Shaping Morphogen Gradients by Proteoglycans

During development, secreted morphogens such as Wnt, Hedgehog (Hh), and BMP emit from their producing cells in a morphogenetic field, and specify different cell fates in a direct concentration-dependent manner. Understanding how morphogens form their concentration gradients to pattern tissues has been a central issue in developmental biology. Various experimental studies from Drosophila have led to several models to explain the formation of morphogen gradients. Over the past decade, one of the main findings in this field is the characterization of heparan sulfate proteoglycan (HSPG) as an essential regulator for morphogen gradient formation. Genetic and cell biological studies have showed that HSPGs can regulate morphogen activities at various steps including control of morphogen movement, signaling, and intracellular trafficking. Here, we review these data, highlighting recent findings that reveal mechanistic roles of HSPGs in controlling morphogen gradient formation.Embryonic development involves many spatial and temporal patterns of cell and tissue organization. These patterning processes are controlled by gradients of morphogens, the “form-generating substances” (Tabata and Takei 2004; Lander 2007). Secreted morphogen molecules, including members of Wnt, Hedgehog (Hh), and transforming growth factor-β (TGF-β) families, are generated from organizing centers and form concentration gradients to specify distinct cell fates in a concentration-dependent manner. Understanding how morphogen gradients are established during development has been a central question in developmental biology. Over the past decade, studies in both Drosophila and vertebrates have yielded important insights in this field. One of the important findings is the characterization of heparan sulfate proteoglycan (HSPG) as an essential regulator for morphogen gradient formation. In this review, we first discuss various models for morphogen movement. Then, we focus on the functions of HSPGs in morphogen movement, signaling, and trafficking.     

Forming and Interpreting Gradients in the Early Xenopus Embryo

The amphibian embryo provides a powerful model system to study morphogen gradients because of the ease with which it is possible to manipulate the early embryo. In particular, it is possible to introduce exogenous sources of morphogen, to follow the progression of the signal, to monitor the cellular response to induction, and to up- or down-regulate molecules that are involved in all aspects of long-range signaling. In this article, I discuss the evidence that gradients exist in the early amphibian embryo, the way in which morphogens might traverse a field of cells, and the way in which different concentrations of morphogens might be interpreted to activate the expression of different genes.The idea that a morphogen gradient activates the expression of different genes at different concentrations was perhaps stated most clearly by Wolpert''s French flag model, in which a graded signal activates the expression of “blue,” “white,” and “red” genes at high, intermediate, and low concentrations (Wolpert 1969). Since that original work, great progress has been made in identifying morphogens and their target genes and it is now clear that the spatial pattern of gene expression in the developing embryo is frequently established by graded signals of this sort. But many questions remain, and in particular little is known about how gradients are established in the embryo with the necessary precision and how cells interpret different concentrations of morphogen to activate different genes. I discuss these issues with respect to mesoderm induction in the developing amphibian embryo.     

Morphogen Gradient Formation

How morphogen gradients are formed in target tissues is a key question for understanding the mechanisms of morphological patterning. Here, we review different mechanisms of morphogen gradient formation from theoretical and experimental points of view. First, a simple, comprehensive overview of the underlying biophysical principles of several mechanisms of gradient formation is provided. We then discuss the advantages and limitations of different experimental approaches to gradient formation analysis.How a multicellular organism develops from a single fertilized cell has fascinated people throughout history. By looking at chick embryos of different developmental stages, Aristotle first noted that development is characterized by growing complexity and organization of the embryo (Balme 2002). During the 19th century, two events were recognized as key in development: cell proliferation and differentiation. Driesch first noted that to form organisms with correct morphological pattern and size, these processes must be controlled at the level of the whole organism. When he separated two sea urchin blastomeres, they produced two half-sized blastula, showing that cells are potentially independent, but function together to form a whole organism (Driesch 1891, 1908). Morgan noted the polarity of organisms and that regeneration in worms occurs with different rates at different positions. This led him to postulate that regeneration phenomena are influenced by gradients of “formative substances” (Morgan 1901).The idea that organisms are patterned by gradients of form-providing substances was explored by Boveri and Hörstadius to explain the patterning of the sea urchin embryo (Boveri 1901; Hörstadius 1935). The discovery of the Spemann organizer, i.e., a group of dorsal cells that when grafted onto the opposite ventral pole of a host gastrula induce a secondary body axis (Spemann and Mangold 1924), suggested that morphogenesis results from the action of signals that are released from localized groups of cells (“organizing centers”) to induce the differentiation of the cells around them (De Robertis 2006). Child proposed that these patterning “signals” represent metabolic gradients (Child 1941), but the mechanisms of their formation, regulation, and translation into pattern remained elusive.In 1952, Turing showed that chemical substances, which he called morphogens (to convey the idea of “form producers”), could self-organize into spatial patterns, starting from homogenous distributions (Turing 1952). Turing’s reaction–diffusion model shows that two or more morphogens with slightly different diffusion properties that react by auto- and cross-catalyzing or inhibiting their production, can generate spatial patterns of morphogen concentration. The reaction–diffusion formalism was used to model regeneration in hydra (Turing 1952), pigmentation of fish (Kondo and Asai 1995; Kondo 2002), and snails (Meinhardt 2003).At the same time that Turing showed that pattern can self-organize from the production, diffusion, and reaction of morphogens in all cells, the idea that morphogens are released from localized sources (“organizers” à la Spemann) and form concentration gradients was still explored. This idea was formalized by Wolpert with the French flag model for generation of positional information (Wolpert 1969). According to this model, morphogen is secreted from a group of source cells and forms a gradient of concentration in the target tissue. Different target genes are expressed above distinct concentration thresholds, i.e., at different distances to the source, hence generating a spatial pattern of gene expression (Fig. 1C).Open in a separate windowFigure 1.Tissue geometry and simplifications. (A) Gradients in epithelia (left) and mesenchymal tissues (right). Because of symmetry considerations, one row of cells (red outline) is representative for the whole gradient. (B) Magnified view of the red row of cells shown in A. Cells with differently colored nuclei (brown, orange, and blue) express different target genes. (C) A continuum model in which individual cells are ignored and the concentration is a function of the positions x. The morphogen activates different target genes above different concentration thresholds (brown and orange).Experiments in the 1970s and later confirmed that tissues are patterned by morphogen gradients. Sander showed that a morphogen released from the posterior cytoplasm specifies anterioposterior position in the insect egg (Sander 1976). Chick wing bud development was explained by a morphogen gradient emanating from the zone of polarizing activity to specify digit positions (Saunders 1972; Tickle, et al. 1975; Tickle 1999). The most definitive example of a morphogen was provided with the identification of Bicoid function in the Drosophila embryo (Nüsslein-Volhard and Wieschaus 1980; Frohnhöfer and Nüsslein-Volhard 1986; Nüsslein-Volhard et al. 1987) and the visualization of its gradient by antibody staining (Driever and Nüsslein-Volhard 1988b, 1988a; reviewed in Ephrussi and St Johnston 2004). Since then, many examples of morphogen gradients acting in different organs and species have been found.In an attempt to understand pattern formation in more depth, quantitative models of gradient formation have been developed. An early model by Crick shows that freely diffusing morphogen produced in a source cell and destroyed in a “sink” cell at a distance would produce a linear gradient in developmentally relevant timescales (Crick 1970). Today, it is known that a localized “sink” is not necessary for gradient formation: Gradients can form if all cells act as sinks and degrade morphogen, or even if morphogen is not degraded at all. Here, we review different mechanisms of gradient formation, the properties of these gradients, and the implications for patterning. We discuss the theory behind these mechanisms and the supporting experimental data.     

Robust Generation and Decoding of Morphogen Gradients

Morphogen gradients play a key role in multiple differentiation processes. Both the formation of the gradient and its interpretation by the receiving cells need to occur at high precision to ensure reproducible patterning. This need for quantitative precision is challenged by fluctuations in the environmental conditions and by variations in the genetic makeup of the developing embryos. We discuss mechanisms that buffer morphogen profiles against variations in gene dosage. Self-enhanced morphogen degradation and pre-steady-state decoding provide general means for buffering the morphogen profile against fluctuations in morphogen production rate. A more specific “shuttling” mechanism, which establishes a sharp and robust activation profile of a widely expressed morphogen, and enables the adjustment of morphogen profile with embryo size, is also described. Finally, we consider the transformation of the smooth gradient profile into sharp borders of gene expression in the signal-receiving cells. The integration theory and experiments are increasingly used, providing key insights into the system-level functioning of the developmental system.In order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues, cells need to receive information about their position within the field. During development, positional information is often conveyed by spatial gradients of morphogens (Wolpert 1989). In the presence of such gradients, cells are subject to different levels of morphogen, depending on their positions within the field, and activate, accordingly, one of several gene expression cassettes. The quantitative shape of the morphogen gradient is critical for patterning, with cell-fate boundaries established at specific concentration thresholds. Although these general features of morphogen-based patterning are universal, the range and form of the morphogen profile, and the pattern of induced target genes, vary significantly depending on the tissue setting and the signaling pathways used.The formation of a morphogen gradient is a dynamic process, influenced by the kinetics of morphogen production, diffusion, and degradation. These processes are tightly controlled through intricate networks of positive and negative feedback loops, which shape the gradient and enhance its reproducibility between individual embryos and developmental contexts. In the past three decades, many of the components comprising the morphogen signaling cascades have been identified and sorted into pathways, enabling one to start addressing seminal questions regarding their functionality: How is it that morphogen signaling is reproducible from one embryo to the next, despite fluctuations in the levels of signaling components, temperature differences, variations in size, or unequal distribution of components between daughter cells? Are there underlying mechanisms that assure a reproducible response? Are these mechanisms conserved across species, similar to the signaling pathways they control?In this review, we outline insights we gained by quantitatively analyzing the process of morphogen gradient formation. We focus on mechanisms that buffer morphogen profiles against fluctuations in gene dosage, and describe general means by which such buffering is enhanced. These mechanisms include self-enhanced morphogen degradation and pre-steady-state decoding. In addition, we describe a more specific “shuttling” mechanism that is used to generate a sharp and robust profile of a morphogen activity from a source that is broadly produced. We discuss the implication of the shuttling mechanism for the ability of embryos to adjust their pattern with size. Finally, we consider the transformation of the smooth gradient profile into sharp borders of gene expression in the signal-receiving cells.     

The Electrophysiological Underpinnings of Processing Gender Stereotypes in Language

Despite the widely documented influence of gender stereotypes on social behaviour, little is known about the electrophysiological substrates engaged in the processing of such information when conveyed by language. Using event-related brain potentials (ERPs), we examined the brain response to third-person pronouns (lei “she” and lui “he”) that were implicitly primed by definitional (passeggera _FEM “passenger”, pensionato _MASC “pensioner”), or stereotypical antecedents (insegnante “teacher”, conducente “driver”). An N400-like effect on the pronoun emerged when it was preceded by a definitionally incongruent prime (passeggera _FEM – lui; pensionato _MASC – lei), and a stereotypically incongruent prime for masculine pronouns only (insegnante – lui). In addition, a P300-like effect was found when the pronoun was preceded by definitionally incongruent primes. However, this effect was observed for female, but not male participants. Overall, these results provide further evidence for on-line effects of stereotypical gender in language comprehension. Importantly, our results also suggest a gender stereotype asymmetry in that male and female stereotypes affected the processing of pronouns differently.     

Forming Patterns in Development without Morphogen Gradients: Scattered Differentiation and Sorting Out

Few mechanisms provide alternatives to morphogen gradients for producing spatial patterns of cells in development. One possibility is based on the sorting out of cells that initially differentiate in a salt and pepper mixture and then physically move to create coherent tissues. Here, we describe the evidence suggesting this is the major mode of patterning in Dictyostelium. In addition, we discuss whether convergent evolution could have produced a conceptually similar mechanism in other organisms.A limited number of processes are thought to regulate the differentiation of specialized cell types and their organization to form larger scale structures, such as organs or limbs, during embryonic development. First, early embryological experiments revealed a patterning process that depends on special “organizing” regions in the embryo. This idea was encapsulated as “positional information” and led to the concept of morphogen gradients (Fig. 1) (Wolpert 1996). In addition, cytoplasmic determinants have been shown to direct development along different lines when they are partitioned unequally between daughter cells by asymmetric cell division (Betschinger and Knoblich 2004). Finally, short-range inductive signaling can specify cells at a local level and when reiterated produces highly ordered structures (Simpson 1990; Freeman 1997; Meinhardt and Gierer 2000).Open in a separate windowFigure 1.Alternative ways of patterning cells during development. (A) Patterning by “positional information”: A group of undifferentiated cells is patterned by a morphogen diffusing from a pre-established source, producing a concentration gradient. Cells respond according to the local morphogen concentration, becoming red, white, or blue. (B, C) Patterning without positional information: This is a two-step process in which different cell types first differentiate mixed up with each other, and then sort out. The initial differentiation can be controlled by strictly local interactions between the cells, as in lateral inhibition (B), or by a global signal to which cells respond with different sensitivities and whose concentration they regulate by negative feedback (C). Once sorting has occurred, the global inducer forms a reverse gradient, which could then convey positional information for further patterning events.The question then arises of whether evolution has devised any further global patterning mechanisms. One possibility that has been repeatedly considered, but not firmly established as a general mechanism, is based on sorting out. In this process, pattern is produced in two steps: (1) Different cell types are initially specified from a precursor pool independent of their position to produce a salt and pepper mixture and (2) the mixture of cell types is resolved into discrete tissues by the physical movement and sorting out of the cells (Fig. 1). Consequently, this mechanism does not involve positional information. However, it can actually provide the conditions under which positional signaling and morphogen gradients can arise, if the resolved tissues then act as sources and sinks for signal molecules.We first describe the powerful evidence that this alternative patterning process is used during the developmental cycle of the social amoeba Dictyostelium discoideum, and then consider the possibility that this patterning strategy may be used more widely.     

Twin Town in South Brazil: A Nazi's Experiment or a Genetic Founder Effect?

Cândido Godói (CG) is a small municipality in South Brazil with approximately 6,000 inhabitants. It is known as the “Twins'' Town” due to its high rate of twin births. Recently it was claimed that such high frequency of twinning would be connected to experiments performed by the German Nazi doctor Joseph Mengele. It is known, however, that this town was founded by a small number of families and therefore a genetic founder effect may represent an alternatively explanation for the high twinning prevalence in CG. In this study, we tested specific predictions of the “Nazi''s experiment” and of the “founder effect” hypotheses. We surveyed a total of 6,262 baptism records from 1959–2008 in CG catholic churches, and identified 91 twin pairs and one triplet. Contrary to the “Nazi''s experiment hypothesis”, there is no spurt in twinning between the years (1964–1968) when Mengele allegedly was in CG (P = 0.482). Moreover, there is no temporal trend for a declining rate of twinning since the 1960s (P = 0.351), and no difference in twinning among CG districts considering two different periods: 1927–1958 and 1959–2008 (P = 0.638). On the other hand, the “founder effect hypothesis” is supported by an isonymy analysis that shows that women who gave birth to twins have a higher inbreeding coefficient when compared to women who never had twins (0.0148, 0.0081, respectively, P = 0.019). In summary, our results show no evidence for the “Nazi''s experiment hypothesis” and strongly suggest that the “founder effect hypothesis” is a much more likely alternative for explaining the high prevalence of twinning in CG. If this hypothesis is correct, then this community represents a valuable population where genetic factors linked to twinning may be identified.     

Crystal structure of a trimeric form of the KV7.1 (KCNQ1) A‐domain tail coiled‐coil reveals structural plasticity and context dependent changes in a putative coiled‐coil trimerization motif

Coiled-coils are widespread protein–protein interaction motifs typified by the heptad repeat (abcdefg)_n in which “a” and “d” positions are hydrophobic residues. Although identification of likely coiled-coil sequences is robust, prediction of strand order remains elusive. We present the X-ray crystal structure of a short form (residues 583–611), “Q1-short,” of the coiled-coil assembly specificity domain from the voltage-gated potassium channel Kv7.1 (KCNQ1) determined at 1.7 Å resolution. Q1-short lacks one and half heptads present in a previously studied tetrameric coiled-coil construct, Kv7.1 585–621, “Q1-long.” Surprisingly, Q1-short crystallizes as a trimer. In solution, Q1-short self-assembles more poorly than Q1-long and depends on an R-h-x-x-h-E motif common to trimeric coiled-coils. Addition of native sequences that include “a” and “d” positions C-terminal to Q1-short overrides the R-h-x-x-h-E motif influence and changes assembly state from a weakly associated trimer to a strongly associated tetramer. These data provide a striking example of a naturally occurring amino sequence that exhibits context-dependent folding into different oligomerization states, a three-stranded versus a four-stranded coiled-coil. The results emphasize the degenerate nature of coiled-coil energy landscapes in which small changes can have drastic effects on oligomerization. Discovery of these properties in an ion channel assembly domain and prevalence of the R-h-x-x-h-E motif in coiled-coil assembly domains of a number of different channels that are thought to function as tetrameric assemblies raises the possibility that such sequence features may be important for facilitating the assembly of intermediates en route to the final native state.     

Health Risk or Resource? Gradual and Independent Association between Self-Rated Health and Mortality Persists Over 30 Years

Background

Poor self-rated health (SRH) is associated with increased mortality. However, most studies only adjust for few health risk factors and/or do not analyse whether this association is consistent also for intermediate categories of SRH and for follow-up periods exceeding 5–10 years. This study examined whether the SRH-mortality association remained significant 30 years after assessment when adjusting for a wide range of known clinical, behavioural and socio-demographic risk factors.

Methods

We followed-up 8,251 men and women aged ≥16 years who participated 1977–79 in a community based health study and were anonymously linked with the Swiss National Cohort (SNC) until the end of 2008. Covariates were measured at baseline and included education, marital status, smoking, medical history, medication, blood glucose and pressure.

Results

92.8% of the original study participants could be linked to a census, mortality or emigration record of the SNC. Loss to follow-up 1980–2000 was 5.8%. Even after 30 years of follow-up and after adjustment for all covariates, the association between SRH and all-cause mortality remained strong and estimates almost linearly increased from “excellent” (reference: hazard ratio, HR 1) to “good” (men: HR 1.07 95% confidence interval 0.92–1.24, women: 1.22, 1.01–1.46) to “fair” (1.41, 1.18–1.68; 1.39, 1.14–1.70) to “poor”(1.61, 1.15–2.25; 1.49, 1.07–2.06) to “very poor” (2.85, 1.25–6.51; 1.30, 0.18–9.35). Persons answering the SRH question with “don''t know” (1.87, 1.21–2.88; 1.26, 0.87–1.83) had also an increased mortality risk; this was pronounced in men and in the first years of follow-up.

Conclusions

SRH is a strong and “dose-dependent” predictor of mortality. The association was largely independent from covariates and remained significant after decades. This suggests that SRH provides relevant and sustained health information beyond classical risk factors or medical history and reflects salutogenetic rather than pathogenetic pathways.     

Milk intake and total dairy consumption: associations with early menarche in NHANES 1999-2004

Background

Several components of dairy products have been linked to earlier menarche.

Methods/Findings

This study assessed whether positive associations exist between childhood milk consumption and age at menarche or the likelihood of early menarche (<12 yrs) in a U.S sample. Data derive from the National Health and Nutrition Examination Survey (NHANES) 1999–2004. Two samples were utilized: 2657 women age 20–49 yrs and 1008 girls age 9–12 yrs. In regression analysis, a weak negative relationship was found between frequency of milk consumption at 5–12 yrs and age at menarche (daily milk intake β = −0.32, P<0.10; “sometimes/variable milk intake” β = −0.38, P<0.06, each compared to intake rarely/never). Cox regression yielded no greater risk of early menarche among those who drank milk “sometimes/varied” or daily vs. never/rarely (HR: 1.20, P<0.42, HR: 1.25, P<0.23, respectively). Among the 9–12 yr olds, Cox regression indicated that neither total dairy kcal, calcium and protein, nor daily milk intake in the past 30 days contributed to early menarche. Girls in the middle tertile of milk intake had a marginally lower risk of early menarche than those in the highest tertile (HR: 0.6, P<0.06). Those in the lowest tertiles of dairy fat intake had a greater risk of early menarche than those in the highest (HR: 1.5, P<0.05, HR: 1.6, P<0.07, lowest and middle tertile, respectively), while those with the lowest calcium intake had a lower risk of early menarche (HR: 0.6, P<0.05) than those in the highest tertile. These relationships remained after adjusting for overweight or overweight and height percentile; both increased the risk of earlier menarche. Blacks were more likely than Whites to reach menarche early (HR: 1.7, P<0.03), but not after controlling for overweight.

Conclusions

There is some evidence that greater milk intake is associated with an increased risk of early menarche, or a lower age at menarche.     

Breastfeeding and infant temperament at age three months

Background & Methods

To examine the relationship between breastfeeding and maternally-rated infant temperament at age 3 months, 316 infants in the prospective Cambridge Baby Growth Study, UK had infant temperament assessed at age 3 months by mothers using the Revised Infant Behavior Questionnaire, which produces scores for three main dimensions of temperament derived from 14 subscales. Infant temperament scores were related to mode of infant milk feeding at age 3 months (breast only; formula milk only; or mixed) with adjustment for infant''s age at assessment and an index of deprivation.

Results

Infant temperament dimension scores differed across the three infant feeding groups, but appeared to be comparable between exclusive breast-fed and mixed-fed infants. Compared to formula milk-fed infants, exclusive breast-fed and mixed-fed infants were rated as having lower impulsivity and positive responses to stimulation (adjusted mean [95% CI] “Surgency/Extraversion” in formula-fed vs. mixed-fed vs. breast-fed groups: 4.3 [4.2–4.5] vs. 4.0 [3.8–4.1] vs. 4.0 [3.9–4.1]; p-heterogeneity = 0.0006), lower ability to regulate their own emotions (“Orienting/Regulation”: 5.1 [5.0–5.2], vs. 4.9 [4.8–5.1] vs. 4.9 [4.8–5.0]; p = 0.01), and higher emotional instability (“Negative affectivity”: 2.8 [2.6–2.9] vs. 3.0 [2.8–3.1] vs. 3.0 [2.9–3.1]; p = 0.03).

Conclusions

Breast and mixed-fed infants were rated by their mothers as having more challenging temperaments in all three dimensions; particular subscales included greater distress, less smiling, laughing, and vocalisation, and lower soothability. Increased awareness of the behavioural dynamics of breastfeeding, a better expectation of normal infant temperament and support to cope with difficult infant temperament could potentially help to promote successful breastfeeding.