An Early Cortical Progenitor-Specific Mechanism Regulates Thalamocortical Innervation.

The cortical subplate is critical in regulating the entry of thalamocortical sensory afferents into the cortex. These afferents reach the subplate at embryonic day (E)15.5 in the mouse, but "wait" for several days, entering the cortical plate postnatally. We report that when transcription factor LHX2 is lost in E11.5 cortical progenitors, which give rise to subplate neurons, thalamocortical afferents display premature, exuberant ingrowth into the E15.5 cortex. Embryonic mutant subplate neurons are correctly positioned below the cortical plate, but they display an altered transcriptome and immature electrophysiological properties during the waiting period. The sensory thalamus in these cortex-specific Lhx2 mutants displays atrophy and by postnatal day (P) 7, sensory innervation to the cortex is nearly eliminated leading to a loss of the somatosensory barrels. Strikingly, these phenotypes do not manifest if LHX2 is lost in postmitotic subplate neurons, and the transcriptomic dysregulation in the subplate resulting from postmitotic loss of LHX2 is vastly distinct from that seen when LHX2 is lost in progenitors. These results demonstrate a mechanism operating in subplate progenitors that has profound consequences on the growth of thalamocortical axons into the cortex.SIGNIFICANCE STATEMENT Thalamocortical nerves carry sensory information from the periphery to the cortex. When they first grow into the embryonic cortex, they "wait" at the subplate, a structure critical for the guidance and eventual connectivity of thalamic axons with their cortical targets. How the properties of subplate neurons are regulated is unclear. We report that transcription factor LHX2 is required in the progenitor "mother" cells of the cortical primordium when they are producing their "daughter" subplate neurons, in order for the thalamocortical pathway to wait at the subplate. Without LHX2 function in subplate progenitors, thalamocortical axons grow past the subplate, entering the cortical plate prematurely. This is followed by their eventual attrition and, consequently, a profound loss of sensory innervation of the mature cortex.

An evolutionarily conserved Lhx2-Ldb1 interaction regulates the acquisition of hippocampal cell fate and regional identity.

The protein co-factor Ldb1 regulates cell fate specification by interacting with LIM-homeodomain (LIM-HD) proteins in a tetrameric complex consisting of an LDB:LDB dimer that bridges two LIM-HD molecules, a mechanism first demonstrated in the Drosophila wing disc. Here, we demonstrate conservation of this interaction in the regulation of mammalian hippocampal development, which is profoundly defective upon loss of either Lhx2 or Ldb1 Electroporation of a chimeric construct that encodes the Lhx2-HD and Ldb1-DD (dimerization domain) in a single transcript cell-autonomously rescues a comprehensive range of hippocampal deficits in the mouse Ldb1 mutant, including the acquisition of field-specific molecular identity and the regulation of the neuron-glia cell fate switch. This demonstrates that the LHX:LDB complex is an evolutionarily conserved molecular regulatory device that controls complex aspects of regional cell identity in the developing brain.

Hierarchical genetic interactions between FOXG1 and LHX2 regulate the formation of the cortical hem in the developing telencephalon.

During forebrain development, a telencephalic organizer called the cortical hem is crucial for inducing hippocampal fate in adjacent cortical neuroepithelium. How the hem is restricted to its medial position is therefore a fundamental patterning issue. Here, we demonstrate that Foxg1-Lhx2 interactions are crucial for the formation of the hem. Loss of either gene causes a region of the cortical neuroepithelium to transform into hem. We show that FOXG1 regulates Lhx2 expression in the cortical primordium. In the absence of Foxg1, the presence of Lhx2 is sufficient to suppress hem fate, and hippocampal markers appear selectively in Lhx2-expressing regions. FOXG1 also restricts the temporal window in which loss of Lhx2 results in a transformation of cortical primordium into hem. Therefore, Foxg1 and Lhx2 form a genetic hierarchy in the spatiotemporal regulation of cortical hem specification and positioning, and together ensure the normal development of this hippocampal organizer.

Novel functions of LHX2 and PAX6 in the developing telencephalon revealed upon combined loss of both genes.

Patterning of the telencephalic neuroepithelium is a tightly regulated process controlled by transcription factors and signalling molecules. The cortical primordium is flanked by two signalling centres, the hem medially, and the antihem laterally. The hem induces the formation of the hippocampus in adjacent neuroepithelium. Therefore, the position of the hem defines the position of the hippocampus in the brain. The antihem is positioned at the boundary between the dorsal and ventral telencephalon and proposed to provide patterning cues during development. LIM-homeodomain (LIM-HD) transcription factor LHX2 suppresses both hem and antihem fate in the cortical neuroepithelium. Upon loss of Lhx2, medial cortical neuroepithelium is transformed into hem, whereas lateral cortical neuroepithelium is transformed into antihem. Here, we show that transcription factor PAX6, known to regulate patterning of the lateral telencephalon, restricts this tissue from transforming into hem upon loss of Lhx2. When Lhx2 and Pax6 are both deleted, the cortical hem expands to occupy almost the complete extent of the cortical primordium, indicating that both factors act to suppress hem fate in the lateral telencephalon. Furthermore, the shift in the pallial-subpallial boundary and absence of the antihem, observed in the Pax6 mutant, are both restored in the Lhx2; Pax6 double mutant. Together, these results not only reveal a novel function for LHX2 in regulating dorsoventral patterning in the telencephalon, but also identify PAX6 as a fundamental regulator of where the hem can form, and therefore implicate this molecule as a determinant of hippocampal positioning.

Decrease in Expression of HOXA10 in the Decidua After Embryo Implantation Promotes Trophoblast Invasion.

An important step toward successful pregnancy involves invasion of the trophoblast cells into the decidua for placentation. Herein, we show that in the human and baboon decidua HOXA10 expression is downregulated after implantation and that this reduction is most prominent in the decidual cells juxtaposed to the invading placental villi. The supernatants derived from HOXA10-depleted human decidual cells increase the invasiveness of the trophoblast cell lines ACH-3P and JEG3 in vitro; this increase is due to higher expression and activity of matrix metalloproteases (MMPs) and reduced expression of tissue inhibitors of MMPs in both the cell lines. The proinvasive ability of HOXA10-depleted decidual cells is due to increased levels and secretion of leukemia inhibitor factor (LIF) and interleukin (IL)-6. Both these cytokines individually promote invasion of ACH-3P and JEG3 cell by increasing the activities of MMPs and decreasing mRNA levels of TIMPs. Finally, we demonstrate that the supernatants derived from HOXA10-depleted decidual cell-phosphorylated STAT3 (Tyr 705) and knocking down STAT3 in ACH-3P and JEG3 cells restrained the invasion mediated by supernatants derived from HOXA10-depleted decidual cells. These results imply that STAT3 activity is essential and sufficient to promote invasion in response to downregulation of HOXA10 in decidual cells. We propose that downregulation of HOXA10 in the decidual cells promotes the expression of LIF and IL-6, which, in a paracrine manner, activates STAT3 in the trophoblast cells, leading to an increase in MMPs to facilitate invasion.

Decidual Control of Trophoblast Invasion.

At the time of implantation, the trophoblast cells of the embryo adhere and then invade into the maternal endometrium and eventually establish placentation. The endometrium at the same time undergoes decidualization, which is essential for successful pregnancy. While the NK cells of the decidua have been implicated to play a key role in trophoblast invasion, few evidence are now available to demonstrate a pro-invasive property of decidual stromal cells. Secretions from decidualized endometrial stromal cells promote invasion of primary trophoblasts and model cell lines by activating proteases and altering expression of adhesion-related molecules. The decidual secretions contain high amounts of pro-invasive factors that include IL-1ß, IL-5, IL-6, IL-7, IL-8, IL-9, IL-13, IL-15, Eotaxin CCL11, IP-10 and RANTES, and anti-invasive factors IL-10, IL-12 and VEGF. It appears that these decidual factors promote invasion by regulating the protease pathways and integrin expression utilizing the STAT pathways in the trophoblast cells. At the same time the decidua also seem to secrete some anti-invasive factors that are antagonist to the matrix metalloproteinases and/or are activators of tissue inhibitors of metalloproteinases. This might be essential to neutralize the effects of the invasion-promoting factors and restrain overinvasion. It is tempting to propose that during the course of pregnancy, the decidua must balance the production of these pro and anti-invasive molecules and such harmonizing production would allow a timely and regulated invasion.

Lhx2 regulates a cortex-specific mechanism for barrel formation.

LIM homeodomain transcription factors are critical regulators of early development in multiple systems but have yet to be examined for a role in circuit formation. The LIM homeobox gene Lhx2 is expressed in cortical progenitors during development and also in the superficial layers of the neocortex in maturity. However, analysis of Lhx2 function at later stages of cortical development has been hampered by severe phenotypes associated with early loss of function. We identified a particular Cre-recombinase line that acts in the cortical primordium after its specification is complete, permitting an analysis of Lhx2 function in neocortical lamination, regionalization, and circuit formation by selective elimination of Lhx2 in the dorsal telencephalon. We report a profound disruption of cortical neuroanatomical and molecular features upon loss of Lhx2 in the cortex from embryonic day 11.5. A unique feature of cortical circuitry, the somatosensory barrels, is undetectable, and molecular patterning of cortical regions appears disrupted. Surprisingly, thalamocortical afferents innervate the mutant cortex with apparently normal regional specificity. Electrophysiological recordings reveal a loss of responses evoked by stimulation of individual whiskers, but responses to simultaneous stimulation of multiple whiskers were present, suggesting that thalamic afferents are unable to organize the neurocircuitry for barrel formation because of a cortex-specific requirement of Lhx2. We report that Lhx2 is required for the expression of transcription factor paired box gene 6, axon guidance molecule Ephrin A5, and the receptor NMDA receptor 1. These genes may mediate Lhx2 function in the formation of specialized neurocircuitry necessary for neocortical function.

AP-1 transcription factors, mucin-type molecules and MMPs regulate the IL-11 mediated invasiveness of JEG-3 and HTR-8/SVneo trophoblastic cells.

This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo cells.

Endometrial biology during trophoblast invasion.

Attainment of successful implantation depends upon the synchronized changes in the endometrium before and after the arrival of blastocyst into the uterine cavity. The cues obtained from the receptive endometrium helps in proliferation and differentiation of the trophoblast cells. During the course of invasive differentiation, the trophoblast cells undergo several morphological, biochemical and molecular changes to gain the invasive capabilities. In turn, close apposition of the developing embryo brings out functional and morphological changes into the hormone primed receptive endometrium. Global gene expression profiling of the endometrium in response to the developing embryo or in response to the pregnancy hormone, human chorionic gonadotropin, in primate and human models, suggest that the endometrial-embryo cross-talk mainly influences three biological processes. Biological processes getting influenced by the blastocyst "signals" are associated with immunomodulation, biosensing and invasion. Pro- and anti-invasive paracrine factors expressed by different endometrial cell populations regulate the trophoblast invasion through activation of diverse signaling pathways. Identification of the gene signatures involved in embryo-endometrial dialogue would enhance our understanding about the pathologies like miscarriages and endometriosis.

Decidualized endometrial stromal cell derived factors promote trophoblast invasion.

OBJECTIVE: To evaluate the effects of decidua-derived factors on trophoblast invasion. DESIGN: Experimental study. SETTINGS: Research institute. PATIENT(S): In vitro decidualized human endometrial cells, trophoblast cell lines JEG-3, and ACH-3P. INTERVENTION(S): The effect of decidual conditioned medium (DCM) on the invasion of trophoblast cells lines via JEG-3 and ACH-3P was investigated using a Matrigel invasion assay. The changes in expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrins in response to DCM in the trophoblast cells was also evaluated. MAIN OUTCOME MEASURE(S): Response of the trophoblast cells to the conditioned medium from decidual cells in terms of their invasive capability, and expression on invasion related molecules was measured. RESULT(S): DCM increased the invasion of both the cell lines by approximately 1.8-2.2-fold, compared with control condition medium. The increase in invasion was associated with elevated levels of MMP2, MMP3, and MMP9 mRNA and increased activity of MMP2 and MMP9 in DCM-treated ACH-3P, but not JEG-3 cells. DCM treatment led to a reduction in TIMP1 and TIMP3 and increased TIMP2 mRNA in JEG-3, cells but not ACH-3P cells. Compared with CCM-treated controls, DCM treatment led to a significant increase in the mRNA expression of integrin α5 and α6, but not integrin αV subunit in both cell lines. CONCLUSION(S): Decidua-derived factors increase the invasiveness of trophoblast cell lines and alter the expression of integrins, MMPs, and TIMPs.

Regulation of decidualization, interleukin-11 and interleukin-15 by homeobox A 10 in endometrial stromal cells.

Cytokine production by the endometrial stromal and decidual cells is essential for successful differentiation of the endometrial stromal cells and uterine leukocytes to sustain pregnancy. Interleukin-11 and -15 (IL-11 and IL-15) secreted by the stromal and decidual cells are two key modulators of the process of decidualization and natural killer cell (NK) activity in the uterus and are essential for pregnancy. However, limited information exists on the maternal factors that regulate the production of these cytokines by the stromal cells. In this study, we investigated the role of homeobox A10 (HOXA10) in the regulation of expression of genes encoding the decidualization markers insulin-like growth factor binding protein-1 (IGFBP1), prolactin and the cytokines IL-11 and IL-15 by endometrial stromal and decidual cells in vitro. The results demonstrated that the expression of IGFBP1, Prolactin (PRL), HOXA10, IL11, and IL15 are co-regulated during steroid hormone-mediated decidualization of human endometrial stromal cells in vitro. In the predecidual cells, downregulation of HOXA10 by siRNA suppresses IGFBP1 and IL15, but increases IL11 expression. In the decidualized cells, knocking down HOXA10 inhibits IGFBP1 and PRL expression but elevates the expression of IL11 and IL15. In addition, our data also demonstrate that transient inhibition of HOXA10 expression in the predecidual cells does not influence its ability to subsequently decidualize or affect cytokine expression, suggesting that steroid hormone-mediated decidualization and cytokine production in vitro does not require HOXA10 preconditioning.

HOXA10 signals on the highway through pregnancy.

Implantation represents the remarkable synchronization between the development of the embryo and the differentiation of the endometrium. It depends on uterine-dependent and embryo-specific events, which are critically and sequentially coordinated. A plethora of molecules have been identified which play major roles before and after embryo implantation. In recent years HomeoboxA10 (HOXA10) has emerged as one of the most promising candidates which regulate the events occurring in the maternal compartment for successful establishment of pregnancy. HOXA10 is a transcription factor that is crucial for development and patterning of the uterus during embryogenesis. In the adult endometrium, HOXA10 is expressed in a menstrual cycle dependent manner and it is regulated by ovarian steroid hormones and embryonic signals, HOXA10 is required for uterine receptivity and implantation, and is a key regulator of decidualization. In the decidua, HOXA10 is involved in regulation of cell cycle and local immunomodulation. The present review summarizes the events that are regulated by HOXA10 in embryo implantation and decidualization.