This work was supported by the University of Zurich, an IEF Marie Curie Grant (grant no. TransEpigen-254797 to A.H.), an Advanced Grant of the European Research Council (grant no. MEDEA-250358 to U.G.), and a Research and Technology Development project (grant MecanX to U.G.) from SystemsX.ch, the Swiss Initiative in Systems Biology.

1. Flower and Silique Fixation
<ol>
	<li>Harvest flowers and siliques from plants synchronized at the opening of the first flower. 
	NOTE: Under the experimental conditions used here, plants start flowering approximately 21 days after transplantation from Murashige and Skoog (MS) plates to soil. Seeds are stratified for 3&#8211;4 days at 4 &#176;C and germinated/grown on MS plates at 22 &#176;C/16 h light and 18 &#176;C/8 h dark for 8 to 10 days, before transplanting the seedlings on nutrient-rich soil in pots kep...

The existence of many flower buds within a single inflorescence of Arabidopsis, spanning all flower developmental stages, offers a unique opportunity for studies aimed at characterizing an effect of a treatment or a developmental feature simultaneously across the different stages of flower development. A good reference point between different individual plants is the opening of the first flower of the main inflorescence. Plants are treated in such a way that flowering is synchronized as much as possible (e.g...

Flowers are among the most important defining organs of angiosperms. Flowering plants appeared some 90&#8211;130 million years ago1, and diversified so fast that their rapid appearance was described as an &#34;abominable mystery&#34; by Charles Darwin2. The interests of plant researchers in flower development are diverse. Some research has focused on understanding the evolutionary origin of the flower as a whole, or the evolution of specific anatomical, structural, and functional properties of flowers3,4,5,6. The high variation in floral form and structure, as well as the modes of sexual and asexual reproduction relying on them, make the flower a highly complex structure. This has led to diverse efforts to characterize the anatomy and structural features of floral organs, using light and electron microscopical techniques that could be combined with genetic and molecular investigations7. Furthermore, as the source of fruits and seeds, flowers are of paramount importance for human and animal nutrition. Therefore, the characterization of flower and fruit development has many implications for applied research, including food security for an ever-increasing human population and ecological conservation strategies under a changing environment8,9,10.
Flower development in Arabidopsis starts with flower induction and the transformation of the vegetative meristem to an inflorescence (group of flowers) meristem. Flower primordia are initiated laterally on the flank of the inflorescence meristem11. The floral organ primordia form progressively in concentric whorls from the outside to the center of the flower, and eventually develop into sepals, petals, stamens, and carpels7. These floral organs fulfill distinct nutritive, protective, and functional (e.g., pollinator attraction) roles in different plant species, with the sexual organs sustaining the development of male and female gametophytes, respectively12,13. The gametophytes, in turn, each differentiate a pair of male (sperm) and female gametes (egg and central cell), which unite upon double fertilization to form the next generation, the zygote, and the primary endosperm, a terminal tissue supporting the development of the embryo14,15. Fruit and seed development support the growth, maturation, and preservation of the embryo and, eventually, its dispersal. Extensive research has been performed to characterize flower and embryo development in diverse plant species, especially in the model species Arabidopsis7,16,17.
Early microscopic analyses of flower development were based on time-consuming sample processing and observation techniques, such as paraffin or resin embedding and sectioning, combined with light or electron microscopy. These traditional microscopic techniques were often used in combination with molecular genetic investigations, such as microscopical analyses of mutants, the localization of RNA by in situ hybridization, or the immuno-detection of proteins. Recent progress in wide-field and confocal fluorescence microscopy, in state-of-the-art 3-D computer-aided image analyses, and the continuous refinement of molecular methods that can be used on minimally processed whole-mount specimens, has led to a need for efficient and minimal sample processing techniques that are preferentially amenable to quantitative analyses. In recent years, significant progress has been made in developing clearing techniques on whole-mount animal specimen. They render the sample transparent either by using aqueous urea- or sugar-based reagents (e.g., SCALE, SeeDB, CUBIC)18,19,20, or by selectively removing lipids (using the detergent SDS) after embedding samples in stable hydrogels; the removal of lipids can be achieved either by passive diffusion (e.g., modified CLARITY protocol21, PACT-PARS-RIMS22) or actively by electrophoresis (original CLARITY protocol23 and ACT-PRESTO24). Encouraged by this fast progress, some derived techniques are also emerging for use in plants.
In this methods paper focused on the model Arabidopsis, we describe the procedure for the proper dissection of flower buds, flowers, and young siliques, and the clearing of whole-mount samples for diverse staining and observation procedures using classical or a recent SDS-based clearing method. Examples for starch, callose, and chromatin staining are given. Although these procedures may need further improvements and adaptations when used with other species, we hope they will set the stage for further research on these simple but critical methods that are the starting point of many research projects.

<table>
	<tbody>
		<tr>
			<td>Reagents and Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Scharlau</td>
			<td>ET00102500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Applichem</td>
			<td>A3686,2500</td>
			<td>100% Molecular biology grade</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Scharlau</td>
			<td>ME03062500</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Solution</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Sigma-Aldrich</td>
			<td>81910-250 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>15307</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Fluka</td>
			<td>51198</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>Fluka</td>
			<td>69773</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma-Aldrich</td>
			<td>77607-250ML</td>
			<td>We used liquid phenol (use the density to find the required volume for your solution)</td>
		</tr>
		<tr>
			<td>Clove oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8392-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Roth</td>
			<td>4436.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Fluka</td>
			<td>57665</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Merck</td>
			<td>5043</td>
			<td></td>
		</tr>
		<tr>
			<td>Malachite Green</td>
			<td>Fluka</td>
			<td>63160</td>
			<td></td>
		</tr>
		<tr>
			<td>Fuchsin acid</td>
			<td>Fluka</td>
			<td>84600</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma</td>
			<td>7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>71690</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium di-Hydrogen Phosphate</td>
			<td>Applichem</td>
			<td>A1047,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S9763-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>04347</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Applichem</td>
			<td>A2937,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcofluor</td>
			<td>Sigma</td>
			<td>F6259</td>
			<td>Fluorescent brightener 28</td>
		</tr>
		<tr>
			<td>Auramine</td>
			<td>Chroma</td>
			<td>10120</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Merck</td>
			<td>1275</td>
			<td></td>
		</tr>
		<tr>
			<td>MS medium</td>
			<td>Carolina</td>
			<td>19-57030</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient-rich substrate</td>
			<td>Einheitserde</td>
			<td>ED73</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch maker&#39;s glass</td>
			<td></td>
			<td></td>
			<td>No specific brand</td>
		</tr>
		<tr>
			<td>15 ml falcon centrifuge tubes</td>
			<td>VWR</td>
			<td>62406-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumond Forceps</td>
			<td>Actimed</td>
			<td>0208-5SPSF-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>DUMONT BIOLOGY</td>
			<td>0108-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>BD Plastipak 300013</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Preparation needle</td>
			<td>BD</td>
			<td>BD Microlance 304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Scientific</td>
			<td>10143562CE</td>
			<td>cut edges</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Scientific</td>
			<td>DV40008</td>
			<td></td>
		</tr>
		<tr>
			<td>Humid box</td>
			<td></td>
			<td></td>
			<td>A plastic box with damp paper towel and slide supports inside</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixatives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carnoy&#39;s (Farmer&#39;s) fixative</td>
			<td></td>
			<td></td>
			<td>Absolute ethanol : glacial acetic acid, 3:1 (ml:ml)</td>
		</tr>
		<tr>
			<td>Methanol/acetic acid fixative</td>
			<td></td>
			<td></td>
			<td>50 % (v/v) methanol, 10 % (v/v) glacial acetic acid in deionized water</td>
		</tr>
		<tr>
			<td>FPA50 fixative</td>
			<td></td>
			<td></td>
			<td>Formalin, propionic acid, 50% ethanol; 5:5:90 (ml:ml:ml)</td>
		</tr>
		<tr>
			<td>Clearing solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate/glycerol</td>
			<td></td>
			<td></td>
			<td>Chloral hydrate : glycerol : water, 8:1:2 (g:ml:ml). Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td>
		</tr>
		<tr>
			<td>Modified Hoyer</td>
			<td></td>
			<td></td>
			<td>Gum arabic 7.5 g, chloral hydrate 100 g, glycerol 5 ml , water 30 ml. Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td>
		</tr>
		<tr>
			<td>Herr&#39;s 4&#189; clearing fluid</td>
			<td></td>
			<td></td>
			<td>Lactic acid, chloral hydrate, phenol crystals, clove oil, xylene; 2:2:2:2:1, by weight. Can be used for all flower developmental stages (especially for stamen development) and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td>
		</tr>
		<tr>
			<td>SDS/NaOH solution</td>
			<td></td>
			<td></td>
			<td>Mix-dilute the the SDS and the NaOH stock solution to 1% SDS / 0.2 N NaOH (10x dilution). For all stages of flower and silique developmental stages. The best fixative is the methano/acetic acid fixative; the other two fixatives can also be used. Can be combined with calcofluor, auramine, DAPI, and aniline blue staining solution.</td>
		</tr>
		<tr>
			<td>SDS stock solution</td>
			<td></td>
			<td></td>
			<td>10 % (w/v) sodium dodecyl sulphate. Dissolve 10 g sodium dodecyl sulphate in 80 ml deionized water and make up to 100 ml with deionized water.</td>
		</tr>
		<tr>
			<td>NaOH stock solution</td>
			<td></td>
			<td></td>
			<td>2 N NaOH solution: dissolve 4 g of NaOH in 40 ml of deionized water and make up to 100 ml with deionized water</td>
		</tr>
		<tr>
			<td>Combined clearing and staining solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Herr&#39;s IKI-4&#189;</td>
			<td></td>
			<td></td>
			<td>To a standard 4&#189; (9 g in total) add: 100 mg iodine, 500 mg potassium iodide. This clearing solution can be used for all flower developmental stages and for silique development, either for increasing contrast or for characterizing starch dynamics. Use FPA50 for structural analysis and Carnoy&#39;s fixative for quantitative starch analysis.</td>
		</tr>
		<tr>
			<td>Alexander staining</td>
			<td></td>
			<td></td>
			<td>Ethanol 95% 10ml, malachite green (1% in 95% EtOH) 1 ml, fuchsin acid (1% in ddH2O) 5ml, orange G (1% in ddH2O) 0.5ml, phenol 5g, chloral hydrate 5g, glacial acetic acid 2ml, glycerol 25ml . This clearing/staining alone or in combination with Herr&#39;s 4&#189; solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It&#39;s used on freshly harved non-fixed material.</td>
		</tr>
		<tr>
			<td>Staining solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcofluor solution</td>
			<td></td>
			<td></td>
			<td>Calcofluor 0.007% in water (g:ml). Originally used as an optical brightner. Can be used for staining cellulose, carboxylated polysaccharides and callose in cell walls. Frequently used to stain the intine of the pollen grain. All three fixatives can be used with this solution.</td>
		</tr>
		<tr>
			<td>Auramine solution</td>
			<td></td>
			<td></td>
			<td>Auramine 0.01% in water (g:ml). This lipophilic fluorscent dye can be used for staining cuticles, cutin, and exine among others. All three fixatives can be used with this solution</td>
		</tr>
		<tr>
			<td>Calcofluor-Auramine mixture</td>
			<td></td>
			<td></td>
			<td>Auramine solution : Calcofluor solution, 3:1. Can be used for a combined staining by both solutions. Other proportions can be assayed maintaining a smaller proportion of calcofluor with respect to auramine.</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td></td>
			<td></td>
			<td>DAPI 0.4 ug/ml, 0.1 M sodium phosphate buffer (pH 7), 0.1% Triton-X-100, 1 mM EDTA. This solution can be used for staining chromosome spreads during male and female meiosis, and cell nuclei of any tissue. Frequently used for studying pollen grain development. Carnoy&#39;s and methanol/ acetic acid are the best fixatives for this solution. Formaldehyde-based fixatives such as FPA50 may interfere with the staining. Excitation in the UV and maximum emission around 461 nm.</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer (0.1 M)</td>
			<td></td>
			<td></td>
			<td>Proton receptor: 0.2 M Na2HPO4, proton donor: 0.2 M NaH2PO4, ratio proton donor / proton receptor: 1.364 ( for a pH 7)</td>
		</tr>
		<tr>
			<td>Aniline blue solution</td>
			<td></td>
			<td></td>
			<td>0.1% (w/v) aniline blue, 108 mM K3PO4 (pH 11), 2% glycerol. This solution can be used for staining callose and cellulose of many stages of development (e.g callose deposition in male and female terads, callose plugs in pollen tubes). Excitation in the UV and maximum emission around 455. It can also be excited at 514 nm with emission in the red for cell content staining.</td>
		</tr>
	</tbody>
</table>

whole-mount clearing and staining of arabidopsis flower organs and siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Arabidopsis belongs to the Brassicacea family, bearing inflorescences with bisexual flowers arranged in a corymb (Figure 1). Each flower has four sepals, four petals, six stamens (four long and two short), and a syncarpous gynoecium consisting of two congenitally fused carpels (Figure 1F-H) arranged in four concentric whorls25,26. Arabidopsis...

Watch this Scientific Journal Video about Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques at JoVE.com

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

This protocol describes how to properly dissect flower buds, flowers and young siliques. And the subsequent whole-mounting clearing for diverse staining and observation techniques. This method can help answer key questions in the sexual plant production field.Such as a steadily mutant analysis or any environmentally and experimentally induced failure in flower development and function. The main advantage of this technique is to allow for easy and quick screening of many developmental stages simultaneously. Visual demonstration of this method is critical, as flower organ dissection steps are difficult to learn because they require prior knowledge of flower anatomy and dissection skills.To begin, use small scissors to remove flowers from synchronized plants. And immediately place them into individual microcentrifuge tubes, containing an appropriate fixative. Next, remove the fixative and add enough 70%ethanol to completely cover the samples.Return the samples to four degree Celsius for at least 24 hours. Then fill a watchmaker's glass with freshly made 70%ethanol and place it into a small Petri dish for support. Then place the inflorescence into the watchmaker's glass and ensure it is totally covered by the ethanol.Using forceps and a syringe equipped with a needle, dissect the flower under the stereo microscope by tearing apart the sepals, petals and stamen. Then place the watchmaker's glass aside and use a glass slide for the final dissection. Move the sample to slide and add 10 microliters of freshly made 70%ethanol.Make cuts on each side of the carpal, rotating it as needed. Then gently remove the valves to expose the ovules. Make a sharp small cut to detach half of the transmitting tissue from the receptacle and use forceps and a needle to tear apart the two halves.Remove the stigma style and the peduncle with sharp cuts. Using the forceps and needle gently flip and arrange the still attached ovules to obtain two parallel rows. Use a small piece of blotting paper to remove ethanol from the sample on the glass slide.Then add 20 microliters of clearing solution to the slide. Using a pair of syringes with hypodermic needles position the specimens and remove any remaining air bubbles. Next, gently place a cover slip onto the slide and wait until the clearing solution fills the space between the cover slip and the sample.Place the slide in a slide holder and leave it under the fume hood. So let's freshly harvested flower buds that are about to open with non-dehiscent anthers. Then prepare a 96 well plate with enough Alexander solution to fully submerge the flower buds.Remove the sepals and petals to expose the anthers and dip them into the Alexander solution. Allow the plate to sit under the fume hood for one to three hours. Then replace the Alexander solution with Herr's four and a half solution and incubate the plate under the fume hood overnight.The next day use forceps to carefully move the cleared buds to a new slide. Next, dissect the stamens and remove all other tissues from the samples. Then proceed with chloral hydrate based clearing and combined clearing staining using Herr's four and a half solution.First move the flower from the watchmaker's glass to the slide and use a blotting paper to remove excess ethanol. Add five to 10 microliters of DAPI solution. Using two syringes with hypodermic needles, dissect the stamens and remove all other flower organs.Then release the pollen grains into the DAPI solution. Remove all debris from the stamen from the slide ensuring that only the pollen grains are left on the slide. Removing all the stamen debris from the slide is the most critical step for exine removal.Only pollen grains should be left between the slide and cover slip. Next, place a cover slip on the specimen bearing slide and add the minimum amount of DAPI solution needed to fill the space between the slide and the cover slip. Place an index finger gently on the cover slip and make a quick short sliding movement.Place the slide in a human box in the dark at four degrees Celsius for at least 15 minutes. Then observe the slide under wide field florescence or confocal microscopy within 24 hours. Move the dissected sample from the watchmaker's glass to a glass plate with cavities filled with 1%SDS and a 0.2 sodium hydroxide solution.Incubate overnight at room temperature. The SDS sodium hydroxide solution will quickly clear the samples, making them appear transparent within a few minutes. Carefully rinse the sample with water and place it onto a clean slide.Then add 20 to 40 microliters of staining solution to the slide. Depending on the floral organ to be analyzed and on the researcher's skills this DS treatment can be carried out before, for experienced researchers or after the dissection step for less experienced researchers. Next place a cover slip onto the slide taking care not to crush the preparation.Then place all the prepared slides into a human box covered with aluminum foil and incubate at four degrees Celsius for one hour. Finally observe the specimen under wide field florescence or confocal microscopy. Following the dissection procedure, described in the protocol, unnecessary tissues that might hinder observations are removed and more detailed images are achieved.Depending on the objective, chloral hydrate staining can be used to characterize early flower development at the whole flower bud level, meiosis and micro-gomedal genesis in the anther, early ovule development and the specification of the megaspore mother cell, female go-med-ified development, or even pollen tube invasion of the center grid. Combined Alexander staining and Herr's four and a half clearing, enables the evaluation of pollen abortion within a given locule or an anther as a whole. While viable pollen grains show a red cytoplasm, aborted ones show an empty cytoplasm and eventually they're irregularly shaped and totally crushed.Removing the exine results in an increased staining intensity of nuclei and higher details of chromatin organization. Using and SDS sodium hydroxide clearing prior to staining made inflorescence transparent. And resulted in higher staining of callose within floral tissues.It revealed usually difficult to see callose accumulations. Such as the Callose layer that separates the generative cell from the vegetative cell. Even when pollen grains were still contained within the anther.Once mastered, these techniques can be done in less than 48 hours, if they are performed properly. While attempting this procedure, it's important to remember that the results largely depend upon a good sample preparation, such as using the proper fixative, performing the dissection properly and isolating the tissue of interest on the slide.

whole-mount-clearing-staining-arabidopsis-flower-organs

Research

JoVE Journal

Developmental Biology

16.3K vistas.  University of Zurich. This protocol describes how to properly dissect flower buds, flowers and young siliques. And the subsequent whole-mounting clearing for diverse staining and observation techniques. This method can help answer key questions in the sexual plant production field.Such as a steadily mutant analysis or any environmentally and experimentally induced failure in flower development and function. The main advantage of this technique is to allow for easy and quick screening of many developmental s...