac and gut looping. We report that disruption of pitx3 activity on one side of an embryo relative to the other, either by over- or underexpression of pitx3, elicits a crooked dorsal axis in embryos that is a consequence of a retarded progression through somitogenesis. Unlike in amniotes, Xenopus somites form as cohorts of presomitic cells that rotate perpendicular to the dorsal axis. Since no vertebral anomalies have been reported in mouse and human Pitx3 mutants, we attempt to distinguish whether the segmentation clock is uniquely affected in frog or if the pitx3 perturbation inhibits the cellular changes that are necessary to rotation of presomitic cells. In Xenopus, pitx3 appears to inhibit the rotation of presomitic cell cohorts and to be necessary to the bilaterally symmetric expression of pitx2 in somites.

Background: Unexpected phenotypes resulting from morpholino-mediated translational knockdown of Pitx3 in Xenopus laevis required further investigation regarding the genetic networks in which the gene might play a role. Microarray analysis was, therefore, used to assess global transcriptional changes downstream of Pitx3. Results: From the large data set generated, selected candidate genes were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Conclusions: We have identified four genes as likely direct targets of Pitx3 action: Pax6, β Crystallin-b1 (Crybb1), Hes7.1, and Hes4. Four others show equivocal promise worthy of consideration: Vent2, and Ripply2 (aka Ledgerline or Stripy), eFGF and RXRα. We also describe the expression pattern of additional and novel genes that are Pitx3-sensitive but that are unlikely to be direct targets. Developmental Dynamics 241:1487–1505, 2012. © 2012 Wiley Periodicals, Inc.

Cardiac troponin I (troponin Ic) expression is restricted to the heart at all stages of Xenopus development. Whole-mount in situ hybridization and Northern blot analysis indicates that troponin Ic is first expressed in tailbud embryos (stage 28) about the time of the first cytological heart differentiation and about 24 hr before beating tissue is observed. We have used this marker to examine abnormal heart morphogenesis in embryos treated with retinoic acid and lithium. When retinoic acid is administered to embryos prior to heart specification, heart tissue is reduced and often completely ablated. When embryos are treated after heart specification, but before the heart primordium migrates to the ventral midline, the migration is unaffected but smaller, abnormal hearts result. Lithium treatment of cleavage stage embryos causes an increase in heart tissue. In severely dorsalized embryos, heart tissue can be found around the entire embryo with the exception of a small gap at the most dorsal point. This gap indicates that migration of the heart to the ventral midline does not occur in these embryos. Later in development, a centrally located, beating heart is observed in dorsalized embryos. The timing of its appearance suggests that it is formed by movements normally associated with heart morphogenesis rather than migration.

SummaryA central hypothesis for the limited capacity for adult central nervous system (CNS) axons to regenerate is the presence of myelin-derived axon growth inhibitors, the role of which, however, remains poorly understood. We have conducted a comprehensive genetic analysis of the three major myelin inhibitors, Nogo, MAG, and OMgp, in injury-induced axonal growth, including compensatory sprouting of uninjured axons and regeneration of injured axons. While deleting any one inhibitor in mice enhanced sprouting of corticospinal or raphespinal serotonergic axons, there was neither associated behavioral improvement nor a synergistic effect of deleting all three inhibitors. Furthermore, triple-mutant mice failed to exhibit enhanced regeneration of either axonal tract after spinal cord injury. Our data indicate that while Nogo, MAG, and OMgp may modulate axon sprouting, they do not play a central role in CNS axon regeneration failure.

We have cloned and characterized a second member of the Xenopus CAP (cyclase associated protein) gene family. xCAP2 demonstrates greater restriction of expression than its homolog, xCAP1, and is differentially expressed throughout early embryogenesis. Although present as a maternal transcript, CAP2 comes to be expressed in the anterior-most mesoderm/endoderm during late gastrulation, in paraxial mesoderm during late neurula stages, and later expresses in lens, cardiac primordia, somites, otic vesicles, retina, and in the optic and craniofacial musculature. The gene is also expressed in the leading edge of myotome.

In contrast to the pattern of limb emergence in mammals, chicks, and the newt N. viridescens, embryos such as Xenopus laevis and Eleutherodactylus coqui initiate pelvic limb buds before they develop pectoral ones. We studied the expression of Pitx1 in X. laevis and E. coqui to determine if this paired-like homeodomain transcription factor directs differentiation specifically of the hindlimb, or if it directs the second pair of limbs to form, namely the forelimbs. We also undertook to determine if embryonic expression patterns were recapitulated during the regeneration of an amputated limb bud. Pitx1 is expressed in hindlimbs in both X. laevis and E. coqui, and expression is similar in both developing and regenerating limb buds. Expression in hindlimbs is restricted to regions of proliferating mesenchyme.Key words: regeneration, Xenopus laevis, limb bud, Pitx1 protein, specification.

The first full single-photon emission computed tomography (SPECT) imager to exploit eight compact high-intrinsic-resolution cadmium zinc telluride (CZT) detectors, called SemiSPECT, has been completed. Each detector consists of a CZT crystal and a customized application-specific integrated circuit (ASIC). The CZT crystal is a 2.7 cm x 2.7 cm x -0.2 cm slab with a continuous top electrode and a bottom electrode patterned into a 64 x 64 pixel array by photolithography. The ASIC is attached to the bottom of the CZT crystal by indium-bump bonding. A bias voltage of -180 V is applied to the continuous electrode. The eight detectors are arranged in an octagonal lead-shielded ring. Each pinhole in the eight-pinhole aperture placed at the center of the ring is matched to each individual detector array. An object is imaged onto each detector through a pinhole, and each detector is operated independently with list-mode acquisition. The imaging subject can be rotated about a vertical axis to obtain additional angular projections. The performance of SemiSPECT was characterized using 99mTc. When a 0.5 mm diameter pinhole is used, the spatial resolution on each axis is about 1.4 mm as estimated by the Fourier crosstalk matrix, which provides an algorithm-independent average resolution over the field of view. The energy resolution achieved by summing neighboring pixel signals in a 3 x 3 window is about 10% full-width-at-half-maximum of the photopeak. The overall system sensitivity is about 0.5 x 10(-4) with the energy window of +/-10% from the photopeak. Line-phantom images are presented to visualize the spatial resolution provided by SemiSPECT, and images of bone, myocardium, and human tumor xenografts in mice demonstrate the feasibility of preclinical small-animal studies with SemiSPECT.

Pitx3 is expressed in tissues fated to contribute to eye development, namely, neurula stage ectoderm and prechordal mesoderm, then presumptive lens ectoderm, placode, and finally lens. Pitx3 overexpression alters lens, optic cup, optic nerve, and diencephalon development. Many of the induced anomalies are attributable to midline deficits; however, as assessed by molecular markers, ectopic Pitx3 appears to temporarily enlarge the lens field. These changes are usually insufficient to generate either ectopic lenses to enlarge the eye that eventually differentiates. Conversely, use of a repressor chimera or of antisense morpholinos alters early expression of marker genes, and later inhibits lens development, thereby abrogating retinal induction. Reciprocal grafting experiments using wild-type and morpholino-treated tissues demonstrate that Pitx3 expression in the presumptive lens ectoderm is required for lens formation. Contradictory to recent assertions that retina can form in the absence of a lens, the expression of Pitx3 in the presumptive lens ectoderm is critical for retina development. Developmental Dynamics 234:577–589, 2005. © 2005 Wiley-Liss, Inc.

In vertebrates, clusters of Hox genes express in a nested and hierarchical fashion to endow the embryo’s segments with discrete identities. Later in development, members of the same gene family are employed again to pattern the limb, intestinal, and reproductive systems. A careful analysis of the morphologies of Hox mutant mice suggests that the genes provide qualitatively different cues during the specification of segments than they do during the development of more recently derived structures. In addition to the regulatory differences noted by others, the activity of Hox genes during specification of the vertebrate metameres in some recent deletion experiments is inconsistent with a role for them as strictly spatial determinants. On the contrary, the phenotypes observed are suggestive of a role for them as elements of a generic time-keeping mechanism. By contrast, the specification of more recent evolutionary structures appears to be more spatial andgene-specific. These differences in role and effect may suggest some simple mechanisms by which the Hox clusters operate, and rules by which gene networks can be diverted to create new structures over the course of evolution. Specific predictions and experiments are proposed.

We have cloned and characterized a Xenopus member of the cyclase associated protein (CAP) gene family. xCAP1 is expressed as a maternal transcript, but is up-regulated prior to gastrulation and subsequently localizes to head mesenchyme, lens, otic vesicle, and trunk mesoderm including the pronephros. At different stages, the gone also appears to differentiate surface from deep (sensorial) ectoderm. As in Drosophila, Xenopus CAP1 is expressed in the developing eye, specifically in the differentiating lens. However, in distinction to Drosophila, Xenopus CAP1 does not express in periodically arrayed neural bands. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Ptx1 belongs to an expanding family of bicoid-related vertebrate homeobox genes. These genes, like their Drosophila homolog, seem to play a role in the development of anterior structures and, in particular, the brain and facies. We report the chromosomal localization of mouse Ptx1, and the cloning, sequencing, and chromosomal localization of the human homolog PTX1. The putative encoded proteins share 100% homology in the homeodomain and are 88% and 97% conserved in the N- and C-termini respectively. Intron/exon boundaries are also conserved. Murine Ptx1 was localized, by interspecific backcrossing, to Chr 13 within 2.6 cM of Caml. The gene resides centrally on Chromosome (Chr) 13 in a region syntenic with human Chr 5q. Subsequent analysis by fluorescent in situ hybridization places the human gene, PTX1, on 5q31, a region associated with Treacher Collins Franceschetti Syndrome. Taken together with the craniofacial expression pattern of Ptx1 during early development, the localization of the gene in this chromosomal area is consistent with an involvement in Treacher Collins Franceschetti Syndrome.

In the minds of many, Hox gene null mutant phenotypes have confirmed the direct role that these genes play in specifying the pattern of vertebrate embryos. The genes are envisaged as defining discrete spatial domains and, subsequently, conferring specific segmental identities on cells undergoing differentiation along the antero-posterior axis. However, several aspects of the observed mutant phenotypes are inconsistent with this view. These include: the appearance of other, unexpected transformations along the dorsal axis; the occurrence of mirror-image duplications; and the development of anomalies outside the established domains of normal Hox gene expression. In this paper, Hox gene disruptions are shown to elicit regeneration-like responses in tissues confronted with discontinuities in axial identity. The polarities and orientations of transformed segments which emerge as a consequence of this response obey the rules of distal transformation and intercalary regeneration. In addition, the incidence of periodic anomalies suggests that the initial steps of Hox-mediated patterning occurs in Hensen's node. As gastrulation proceeds, mesoderm cell cycle kinetics impose constraints upon subsequent cellular differentiation. This results in the delayed manifestation of transformations along the antero-posterior axis. Finally, a paradigm is sketched in which temporal, rather than spatial axial determinants direct differentiation. Specific, testable predictions are made about the role of Hox genes in the establishment of segmental identity. In the minds of many, Hox gene null mutant phenotypes have confirmed the direct role that these genes play in specifying the pattern of vertebrate embryos. The genes are envisaged as defining discrete spatial domains and, subsequently, conferring specific segmental identities on cells undergoing differentiation along the antero-posterior axis. However, several aspects of the observed mutant phenotypes are inconsistent with this view. These include: the appearance of other, unexpected transformations along the dorsal axis; the occurrence of mirror-image duplications; and the development of anomalies outside the established domains of normal Hox gene expression. In this paper, Hox gene disruptions are shown to elicit regeneration-like responses in tissues confronted with discontinuities in axial identity. The polarities and orientations of transformed segments which emerge as a consequence of this response obey the rules of distal transformation and intercalary regeneration. In addition, the incidence of periodic anomalies suggests that the initial steps of Hox-mediated patterning occurs in Hensen's node. As gastrulation proceeds, mesoderm cell cycle kinetics impose constraints upon subsequent cellular differentiation. This results in the delayed manifestation of transformations along the antero-posterior axis. Finally, a paradigm is sketched in which temporal, rather than spatial axial determinants direct differentiation. Specific, testable predictions are made about the role of Hox genes in the establishment of segmental identity.

We report the isolation of two retinoic acid receptor isoforms (RAR gamma), which differ only in the 5'untranslated and putative N-terminus A regions. The two isoforms appear to serve as early markers for the presumptive neural axis; however, their expression patterns differ. RAR-gamma 2.1 is first expressed at gastrulation at the dorsal lip and subsequently along the presumptive neural axis. RAR- gamma 2.2 represents the full-length sequence of a receptor cDNA already partially characterized and present as a maternal transcript [Ellinger-Ziegelbauer and Dreyer (1991); Genes Dev 5:94-104, (1993): Mech Dev 41:31-46; Pfeffer and DeRobertis, (1994) Mech Dev: 45:147-153]. Unlike RAR-gamma 2.2, the 2.1 variant is not expressed either in pre-somitic mesoderm or notochord. RAR-gamma 2.1 is strongly expressed in branchial arches and to a lesser extent in the neural floor plate. The two isoforms also exhibit differential sensitivity to retinoic acid. Constitutive expression of RAR gamma 2.2 following neurulation appears to be depressed by treatment with retinoic acid, but domains of highest expression, namely, the head and tail, remain relatively unaffected, as do patterns of expression prior to late neurulation. By contrast, RAR-gamma 2.1 is not transcribed in retinoid-inhibited structures. Using microinjection techniques, we show that changes of RAR-gamma 2.1 expression in presumptive head structures occur as an early and local consequence of retinoic acid administration. Since RAR-gamma 2.1 expression is inhibited by retinoic acid, we tested to see if other treatments that perturb axis formation had any effect. Surprisingly, UV irradiation did not suppress that its inhibition by retinoic acid is not due solely to inhibition of anterior neural development. These experiments demonstrate a new subdivision of isoforms that undergo differential expression during development and that exhibit differential sensitivity to retinoic acid and to UV. This sensitivity and the presence of this isoform variant in regions that are known to exhibit polarizing activity strengthen the hypothesis that these receptors play a primary role during morphogenesis.

In order to more accurately determine the mechanism by which retinoic acid causes embryonic defects, we have developed a simple method of locally applying retinoic acid rather than immersing the whole embryo in retinoic acid solutions. Retinoic acid was suspended in corn oil and then injected between the surface and the deep ectodermal layers of an early gastrula Xenopus embryo. When droplets containing retinoic acid were injected into the presumptive head region, the embryos exhibited inhibited development of anterior structures near the injection site. Development of the eye, cement gland, hatching gland, olfactory pits, and expression of engrailed protein were all disrupted near the injection site. Inhibited development of anterior structures was far greater on the injected side of the embryo than on the uninjected side. The retinoic acid droplet did not cause an anterior shift of structures on the injected side relative to the uninjected side. These experiments suggest that retinoic acid does not cause global respecification of axial level in the head, but rather suppresses development of anterior structures. Retinoic acid injected into presumptive trunk regions had no discernible effect.