Charmaine Pira

Efficient ectopic gene expression targeting chick mesoderm

Abstract

Detection of genes regulated by Lmx1b during limb dorsalization.

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral–ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wild type mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes that were differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix‐related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism that includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to‐date and provides targets for further investigation.

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition.

BackgroundNogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition.ResultsWe isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment.ConclusionThese data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS.

Lmx1b-targeted cis-regulatory modules involved in limb dorsalization

Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% (n=617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential cis-regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related Gdf5 that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with Lmx1b that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization. Summary: Correlating Lmx1b-binding sites with Lmx1b-regulated genes during mouse limb development uncovers cis-regulatory modules and their gene targets involved in limb dorsal-ventral identity.

IDENTIFICATION OF DEVELOPMENTAL ENHANCERS USING TARGETED REGIONAL ELECTROPORATION (TREP) OF EVOLUTIONARILY CONSERVED REGIONS

INTRODUCTION During development, precise temporal and spatial regulation of critical genes is required to orchestrate body plan morphology. Preservation of a generalized developmental process and body plan across divergent species suggests that regulation has also been conserved. Thus, evolutionarily conserved regions (ECRs) in association with developmentally important genes are likely candidates as regulatory elements. Screening of ECRs has recently been described during early chick development, using in vitro whole embryo electroporation of ECR constructs containing green fluorescent protein (GFP). The major limitation of this technique is that the chick embryos only survive in vitro for about 48 hrs after electroporation and thus enhancers involved in later development and organogenesis cannot be determined. We previously reported on a method to deliver expression vectors at targeted locations during in ovo development by confined microelectroporation (CMEP). We have modified this technique to broaden the targeted region of electroporation and vector delivery, i.e., targeted regional electroporation (TREP). Herein, we demonstrate the ability of this technique to screen for ECR activity at later stages of development.

LHX2 Mediates the FGF-to-SHH Regulatory Loop during Limb Development

During limb development, fibroblast growth factors (Fgfs) govern proximal–distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximal–distal and anterior–posterior axes by maintaining Sonic hedgehog (Shh) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER) that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application), while prolonged exposure (24 h) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER.

SONIC HEDGEHOG ESTABLISHES THE FIBROBLAST GROWTH FACTORS RESPONSIVENESS IN THE LIMB BUD SHH-FGF FEEDBACK LOOP.: 176

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), whereas progressive limb pattern formation is regulated by SHH emanating from the zone of polarizing activity (ZPA). SHH also up-regulates factors that stimulate posteriorly accentuated FGFs in the AER. In turn, these FGFs maintain SHH expression in the adjacent distal mesoderm to complete the SHH-FGF feedback loop. Interestingly, application of FGF to the anterior mesoderm fails to up-regulate SHH, indicating a requirement for additional factors. We hypothesized that SHH induces this FGF responsiveness in limb mesoderm. To test this hypothesis, we ectopically expressed a SHH-GFP construct in the distal-anterior mesoderm by confined microelectroporation (CMEP) at HH 18-19. We demonstrated subsequent expression of SHH and its downstream target, the patched receptor (PTCH), by whole mount in situ hybridization (WISH). After 18 hours (HH 23-24) of exposure to SHH, we applied an FGF-soaked bead to the anterior margin. After an additional 24 hours, chicks were harvested and the SHH expression was determined by WISH. SHH and PTCH expression were up-regulated 24 hours after CMEP. The initial ectopic expression of SHH correlates with the site of transfection indicated by GFP fluorescence. Interestingly, by 42 hours the expression of SHH has shifted to lie immediately underneath the distal anterior AER and does not correlate with GFP fluorescence. These data signify that endogenous expression and FGF-regulated maintenance of SHH has been established. Furthermore, anteriorly placed FGF-soaked beads locally up-regulate SHH expression. Collectively, these data indicate that exposure of limb mesoderm to SHH activates a unique mechanism to produce SHH in response to FGF and thus link SHH expression tightly to the AER during limb outgrowth.

THE ROLE OF SONIC HEDGEHOG IN THE INDUCTION OF CHICK LIMB REGENERATION/REDEVELOPMENT.: 178

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), tightly linked to progressive limb pattern formation regulated by SHH emanating from the posteriorly positioned zone of polarizing activity (ZPA). The reciprocal induction of SHH and FGF during limb development has been termed the SHH-FGF loop. Amputation of a chick wing bud during early development results in removal of the AER and the ZPA with truncation of the distal limb. Addition of FGF to the posterior, but not the anterior, stump immediately after amputation induces tissue regeneration and reactivates development of the distal limb structures. SHH expression is up-regulated adjacent to posteriorly applied FGF-soaked beads but not anterior applied FGF and correlates with regenereative capacity. We wondered whether SHH was sufficient to induce regenerative capacity. To determine the role of SHH in limb regenerative capacity, we amputated developing wings (HH stage 23) and ectopically expressed SHH by confined microeclectroporation (CMEP) in the anterior and posterior stump margins in the presence or absence of FGF-soaked beads. Embryos were harvested at day 10 (6 days after amputation) and the limb morphology analyzed by alcian green staining of the skeletal elements. In other experiments, chicks were harvested 30 to 40 hours after electroporation, and whole-mount in situ hybridization (WMISH) to FGF4 was used to identify any ectopic ectodermal FGF expression. Ectopic expression of SHH in the posterior stump alone could not reestablish segmented limb development. In contrast, expression of ectopic SHH in the anterior stump induced segmented regeneration/redevelopment in the absence of FGF (9/20 ≈ 40%). However, WMISH for FGF4 identified ectopic FGF4 expression in the ectoderm overlying ectopic anterior SHH expression, indicating the local presence of both factors. Thus, it appears that both SHH and FGF are required to regenerate lost tissue and initiate redevelopment. Our data suggest that SHH induces both positive and negative regulators of FGF expression. Initial SHH expression in naive limb mesoderm (anterior stump cells) up-regulates FGFs in the overlying ectoderm, while cells with prior exposure to SHH (posterior stump cells) no longer have the ability to up-regulate FGF in response to SHH. This shutdown of the SHH-FGF-loop, overcome in amphibian regeneration, may be a critical step in promoting mammalian limb regeneration.

248 THE INTERACTION BETWEEN FIBROBLAST GROWTH FACTOR AND SONIC HEDGEHOG IS NECESSARY FOR CHICK LIMB REGENERATION.

The ability to regenerate a lost body part during wound healing is largely restricted to amphibians and lower vertebrates. However, regenerative wound healing following human fetal surgery has exposed the potential for regeneration in higher vertebrates. Regeneration is dependent upon the activation of growth factors that drive cell proliferation and patterning factors that shape the growing tissue into the missing body part. Recently, an embryonic chick model has demonstrated that fibroblast growth factors (FGF) can drive regeneration of an amputated wing bud during development if applied to the posterior stump. Furthermore, the reactivation of sonic hedgehog (SHH), a potent patterning factor, surrounding the FGF-soaked bead was an early predictor of regeneration. In contrast, when FGF was introduced in the anterior stump no SHH induction or regeneration occurred. We hypothesized that the combined interaction between FGF and SHH was necessary for regeneration. To test this hypothesis we ectopically expressed both SHH (by electroporation) and FGF2-soaked beads to the anterior margin of stage 23 (HH) forelimb (wing) buds following amputation of the distal 500 μm. The chicks were grown to embryonic day 10; the wings were harvested and stained with alcian green for assessment of the skeleton. In limbs treated with ectopic SHH and FGF, 4 of 18 demonstrated regeneration with ulnarization of the radius, pattern reversal of the wrist, and a subsequent change in digit identity. The limbs treated with only SHH (10) or only FGF (11) failed to regenerate. These data demonstrate that both FGF and SHH are required for regeneration following wound healing and may indicate targets for reactivation in organisms that lack tissue regeneration.

39 ISOLATION AND EXPRESSION OF NOGO-A IN THE DEVELOPING CENTRAL NERVOUS SYSTEM

Purpose NOGO-A is a myelin associated protein best known for its ability to inhibit CNS regeneration in adults and may be involved in the maintenance of established neural pathways. Recent experiments have identified NOGO-A during development, when regeneration is less relevant. Thus, its role during development is unclear. We hypothesize that NOGO-A has a role in neural tube induction or early neuronal differentiation. Methods To test our hypothesis we isolated chicken NOGO-A using RT-PCR. We utilized a protocol for difficult templates to isolate and replicate the GC rich NOGO-A transcript. A 219 base pair probe was synthesized and used to perform whole mount in situ hybridization (WISH) to localize NOGO-A expression in Hamburger and Hamilton (HH) stages 4-17 chick embryos. Regulation and induction of NOGO-A was tested by Shh, Fgf8b and RA bead implantation in HH4 embryos. These were harvested after 4 and 8 hours and processed by WISH. Results We identified two different isoforms of NOGO-A in the chick embryo; 3.8KB and 4.3KB isoforms that we have sequenced. WISH showed initial NOGO-A expression in the neural plate at induction (HH4). Expression then followed formation of the neural tube (HH8), primary brain vesicles (HH12) and somites (HH17). Ectopic Shh increased local NOGO-A expression at 4 hours, Fgf8b showed increased local expression at 8 hours, and RA showed no increased expression. Conclusions Our WISH experiments demonstrated that NOGO-A expression begins early in development and is induced by Shh and Fgf-8, known mediators of neural tube development. Additionally, we identified two isoforms of NOGO-A in the chicken embryo. It remains to be determined if these two isoforms are splice variants, and if they have distinct roles. Our results support our initial hypothesis that NOGO-A has a role beyond inhibition of CNS regeneration and is involved in CNS development.