Exogenous FGF8 signaling in osteocytes leads to mandibular hypoplasia in mice.

OBJECTIVE: Fibroblast growth factor 8 (FGF8) signaling is essential in regulating craniofacial osteogenesis. This study aims to explore the effect of altered FGF8 signaling in maxillomandibular development during embryogenesis. MATERIALS AND METHODS: Dmp1Cre ;R26RmTmG mice were generated to trace Dmp1+ cell lineage, and Dmp1Cre ;R26RFgf8 mice were generated to explore the effects of augmented FGF8 signaling in Dmp1+ cells on osteogenesis with a focus on maxillomandibular development during embryogenesis, as assessed by whole mount skeletal staining, histology, and immunostaining. Additionally, cell proliferation rate and the expression of osteogenic genes were examined. RESULTS: Osteocytes of maxillomandibular bones were found Dmp1-positive prenatally, and Fgf8 over-expression in Dmp1+ cells led to mandibular hypoplasia. While Dmp1Cre allele functions in the osteocytes of the developing mandibular bone at as early as E13.5, and enhanced cell proliferation rate is observed in the bone forming region of the mandible in Dmp1Cre ;R26RFgf8 mice at E14.5, histological examination showed that osteogenesis was initially impacted at E15.5, along with an inhibition of osteogenic differentiation markers. CONCLUSIONS: Augmented FGF8 signaling in Dmp1+ cells lead to osteogenic deficiency in the mandibular bones, resulting in mandibular hypoplasia.

Roles of FGF8 subfamily in embryogenesis and oral­maxillofacial diseases (Review).

Fibroblast growth factors (FGFs) are diffusible polypeptides released by a variety of cell types. FGF8 subfamily members regulate embryonic development processes through controlling progenitor cell growth and differentiation, and are also functional in adults in tissue repair to maintain tissue homeostasis. FGF8 family members exhibit unique binding affinities with FGF receptors and tissue distribution patterns. Increasing evidence suggests that, by regulating multiple cellular signaling pathways, alterations in the FGF8 subfamily are involved in craniofacial development, odontogenesis, tongue development and salivary gland branching morphogenesis. Aberrant FGF signaling transduction, caused by mutations as well as abnormal expression or isoform splicing, plays an important role in the development of oral diseases. Targeting FGF8 subfamily members provides a new promising strategy for the treatment of oral diseases. The aim of this review was to summarize the aberrant regulations of FGF8 subfamily members and their potential implications in oral­maxillofacial diseases.

A model of neocortical area patterning in the lissencephalic mouse may hold for larger gyrencephalic brains.

In the mouse, two telencephalic signaling centers orchestrate embryonic patterning of the cerebral cortex. From the rostral patterning center in the telencephalon, the Fibroblast Growth Factor, FGF8, disperses as a morphogen to establish the rostral to caudal axis of the neocortical area map. FGF8 coordinates with Wnt3a from the cortical hem to regulate graded expression of transcription factors that position neocortical areas, and control hippocampal development. Whether similar signaling centers pattern the much larger cortices of carnivore and primate species, however, is unclear. The limited dispersion range of FGF8 and Wnt3a is inconsistent with patterning larger cortical primordia. Yet the implication that different mechanisms organize cortex in different mammals flies in the face of the tenet that developmental patterning mechanisms are conserved across vertebrate species. In the present study, both signaling centers were identified in the ferret telencephalon, as were expression gradients of the patterning transcription factor genes regulated by FGF8 and Wnt3a. Notably, at the stage corresponding to the peak period of FGF8 signaling in the mouse neocortical primordium (NP), the NP was the same size in ferret and mouse, which would allow morphogen patterning of the ferret NP. Subsequently, the size of ferret neocortex shot past that of the mouse. Images from online databases further suggest that NP growth in humans, too, is slowed in early cortical development. We propose that if early growth in larger brains is held back, mechanisms that pattern the neocortical area map in the mouse could be conserved across mammalian species.

FGF8 induces therapy resistance in neoadjuvantly radiated rectal cancer.

PURPOSE: Therapy response to neoadjuvant radiochemotherapy (nRCT) of locally advanced rectal cancer varies widely so that markers predicting response are urgently needed. Fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling is involved in pro-survival signaling and thereby may result in radiation resistance. METHODS: In a cohort of 43 rectal cancer patients, who received nRCT, we analyzed protein levels of FGF 8 and its downstream target Survivin by immunohistochemistry to assess their impact on nRCT response. In vitro resistance models were created by exposing colorectal cancer cell lines to fractionated irradiation and selecting long-term survivors. RESULTS: Our findings revealed significantly higher FGF8 and Survivin staining scores in pre-treatment biopsies as well as in surgical specimens of non-responsive compared to responsive patients. Functional studies demonstrated dose-dependent induction of FGF8 mRNA expression in mismatch-incompetent DLD1 cells already after one dose of irradiation. Surviving clones after one or two series of radiation were more resistant to an additional radiation fraction than non-irradiated controls and showed a significant increase in expression of the FGF8 receptor FGFR3 and of Survivin on both the RNA and the protein levels. CONCLUSION: The results of this study suggest that FGF8 and Survivin contribute to radiation resistance in rectal cancer and may serve as markers to select patients who may not benefit from neoadjuvant radiotherapy.

Geminin Orchestrates Somite Formation by Regulating Fgf8 and Notch Signaling.

During somitogenesis, Fgf8 maintains the predifferentiation stage of presomitic mesoderm (PSM) cells and its retraction gives a cue for somite formation. Delta/Notch initiates the expression of oscillation genes in the tail bud and subsequently contributes to somite formation in a periodic way. Whether there exists a critical factor coordinating Fgf8 and Notch signaling pathways is largely unknown. Here, we demonstrate that the loss of function of geminin gave rise to narrower somites as a result of derepressed Fgf8 gradient in the PSM and tail bud. Furthermore, in geminin morphants, the somite boundary could not form properly but the oscillation of cyclic genes was normal, displaying the blurry somitic boundary and disturbed somite polarity along the AP axis. In mechanism, these manifestations were mediated by the disrupted association of the geminin/Brg1 complex with intron 3 of mib1. The latter interaction was found to positively regulate mib1 transcription, Notch activity, and sequential somite segmentation during somitogenesis. In addition, geminin was also shown to regulate the expression of deltaD in mib1-independent way. Collectively, our data for the first time demonstrate that geminin regulates Fgf8 and Notch signaling to regulate somite segmentation during somitogenesis.

Coordinated directional outgrowth and pattern formation by integration of Wnt5a and Fgf signaling in planar cell polarity.

Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. Planar cell polarity (PCP) signaling is emerging as a crucial evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of a Wnt signaling gradient in epithelial tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is regulated in a coordinated manner with embryonic patterning during morphogenesis. Here, in mouse developing limbs, we find that apical ectoderm ridge-derived Fgfs required for limb patterning regulate PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that the Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, and that Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is regulated by coordination of directional growth and patterning through integration of Wnt5a and Fgf signaling.

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

Different regulation of limb development by p63 transcript variants.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus.

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals.

Perinatal midline astrocyte development is impaired in fibroblast growth factor 8 hypomorphic mice.

Our previous studies showed that Fgf8 mutations can cause Kallmann syndrome (KS), a form of congenital hypogonadotropic hypogonadism, in which patients do not undergo puberty and are infertile. Interestingly, some KS patients also have agenesis of the corpus callosum (ACC) suggesting that KS pathology is not limited to reproductive function. Here, we asked whether FGF8 dysfunction is the underlying cause of ACC in some KS patients. Indeed, early studies in transgenic mice with Fgf8 mutations reported the presence of failed or incomplete corpus callosum formation. Additional studies in transgenic mice showed that FGF8 function most likely prevents the prenatal elimination of glial fibrillary acidic protein (GFAP)-immunoreactive (IR) glial cells in the indusium griseum (IG) and midline zipper (MZ), two anterior-dorsal midline regions required for corpus callosum formation (i.e., between embryonic days (E) 15.5-18.5). Here, we tested the hypothesis that FGF8 function is critical for the survival of the GFAP-IR midline glial cells. First, we measured the incidence of apoptosis in the anterior-dorsal midline region in Fgf8 hypomorphic mice during embryonic corpus callosum formation. Second, we quantified the GFAP expression in the anterior-dorsal midbrain region during pre- and postnatal development, in order to study: 1) how Fgf8 hypomorphy disrupts prenatal GFAP-IR midline glial cell development, and 2) whether Fgf8 hypomorphy continues to disrupt postnatal GFAP-IR midline glial cell development. Our results indicate that perinatal FGF8 signaling is important for the timing of the onset of anterior-dorsal Gfap expression in midline glial cells suggesting that FGF8 function regulates midline GFAP-IR glial cell development, which when disrupted by Fgf8 deficiency prevents the formation of the corpus callosum. These studies provide an experimentally-based mechanistic explanation as to why corpus callosum formation may fail in KS patients with deficits in FGF signaling.

Mechanisms of fibroblast growth factor signaling in the ovarian follicle.

Fibroblast growth factors (FGFs) have been shown to alter growth and differentiation of reproductive tissues in a variety of species. Within the female reproductive tract, the effects of FGFs have been focused on the ovary, and the most studied one is FGF2, which stimulates granulosa cell proliferation and decreases differentiation (decreased steroidogenesis). Other FGFs have also been implicated in ovarian function, and this review summarizes the effects of members of two subfamilies on ovarian function; the FGF7 subfamily that also contains FGF10, and the FGF8 subfamily that also contains FGF18. There are data to suggest that FGF8 and FGF18 have distinct actions on granulosa cells, despite their apparent similar receptor binding properties. Studies of non-reproductive developmental biology also indicate that FGF8 is distinct from FGF18, and that FGF7 is also distinct from FGF10 despite similar receptor binding properties. In this review, the potential mechanisms of differential action of FGF7/FGF10 and FGF8/FGF18 during organogenesis will be reviewed and placed in the context of follicle development. A model is proposed in which FGF8 and FGF18 differentially activate receptors depending on the properties of the extracellular matrix in the follicle.

Fibroblast growth factor 8 regulates postnatal development of paraventricular nucleus neuroendocrine cells.

BACKGROUND: Fibroblast growth factors (FGFs) are crucial signaling molecules that direct the development of the vertebrate brain. FGF8 gene signaling in particular, may be important for the development of the hypothalamus-pituitary-adrenal (HPA)-axis. Indeed, newborn Fgf8 hypomorphic mice harbor a major reduction in the number of vasopressin (VP) neurons in the paraventricular nucleus (PVN), the central output component of the HPA-axis. Additionally, recent studies indicated that adult heterozygous ((+/neo)) Fgf8 hypomorphic mice exhibit more anxiety-like behaviors than wildtype (WT) mice. These studies led us to investigate whether Fgf8 hypomorphy abrogated VP and/or corticotropin-releasing hormone (CRH) neuronal development in the postnatal day (PN) 21 and adult mouse PVN. Furthermore, we studied whether Fgf8 hypomorphy disrupted HPA responsiveness in these mice. METHODS: Using immunohistochemistry, we examined the development of VP and CRH neurons located in the PVN of PN 21 and adult Fgf8 (+/neo) mice. Moreover, we used a restraint stress (RS) paradigm and measured corticosterone levels with enzyme immunoassays in order to assess HPA axis activation. RESULTS: The number of VP neurons in the PVN did not differ between WT and Fgf8 (+/neo) mice on PN 21 and in adulthood. In contrast, CRH immunoreactivity was much higher in Fgf8 (+/neo) mice than in WT mice on PN 21, this difference was no longer shown in adult mice. RS caused a higher increase in corticosterone levels in adult Fgf8 (+/neo) mice than in WT mice after 15 min, but no difference was seen after 45 min. CONCLUSIONS: First, Fgf8 hypomorphy did not eliminate VP and CRH neurons in the mouse PVN, but rather disrupted the postnatal timing of neuropeptide expression onset in PVN neurons. Second, Fgf8 hypomorphy may, in part, be an explanation for affective disorders involving hyperactivity of the HPA axis, such as anxiety.

Gremlin1 induces anterior-posterior limb bifurcations in developing Xenopus limbs but does not enhance limb regeneration.

Gremlin1 (grem1) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self-regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anterior posterior and proximal distal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 (G) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anterior posterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs.

Fgf signaling controls the telencephalic distribution of Fgf-expressing progenitors generated in the rostral patterning center.

BACKGROUND: The rostral patterning center (RPC) secretes multiple fibroblast growth factors (Fgfs) essential for telencephalon growth and patterning. Fgf expression patterns suggest that they mark functionally distinct RPC subdomains. We generated Fgf8(CreER) and Fgf17(CreER) mice and used them to analyze the lineages of Fgf8- versus Fgf17-expressing RPC cells. RESULTS: Both lineages contributed to medial structures of the rostroventral telencephalon structures including the septum and medial prefrontral cortex. In addition, RPC-derived progenitors were observed in other regions of the early telencephalic neuroepithelium and generated neurons in the olfactory bulb, neocortex, and basal ganglia. Surprisingly, Fgf8(+) RPC progenitors generated the majority of basal ganglia cholinergic neurons. Compared to the Fgf8 lineage, the Fgf17 lineage was more restricted in its early dispersion and its contributions to the telencephalon. Mutant studies suggested that Fgf8 and Fgf17 restrict spread of RPC progenitor subpopulations. CONCLUSIONS: We identified the RPC as an important source of progenitors that contribute broadly to the telencephalon and found that two molecularly distinct progenitor subtypes in the RPC make different contributions to the developing forebrain.

FGF8-FGFR1 signaling acts as a niche factor for maintaining undifferentiated spermatogonia in the mouse.

In mammalian testes, spermatogonial stem cells (SSCs) maintain spermatogenesis over a long period of time by undergoing self-renewal and differentiation. SSCs are among the most primitive of spermatogenic cells (undifferentiated spermatogonia), and their activities are strictly regulated by extrinsic niche factors. However, the factors that constitute a testicular niche remain poorly understood. In this study, we demonstrate that fibroblast growth factor (FGF) signaling maintains undifferentiated spermatogonia through activating ERK1/2 signaling in vivo. Undifferentiated spermatogonia comprise GFRA1(+) and NANOS3(+) subpopulations, which are likely to undergo self-renewal and enter the differentiation pathway, respectively. In the testis, Fgfr1 was expressed in the entire population of undifferentiated spermatogonia, and deleting FGFR1 in spermatogenic cells partially inactivated ERK1/2 and resulted in reduced numbers of both GFRA1(+) and NANOS3(+) cells. In addition, Fgf8 was expressed in spermatogenic cells, and loss- and gain-of-function models of FGF8 demonstrated that FGF8 positively regulated the numbers of undifferentiated spermatogonia through FGFR1, particularly among NANOS3(+) cells. Finally we show a possible involvement of FGF signaling in the reversion from NANOS3(+) into GFRA1(+) undifferentiated spermatogonia. Taken together, our data suggest that FGF signaling is an important component of the testicular niche and has a unique function for maintaining undifferentiated spermatogonia.

Tbx1 modulates endodermal and mesodermal differentiation from mouse induced pluripotent stem cells.

The T-box transcriptional factor (Tbx) family of transcriptional factors has distinct roles in a wide range of embryonic differentiation or response pathways. Tbx1, a T-box transcription factor, is an important gene for the human congenital disorder 22q11.2 deletion syndrome. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem-cell-based therapies. In this study, we generated iPSCs from Tbx1(-/-) and Tbx1(+/+) fibroblasts and investigated the spontaneous differentiation potential of iPSCs by detailed lineage analysis of the iPSC-derived embryoid bodies. Undifferentiated Tbx1(-/-) and Tbx1(+/+) iPSCs showed similar expression levels of pluripotent markers. The ability of the Tbx1(-/-) iPSCs to generate endodermal and mesodermal lineages was compromised upon spontaneous differentiation into embryonic bodies. Restoration of Tbx1 expression in the Tbx1(-/-) iPSCs to normal levels using an inducible lentiviral system rescued these cells from the potential of defective differentiation. Interestingly, overexpression of Tbx1 in the Tbx1(-/-) iPSCs to higher levels than in the Tbx1(+/+) iPSCs again led to a defective differentiation potential. Additionally, we observed that expression of fibroblast growth factor (FGF) 10 and FGF8 was downregulated in the Tbx1(-/-) iPSC-derived cells, which suggests that Tbx1 regulates the expression of FGFs. Taken together, our results implicated the Tbx1 level as an important determinant of endodermal and mesodermal lineage differentiation during embryonic development.

Local apoptosis modulates early mammalian brain development through the elimination of morphogen-producing cells.

Apoptotic cells are observed in the early developing brain. Apoptosis deficiency is proposed to cause brain overgrowth, but here we show that brain malformations in apoptosis-deficient mutants are due to insufficient brain ventricle expansion as a result of uncompleted cranial neural tube closure. Apoptosis eliminates Fgf8-expressing cells in the anterior neural ridge (ANR), which acts as an organizing center of the forebrain by producing FGF8 morphogen. Deficiency of apoptosis leads to the accumulation of undead and nonproliferative cells in the ventral part of the ANR. The undead cells in apoptosis-deficient mutants express Fgf8 continuously, which perturbs gene expression in the ventral forebrain. Thus, apoptosis within a specific subdomain of the ANR is required for correct temporal elimination of an FGF8-producing region within a limited developmental time window, thereby ensuring proper forebrain development.

Subpopulation of proepicardial cells is derived from the somatic mesoderm in the chick embryo.

RATIONALE: The proepicardium (PE) is a transient structure forming at the venous pole of the heart and gives rise to the epicardium, fibroblasts, and smooth muscle cells. The embryological origin of the PE is presently unclear. Asymmetrical formation of the PE on the right inflow tract is a conserved feature of many vertebrate embryos, and in the chicken is under the control of fibroblast growth factor 8 and snail homolog 1. OBJECTIVE: To gain further insight into the process of asymmetrical PE formation, we studied the role of TWIST1 during PE formation in the chick embryo. METHODS AND RESULTS: TWIST1 is asymmetrically expressed on the right side in the somatic mesoderm under the control of snail homolog 1. Fate mapping experiments revealed a contribution of the somatic mesoderm to the PE. After colonization of the heart, this cell lineage gives rise to the epicardium, smooth muscle cells, and potentially fibroblast. Suppression of TWIST1 function in the right coelomic cavity caused a severe disruption of the villous protrusions of the PE and Wilms tumor 1 and transcription factor 21 expression. Rescue with the corresponding mouse cDNA normalized gene expression and PE morphology. Forced expression of TWIST1 on the left side induced ectopic expression domains of Wilms tumor 1 and transcription factor 21. CONCLUSIONS: A significant proportion of the PE has its origin outside of the currently proposed domain in the splanchnic layer of the lateral plate mesoderm. The phenotype in embryos subjected to TWIST1 loss- or gain-of-function suggests an important contribution of somatic mesoderm to the mesothelial cell layer of the PE.

Molecular analysis of regulative events in the developing chick limb.

The developing chick limb has the remarkable ability to regulate for the loss of large amounts of mesenchyme and maintain a normal limb pattern in early (Hamburger and Hamilton Stage 19; E3) limbs. How the limb can regulate for tissue loss and why this ability is lost as development proceeds (after Hamburger and Hamilton Stage 21; E3.5) is unclear. We have investigated the origins of cells involved in regulative processes and, for the first time, the molecular changes occurring, and find striking differences between developmental time points just 0.5 days apart. We demonstrate that subtle changes in cell dispersal and cell proliferation occur in HH St21 limbs but not in HH St19 limbs and also demonstrate that there is no net replacement of removed tissue at either HH St21 or St19. We further show that changes in the Fgf8/Shh/Bmp4/Gremlin signaling pathway together with the appearance of distal Hox gene activation coincide with the limbs' ability to regulate for large tissue loss. We also demonstrate that following small tissue loss, limbs can regulate for missing tissue to produce normal pattern with no net replacement of missing tissue, as seen in limbs following large tissue loss. Our results indicate the regulative ability of the limb is not due to changes in cell proliferation, cell lineage nor replacement of the missing tissue - regulative ability is reliant upon the signaling environment remaining.