Luc Gillibert
email: luc.gillibert@mines-paristech.fr

Théodore Chabardès
email: theodore.chabardes@mines-paristech.fr

Beatriz Marcotegui
email: beatriz.marcotegui@mines-paristech.fr

Local multiscale blur estimation based on toggle mapping for sharp region extraction

Keywords: Blur estimation, Toggle mapping, DIQA database, LINX database

In this paper a multiscale local blur estimation is proposed based on the existing local focus measure that combines gradient and toggle mapping. This method evaluates the quality of images regardless of their content (not in an autofocus context) and can predict OCR accuracy based on local blur. The resulting approach outperforms state of the art blur detection methods. Quantitative results are given on DIQA database. Moreover we demonstrate its usefulness for extracting a region of interest from partially blurry images. Results are shown on images acquired by a project devoted to smartphone based text extraction for visually impaired people. In this context sharp region extraction is essential since it allows warning the users when their picture is unusable. Moreover it saves computing time.

Introduction

Mobile phones offer a number of increasing capabilities, especially high resolution photography and high computational power that can be used for a new range of image processing applications. Text is omnipresent and brings essential information in our daily life. Visual impairment deprives a significant percentage of the population of this important information. The goal of the LINX project is to develop an automatic reader on smartphone to help visually impaired people in their daily life, making textual information available to them.

In order to reach this goal, several challenging tasks need to be solved: text detection, text extraction and Optical Character Recognition (OCR). Each of these tasks is time consuming and must not be executed on useless pixels. Visually impaired people cannot guarantee the quality of the image they acquire. Images can be degraded by bad lighting conditions, shadows, out-of-focus blur, motion blur, perspective problems or noise. Moreover the use of handheld devices aggravates the blur problem. Therefore extracting regions containing sharp enough structures is an extremely useful functionality for this kind of application.

For several decades, Image Quality Assessment (IQA) techniques have attracted great attention from the scientific community. These techniques can be classified into three categories:

• Full Reference (FR): require the original image as a reference. The most popular FR objective metrics are the mean square error (MSE), Peak Signal to Noise Ratio (PSNR) and crosscorrelation between both images [START_REF] Ponomarenko | Tid2008-a database for evaluation of full-reference visual quality assessment metrics[END_REF]. The structural similarity index (SSI) [START_REF] Wang | Image quality assessment: from error visibility to structural similarity[END_REF], attempts to quantify the visibility of errors based on the degradation of structural information. The use of FR IQA is limited because the reference image is often unavailable.

• Reduced reference (RR): are designed to predict the quality of degraded images with only partial information of them. The comparison is based on extracted features instead of on the whole image. Examples of these techniques can be found in [START_REF] Ma | Reduced-reference image quality assessment using reorganized dct-based image representation[END_REF] where the comparison is based on the statistical modeling of the discrete cosine transform (DCT) and [START_REF] Wang | Reduced-reference image quality assessment using a wavelet-domain natural image statistic model[END_REF] based on wavelets. The original image is still required in order to extract the features to be compared.

• No-reference or blind techniques: the measure is computed on the degraded image itself. This is the most challenging task, in which the quality measure relies exclusively on a single acquired image and corresponds to our application context (LINX project).

A wide variety of approaches for NR blur estimation exist. They operate either in the spatial or in the frequency domain. Among the spatial techniques the simplest metrics rely on gradient or laplacian computation. Indeed, the blur reduces the contrast of contours and tends to weaken the gradient. The higher the gradient, the sharper the image. Thus, the sum of thresholded gradient or the squared gradient [START_REF] Frans Ca Groen | A comparison of different focus functions for use in autofocus algorithms[END_REF][START_REF] Santos | Evaluation of autofocus functions in molecular cytogenetic analysis[END_REF], Brenner function [START_REF] Yeo | Autofocusing for tissue microscopy[END_REF], multiscale gradient [START_REF] Chen | No-reference image blur assessment using multiscale gradient[END_REF] and laplacian [START_REF] Shree | Shape from focus. Pattern analysis and machine intelligence[END_REF] are examples of these techniques. Other authors have considered the variance or other histogram measures such as entropy [START_REF] Ray | Focus optimization criteria for computer image-processing[END_REF][START_REF] John F Schlag | Implementation of automatic focusing algorithms for a computer vision system with camera control[END_REF][START_REF] Ng | Practical issues in pixel-based autofocusing for machine vision[END_REF], assuming that a focused image conveys more information than an unfocused one. Finally, some techniques are based on edge width estimation, after an edge detection step [START_REF] Marziliano | Perceptual blur and ringing metrics: application to jpeg[END_REF][START_REF] Cao | Edge-based blur metric for tamper detection[END_REF]. Concerning frequential approaches, they rely on the assumption that a defocused image results from the convolution of the image which produces a decrease in the high frequencies. Thus, the sum of the spectrum components above a given cut frequency [START_REF] Firestone | Comparison of autofocus methods for automated microscopy[END_REF], the ratio between the high frequency coefficients over low frequency ones or the distribution of null coefficients (blurred images are likely to have all of their high frequency coefficients set to zero) have been used as focus measures. Discrete Cosine Transform (DCT) [START_REF] Marichal | Blur determination in the compressed domain using dct information[END_REF][START_REF] Saad | Blind image quality assessment: A natural scene statistics approach in the dct domain[END_REF] and wavelet transform [START_REF] Yang | Wavelet-based autofocusing and unsupervised segmentation of microscopic images[END_REF][START_REF] Xie | Wavelet-based focus measure and 3-d surface reconstruction method for microscopy images[END_REF] have been used for this purpose. The correlation of wavelet coefficients over scales has also been used [START_REF] Ciancio | No-reference blur assessment of digital pictures based on multifeature classifiers[END_REF][START_REF] Wang | Reduced-and no-reference image quality assessment[END_REF]. In [START_REF] Blanchet | Measuring the global phase coherence of an image[END_REF][START_REF] Blanchet | An explicit sharpness index related to global phase coherence[END_REF] a sharpness index is derived from the phase component of the Fourier Transform, based on the fact that edges correspond to points of maximum phase congruence.

Most previous approaches have been established for autofocus purposes, where the quality of an image can be judged compared to the quality of other images with similar content. Consequently, they do not fit our requirements. In order to lift this restriction some local approaches have been proposed, applying the method to a given neighborhood [START_REF] Pertuz | Analysis of focus measure operators for shape-from-focus[END_REF], that needs to be defined or on the result of an edge detection approach [START_REF] Cao | Edge-based blur metric for tamper detection[END_REF][START_REF] Peng | Automated image quality assessment for camera-captured ocr[END_REF].

Recently, the specific problem of document image quality is becoming a topic of intense research. Several papers attempt to predict the OCR accuracy when applied to an image, based on IQA measures of the given image [START_REF] Luis R Blando | Prediction of ocr accuracy using simple image features[END_REF][START_REF] Peng | Automated image quality assessment for camera-captured ocr[END_REF][START_REF] Ye | Learning features for predicting ocr accuracy[END_REF]. The interesting DIQA dataset, described in section 3.2, has been introduced in this context [START_REF] Kumar | A dataset for quality assessment of camera captured document images[END_REF]. We will benchmark our approach on it.

Lately [START_REF] Van-Cuong | Ocr accuracy prediction method based on blur estimation[END_REF] proposed a local blur estimation based on Fuzzy-C-Means clustering approach after a binarization step. They combine this local estimation with the character size in a learning based strategy, in order to predict the OCR accuracy. The learning approach suits a document oriented application but it is not convenient for our LINX project.

Image quality assessment remains an open problem, in particular for generic scene-text images such as those of our LINX project. This paper is an extended version of [START_REF] Chabardès | Local blur estimation based on toggle mapping[END_REF]. In the original paper, a local blur estimator that does not require a prior contour detection, nor a neighborhood definition was introduced. This paper introduces the following novelties:

• A parameters sensitivity study.

• Multiscale extension of the original method in order to deal with documents with variable font size.

• The selection of a sharp region of interest (possibly empty if the whole image is blur) in order to avoid processing doomed to failure.

The paper is organized as follows: Section 2 summarizes the toggle mapping based blur estimator approach. The sensitivity of this blur estimator to its parameters is studied in section 3 and some experimental results are given. The performance of the approach is evaluated on DIQA dataset.

In section 4 a region of interest is extracted from the local blur information. Then, in section 5, the extraction of the region of interest is generalized for multiscale blur detection.

Section 6 discusses the choice of the scales used and shows relevant sharp regions extracted from LINX database with variable font sizes. Finally section 7 concludes this work.

Toggle mapping based blur estimation

Toggle mapping (TM) operator was introduced in [START_REF] Henry | Iterations of a non-linear transformation for enhancement of digital images[END_REF]. From an image I, two transformations are computed: the dilation, δ B (I) and the erosion B (I), the maximum and the minimum value within the structuring element B. Equation 1 describes the TM operator:

T M B (I) = δ B (I) if δ B (I) -I < I -B (I) B (I) otherwise (1)
Thus, each pixel is replaced by one of these two transformations, selecting the one that is closest to the original pixel value. This principle was generalized in [START_REF] Serra | Toggle mappings. From pixels to features[END_REF], with the use of other transformations involved in the toggle process.

TM was designed as a contrast enhancement operator. It was also used as scene text segmentation tool in [START_REF] Fabrizio | Text segmentation in natural scenes using toggle-mapping[END_REF]: pixels replaced by the dilation are set to 1 and those replaced by the erosion are set to 0.

A local blur estimation based on TM is introduced in [START_REF] Chabardès | Local blur estimation based on toggle mapping[END_REF].

We define the residual image as the absolute difference between the original image and the TM:

T M R B (I) = δ B (I) -I if δ B (I) -I < I -B (I) I -B (I) otherwise (2)
A blur boundary leads to a high residue, and this residue increases with the size of the structuring element involved in the TM. This is true up to a TM size equivalent to the blur edge width. A blur estimation could then be built on the evolution of the TM residues, with a series of structuring elements of increasing size. However a sharp boundary and a homogeneous region would have the same series of low TM residue value. In order to distinguish between these two situations we compare these residues with the gradient value. The morphological gradient is defined as the difference between the maximum and the minimum value in the neighborhood of a pixel (δ B (f ) -B (f )), while the TM replaces each pixel with the closest extremum in the neighborhood, that is either the maximum or the minimum. Thus, by definition, TMR is lower than the gradient. However if the TM uses a larger structuring element its residue can reach the gradient value, in which case, the pixel is classified as blur.

Figure 1 shows a simple 1D example with three objects, from sharp on the left to blurry on the right. Figure 1 residue is shown in green and the gradient in red in figure 1(b). We can observe that the residue of the toggle mapping increases with the blurriness while the gradient decreases with it. Figure 1(c) shows the I Q , the quality measure defined as the difference between the gradient and the toggle mapping residue:

I Q = max (0, gradient B N (I) -T M R B M (I))
, with B N the structuring element B of size N and M > N . As expected, contours are ranked according to their contrast and sharpness, while homogeneous regions have a small I Q value.

Figure 2 shows the flow chart of the algorithm. First, a bilateral filter [START_REF] Tomasi | Bilateral filtering for gray and color images[END_REF] is applied in order to get rid of noise. Then I Q is computed as the positive difference between the gradient and the TMR. It is a local measure. It can be thresholded for identifying sharp regions. Small connected components of the thresholded I Q can be removed with an area opening. Other morphological filters will be introduced in section 4. If a global quality score, Q score , is required for the whole image it can be deduced by averaging the thresholded I Q image, taking into account only the values over the threshold. Thus the quality measure does not depend on the quantity of text in the images. As shown in section 3, the removal of small regions has a low impact on the Q score . However it is important for sharp region selection. Figure 3 shows the impact of the bilateral filter on the process. Black pixels correspond to pixels of I Q under the threshold. We can observe a much cleaner result after the filtering. This will be an essential aspect for the sharp region selection. Moreover, the quantitative contribution of this filter and its parametrization will be studied in section 3.

Results and parameters sensitivity

Qualitative results

A good correspondence between observed quality and our score on LINX database has been noted. The quality of the extracted regions of section 6 will confirm this point.

Quantitative results

Quantitative results are given on DIQA database, an interesting dataset recently published [START_REF] Kumar | A dataset for quality assessment of camera captured document images[END_REF]. It is composed of 175 document images acquired by a mobile phone. Images are directly acquired by a camera, so the blur is not simulated. 25 documents have been acquired, 6 to 8 times each, in different conditions: from perfect focus to completely out-of-focus images. The quality of each image is given by the OCR accuracy on the processed image. Three different OCRs have -(c)). Thus, for this simple example with only three images, our quality score is perfectly correlated with the OCR accuracy: the Spearman correlation between them is then equal to 1.

Some works [START_REF] Kumar | A dataset for quality assessment of camera captured document images[END_REF][START_REF] Marc ¸al Rusiñol | Combining focus measure operators to predict ocr accuracy in mobile-captured document images[END_REF] have tried to estimate the OCR accuracy based on some features extracted from the image. The score used for comparing different methods is the median of the Spearman correlation applied to each document set. The scores reported are over 90%. We think that the proposed score overestimates the quality of the OCR accuracy prediction. Indeed, if the Spearman correlation is computed for each document set, the content of images to be compared is similar. The context is then close to an autofocus situation which is much easier than comparing the quality of images with different content. This problem is pointed out in [START_REF] Marc ¸al Rusiñol | Combining focus measure operators to predict ocr accuracy in mobile-captured document images[END_REF]: a failure is reported for an image with a low focus measure whereas the OCR accuracy is over 90%, provoked by the large white space in the page. Another reported issue concerns an image with a high focus measure but a low OCR accuracy. The huge headline leads to a high focus measure while most of the text is Approach FineReader Tesseract Omni Combination of classical blur descriptors [START_REF] Marc ¸al Rusiñol | Combining focus measure operators to predict ocr accuracy in mobile-captured document images[END_REF] 0.6467 --Phase coherence [START_REF] Blanchet | An explicit sharpness index related to global phase coherence[END_REF] 0.5569 0.6318 0.5174 Our approach 0.8426 0.9016 0.7277 out-of-focus. We address these situations in this paper. Moreover, as stated in [START_REF] Marc ¸al Rusiñol | Combining focus measure operators to predict ocr accuracy in mobile-captured document images[END_REF], by reporting the median value, outlier classes in which the methods might not perform well are disregarded. They propose to compute the Spearman correlation directly for the 175 images of the database. We adopt this proposal that we find more reliable. The authors of this paper have tested all combinations of 24 classical blur descriptors. The best results were obtained with the combination of 4 of them: gaussian derivative, gradient energy, squared gradient and histogram range. They report a Spearman correlation of 0.6467 for the whole database as a single set for FineReader OCR. In the same conditions we obtain a Spearman correlation of 0.8426. For benchmarking purposes table 1 summarizes the Spearman correlation obtained in the same conditions with different state of the art approaches.

A higher Spearman correlation indicates the method's ability to rank images according to the expected OCR accuracy. Therefore, we can state that our method outperforms state of the art approaches for OCR accuracy estimation.

Parameters sensitivity

The parameters have been manually tuned for Tesseract accuracy estimation, as Tesseract has been chosen in the framework of our LINX project. We will now test the sensitivity to each parameter by changing its value without changing the value of the other parameters. If not specified otherwise, parameters used are:

• Structuring element: square.

• Bilateral filter: σ spatial = 2 and σ gray = 20. The sensitivity to these parameters is studied on table 2 and table 3.

• Gradient size B 1 = 1 (3 × 3 pixels).
• TM size B 2 = 2 (5 × 5 pixels).

• Binary threshold on I Q : 3. The sensitivity to this parameter is studied on table 4.

• Area opening of size 5. The sensitivity to this parameter is studied on table 5.

The two parameters of the bilateral filter can vary in a wide range without any significant impact on the Spearman correlation. As shown on table 2, the use of a bilateral filter significantly improves the scores. Furthermore σ spatial can vary from 2 to 5 with less than a 1% variation in the Spearman correlation for the three OCR. The sensitivity to these parameters is low. For all the tested parameters variation, the results of the the Spearman correlation obtained on the DIQA image database are higher than the 0.6467 reported in the literature. Therefore our method outperforms the state of the art for OCR accuracy estimation.

If the goal of the approach is to estimate a global Q score , then the area opening can be ignored, because the sensitivity to the size of the area opening on the threshold I Q is very low. But this area opening is very useful for the extraction of a region of interest in section 4.

Region of interest on heterogenous blur

If the image is heteregeneously blurry, the local blur measure I Q , described in section 2, allows to extract a region sharp enough for the OCR. If there is no such sharp region, the whole image is

rejected.

A naive approach would be to select a rectangular region of interest containing all the nonzero pixels of this thresholded I Q . This naive approach will fail in some cases. For example, spurious detection in noisy regions. Another problem with the thresholded I Q is the presence of thin non-textual objects. Typically: borders of paper sheets, isolated straight lines, straight shadows. They are irrelevant for text extraction. They can be ignored, reducing significantly the area of the extracted region of interest.

Thin objects and small isolated sharp regions on the thresholded I Q can be removed before the extraction of the final rectangular region of interest with some simple morphological filters. This filtering process is the following:

1. F 1 (I) = B 1 (δ B 1 (I)) (closing) 2. F 2 (I) = δ B 2 ( B 2 (F 1 (I))) (opening of the closed image) 3. F 3 (I) = γ S area (F 2 (I)) (area opening of F 2 (I)) Where γ S
area is the area opening of surface S. All connected components of surface smaller than S are removed.

The first morphological closing, F 1 , is used for merging small sharp regions and for filling small objects. Characters and lines are sharp on their edges, but have a low I Q value on their uniform regions.

Thin objects and small isolated objects are removed with the morphological opening, F 2 , and the area opening F 3 . The edges of characters are unaffected by this filter because the morphological closing has filled characters, making them large. This step of the process is illustrated on figure 6(c).

After the morphological filters, the bounding box including all remaining pixels is our region of interest.

Multiscale approach

Large characters may be blur but still readable (figure 5). The method introduced in section 3 will reject them. This problem is not present on the DIQA database, because the character size is very consistent in the database, but is present on the LINX database.

For solving this problem, we introduce a multiscale approach. The input image (scale s = 1) is processed at different scales s. At scale s the image is decimated by a factor 2 s in each coordinate, by using a bilinear filter. The whole process described in section 4 is applied to each scale and the union of resulting bounding boxes is computed (see figure 2. Blue dashed line boxes have been added.

A full example is shown on figure 6 for scale 1 and 1 32 . At scale 1 32 , the morphological filter removes the border of the table, on the right of the image, but the cover of the disk is not suppressed.

At each scale a new bounding box is produced, containing readable text for this scale. The final bounding box is the box containing the union of the bounding boxes of all the scales.

The multiscale approach solves the problem of large blurry characters. The additional computational cost is low because the process is applied to reduced-size images. More details on the computational time are given in table 7 of section 6.

Experimental results for the region of interest

Choice of the parameters

The muti-scale approach can use any arbitrary set of scales. By construction, the largest windows are obtained with the full set of scales ({1, 1 4 , 1 8 , 1 16 , 1 32 }). This result is considered as a reference and the Jaccard index (the area of the intersection divided by the area of the union) in order to study the influence of the scale set selection. Table 6 summarizes the result on the whole LINX database. We can observe that:

• a single scale misses 43% of the surface detected by the full set of scales.

• combining only two scales (1 and 1 16 ) detects 89% of the surface obtained by the full set of scales. ): bounding boxes include all the significant text. Third column shows the full set of scales result, leading to very similar results compared with the previous one.

Experimental results on the LINX dataset show that two conveniently chosen scales, in our case {1, 1 16 }, lead to a good trade-off between quality and computing time.

The parameters for the bilateral filter, the TM operator and the estimation of the I Q do not depend on the scale. But the size of the morphological operations used for filtering unwanted objects in the final crop depends on it. For scale 1 the operations are the following: Closing with a square structuring element of size 5, opening with a square structuring element of size 1 and area opening with a surface of 500.

Set of scales

At smaller scales, the closing tends to merge different structures that the subsequent opening is not able to remove. For this reason, we have heuristically reduced the closing size while increasing the opening size. Due to the increased size of the morphological opening, the area opening is no longer critical. Hence, the operations are the following: closing with a square structuring element of size 2, opening with a square structuring element of size 3 and area opening with a surface of 50.

The processing time with the multiscale approach is very close to the processing time with the monoscale approach, as shown on table 7. The additional cost for the multiscale approach with respect to the simple scale is 1.6%.

The processing time is given in seconds for the whole LINX database: 160 images of size 3264 × 2448 (8 mega-pixels). The processing is done on an Intel Core i7-3800 (Sandy Bridge-E C2). The code is written in C++, based on the SMIL library [START_REF] Faessel | Smil simple morphological image library[END_REF], and uses only one core. The total processing time for the whole LINX database is 95.07 seconds. This time can be decomposed in three: 27 seconds for the bilateral filter, 28 seconds for the scaling, the toggle mapping and the morphological operations, and 40 seconds for the area opening. The average processing time for one image is 0.59 seconds, but this time can be reduced. The current algorithms are not parallelized and not fully vectorized. There is still room for optimizations. In particular, area opening seems to be a good candidate for optimization. 7 Processing time in seconds for the whole LINX database (160 images, 8 mega-pixels each). The processing is done on an Intel Core i7-3800 (Sandy Bridge-E C2).

Qualitative results

Qualitative results on LINX database are given in figure 8. Convincing results are obtained. The uniform background is correctly rejected (images 1, 2, 7-9 images in raster order), readable text is selected in the upper part of images 4 and 6 while blurry one (in the lower part) is rejected, the wall in image 5 is selected because of the details on it (cables and wall socket) and image 3 is rejected, as there is no sharp region on it.

Conclusions and perspectives

A method for selecting a region of interest for characters extraction and recognition has been introduced in this paper. It reduces the data to be processed and therefore the time and the energy needed for achieving these tasks. This will lead to a more reactive and efficient application on smartphones.

Qualitative results on LINX examples show that blurry characters that are large enough for being read by the OCR, despite the blur, are correctly selected by the algorithm. The regions of interest are relevant and ignore large uniform or blurry regions of the images.

From a quantitative perspective, the quality indexes obtained are correlated with the OCR accuracy. On the DIQA images, the Spearman correlation reveals a good correspondence between the Q score and the OCR accuracy.

For FineReader, a Spearman correlation of 0.8426 is reached, much better than the best score reported in the literature (0.6467) [START_REF] Marc ¸al Rusiñol | Combining focus measure operators to predict ocr accuracy in mobile-captured document images[END_REF], combining classical blur descriptors. For Tesseract the results are even better, with a Spearman correlation of 0.9016, to be compared to the 0.6318 obtained with the global phase coherence method described in [START_REF] Blanchet | An explicit sharpness index related to global phase coherence[END_REF]. The method is also robust, the sensitivity to variation of the parameters is low.

In the future we will address problems specific to motion blur. In some cases, a strong motion blur can be considered as sharp by our approach. With a directional approach, such errors can be avoided. Another interesting development would be to process images in real-time on a smartphone for providing a shooting assistance. The shooting process is hard for visually impaired people, and providing them with real-time guidance would be helpful.

Fig. 1 .

 1 Fig. 1. Intermediate steps of local quality estimation on three 1D objects. (a) original objects, from sharp on the left to blurry on the right. (b) TMR increases with blur while the gradient decreases. (c) The final I Q , the difference between TMR and the gradient.

Fig. 2 .

 2 Fig. 2. Algorithm flowchart.

17 Fig. 3 .

 173 Fig. 3. Filter influence in I Q computation. Left column: original image. Middle column: Thresholded I Q computed from original image. Right column:Thresholded I Q from bilateral filtered image.

5 Fig. 4 .Figure 4

 544 Fig. 4. Three DIQA examples. A sharp image (left column), intermediate blur (middle column) and a blur image (right column). First row: original images. Second row: I Q image and the respective OCR accuracy and Q score .

Fig. 5 .

 5 Fig. 5. Example with large characters in a blurry context, but still readable. Left column: original images. Right column: miss-detection of readable characters.

Fig. 6 .

 6 Fig. 6. Example of the extraction of the region of interest (scale 1 et 1 32 ). (a) is the input image (scale 1). (b) is the thresholded I Q . This image is closed (F 1 ), then open with a morphological opening (F 2 ) and an area opening (F 3 ) in (d). The same process is repeated at scale 1 32 (d,e,f). The pixels left in (c) and (f) are used for the extraction of the region of interest.

Figure 7

 7 Figure 7 illustrates qualitative results of these differences. First column illustrates the single scale results: significant text is outside the detected bounding box. Second column shows the result of combining two scales (1 and1 16 ): bounding boxes include all the significant text. Third column shows the full set of scales result, leading to very similar results compared with the previous one.Experimental results on the LINX dataset show that two conveniently chosen scales, in our case {1,1 16 }, lead to a good trade-off between quality and computing time.The parameters for the bilateral filter, the TM operator and the estimation of the I Q do not depend on the scale. But the size of the morphological operations used for filtering unwanted objects in the final crop depends on it. For scale 1 the operations are the following: Closing with a square structuring element of size 5, opening with a square structuring element of size 1 and area

Fig. 7 .

 7 Fig. 7.Comparison between different scale sets on a subset of the LINX database. Left column: monoscale approach. Middle column: multiscale approach with scales {1,1 16 }. Right column: multiscale approach with the full set of scales {1,1 4 , 1 8 , 1 16 , 1 32 }.

Fig. 8 .

 8 Fig. 8. Example of LINX images with extracted region of interest.

Table 1

 1 Spearman correlation between OCR accuracy and quality score obtained in the same conditions by different state of the art approaches.

Table 3

 3 Moreover the sensitivity to the size of the area threshold on the I Q is very low. As shown on table 5, the size of the area opening on the I Q can vary from 2 to 11 with less than one per thousand variation in the Spearman correlation for Tesseract and Omni.

	σ spatial FineReader Tesseract Omni
	0	0.7410	0.7110	0.6219
	1	0.8213	0.82	0.6979
	2	0.8426	0.9016	0.7277
	3	0.8399	0.902	0.7266
	4	0.8396	0.902	0.7263
	5	0.8395	0.9024	0.7262

, studies the sensitivity to σ gray parameter. It can vary from 15 to 35 with less than a 1% variation in the Spearman correlation for Tesseract.

As shown on table 4, the sensitivity to the threshold on the I Q is low if the threshold is greater than 2.

Table 2

 2 Sensitivity to the σ spatial used for the bilateral filter based on Spearman correlation between OCR accuracy and quality score.

	σ gray FineReader Tesseract Omni
	5	0.7476	0.7171	0.631
	10	0.8428	0.8586	0.7155
	15	0.8532	0.9006	0.7305
	20	0.8426	0.9016	0.7277
	25	0.8374	0.9003	0.7247
	30	0.836	0.8986	0.7215
	35	0.8344	0.8961	0.7165

Table 3

 3 Sensitivity to the σ gray used for the bilateral filter. The Spearman correlation between OCR accuracy and quality score is given for FineReader, Tesseract and Omni.

	Binary threshold FineReader Tesseract Omni
	1	0.8254	0.8114	0.6942
	2	0.8499	0.8969	0.7271
	3	0.8426	0.9016	0.7277
	4	0.8385	0.9015	0.7276

Table 4

 4 Sensitivity to the binary threshold on the I Q based on Spearman correlation between OCR accuracy and quality score.

Table 5

 5 Sensitivity to the area opening size on the thresholded I Q based on Spearman correlation between OCR accuracy and quality score.

Table 6

 6 Comparison between reference results (scales {1, 1 4 , 1 8 , 1 16 , 1 32 }) and various combinations of scales.

Acknowledgments.This research was supported by Cap Digital Business Cluster LINX Project.


Stéphane Zaffran

Gaëlle Odelin

Sonia Stefanovic

Fabienne Lescroart

Heather C Etchevers
email: heather.etchevers@inserm.fr

Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations

Keywords: valve, mandibular agenesis, maxillary aplasia, exencephaly, double outlet right ventricle, cleft palate

Members of the large family of Hox transcription factors are encoded by genes whose tightly regulated expression in development and in space within different embryonic tissues confer positional identity from the neck to the tips of the limbs. Many structures of the face, head and heart develop from cell populations expressing few or no Hox genes. Hoxb1 is the member of its Special issue of genesis : Celebrating 150 Years of Neural Crest Research 2 chromosomal cluster expressed in the most rostral domain during vertebrate development, but never by the multipotent neural crest cell population anterior to the cerebellum. We have developed a novel floxed transgenic mouse line, CAG-Hoxb1,-EGFP (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP over-expression. When induced within the neural crest lineage, pups die at birth. A variable phenotype develops from E11.5 on, associating frontonasal hypoplasia/aplasia, micrognathia/agnathia, major ocular and forebrain anomalies, and cardiovascular malformations. Neural crest derivatives in the body appear unaffected. Transcription of effectors of developmental signaling pathways (Bmp, Shh, Vegfa) and transcription factors (Pax3, Sox9) is altered in mutants. These outcomes emphasize that repression of Hoxb1, along with other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for craniofacial, visual, auditory and cardiovascular development.

Introduction

For over 600 million years of animal evolution, the multiple genes encoding the Hox family of transcription factors have been master conductors of segmental positional identity in the body and limbs [START_REF] Pearson | Modulating Hox gene functions during animal body patterning[END_REF]. Genomic duplications and rearrangements have led to the presence in most vertebrates of four homologous clusters of thirteen Hox genes, of which only 39 have been retained over the years in humans and mice and 38 in chickens, such that each cluster contains between 9 and 11 genes. The clusters of genes exhibit collinearity: according to the physical order of genes within each cluster from 3' to 5', the rostral expression limit of each is globally arranged along the axis from posterior head to tail, spaced according to metameric landmarks of the body and limbs, and the genes are also expressed sequentially in time [START_REF] Santagati | Cranial neural crest and the building of the vertebrate head[END_REF].

However, detailed examination in the different germ layers and over time shows that this rule of thumb is not perfectly respected, particularly in the rostral region.

The Hox genes expressed most anteriorly in the developing mouse or chick embryo include those of paralog groups 1 and 2. Extant members in these species are Hoxa1, Hoxb1, and Hoxd1 (homologous to the Drosophila labial gene) and Hoxa2 and Hoxb2 (homologous to Drosophila proboscipedia). The expression domains of labial and proboscipedia correspond to rostral structures of the fruitfly head.

During the evolution of vertebrates, additional head structures developed both anterior and ventral to the corresponding domains expressing the equivalent paralog groups, comprising not only tissues derived from the three original germ layers but also a major mesenchymal contribution from an evolutionarily new cell population known as the neural crest (NC). The new head hypothesis was developed 35 years ago to examine the transition from proto-chordates to the successful vertebrate clade [START_REF] Gans | Neural Crest and the Origin of Vertebrates: A New Head[END_REF]. It emphasized that the NC's striking propensity to function like mesoderm in the anterior head was the primary element enabling vertebrate characteristics such as active predation, particularly in building the rostral skull vault and facial skeleton, protecting brain and sensory organs, and, for gnathostomes, developing jaws. This entire craniofacial skeleton is derived from NC cells of the first pharyngeal arch (PA) and frontonasal bud (Le Lièvre, 1978, and references therein).

The ectoderm, mesoderm and NC cell mesenchyme surrounding the oral endoderm ventrally in the first PA and joined by the neuroectoderm dorsally are each devoid of Hox gene expression. In contrast, the hindbrain neuroectoderm and 2 nd and posterior PAs are the sites of the rostral limits of Hox genes and are, at least temporarily, visibly metameric. Collinearity in these rostral limits is not strictly observed, either within the derivatives of a given germ layer, or across them in structures of mixed embryological origin, like a PA [START_REF] Couly | Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development[END_REF]. In addition, rostral Hox expression limits are dynamic, making it difficult to generalize; some exceptions in specific neuronal cell groups can be found depending on the stages examined [START_REF] Wolf | Coordinated expression of Hoxa2, Hoxd1 and Pax6 in the developing diencephalon[END_REF]. Nonetheless, the importance of paralog group 1 and 2 Hox genes in the identity of specific hindbrain segments (morphologically separated by epithelial constrictions during a brief window as rhombomeres ) is illustrated by several loss-of-function experiments in mice. Null mutation of Hoxa1 leads to the absence of rhombomere (r)5 and a large part of r4 [START_REF] Dollé | Local alterations of Krox-20 and Hox gene expression in the hindbrain suggest lack of rhombomeres 4 and 5 in homozygote null Hoxa-1 (Hox-1.6) mutant embryos[END_REF]. The Hoxb1 homozygote mutant mouse fails to specify r4 motor neurons, destined to contribute to the facial motor nucleus in the hindbrain [START_REF] Goddard | Mice with targeted disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve[END_REF].

Global disruption of the transcription factor gene Egr2 (also known as Krox20), which indirectly represses Hoxb1 (Garcia-Dominguez, [START_REF] Garcia-Dominguez | PIASxbeta acts as an activator of Hoxb1 and is antagonized by Krox20 during hindbrain segmentation[END_REF] but directly activates Hoxb2 expression (Sham et al., 1993), leads to reduction or loss of r3 and 5 [START_REF] Schneider-Maunoury | Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain[END_REF].

Hoxb1 is strongly expressed in r4 and the spinal cord caudal to r8. Alone, r4 provides the majority of NC mesenchyme to PA2 (contributing to the hyoid, stapes and styloid) and a small amount to PA1 (proximal jaw bones such as the squamosal, palatoquatrate, malleus and incus) [START_REF] Grapin-Botton | Plasticity of transposed rhombomeres: Hox gene induction is correlated with phenotypic modifications[END_REF][START_REF] Köntges | Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny[END_REF]. Hoxa2 and Hoxb1 are normally maintained in PA2 NC mesenchyme but downregulated in the small subpopulation of cells from r2-r4 populating the caudal portion of PA1 [START_REF] Couly | Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development[END_REF][START_REF] Prince | Hoxa-2 expression in normal and transposed rhombomeres: independent regulation in the neural tube and neural crest[END_REF].

Hoxa2-homozygous mutant mice acquire mirror-image proximal jaw bones in place of those normally formed from PA2, but no distal or anterior elements such as the prominent Meckel's or palatine cartilages [START_REF] Rijli | A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene[END_REF]. Only NC cells that had once expressed Hoxb1 are responsive to a change in positional identity in the absence of Hoxa2.

How would naïve NC, never having expressed Hox genes, respond to their ectopic induction? Previous work has only indirectly addressed this question. Transplantations of quail neural tube segments to more caudal positions in chick hosts, for example from Hoxb1-negative r5/6 to a caudal r8 position, come to express Hoxb1 in the grafted neuroepithelium, while the corresponding NC assume a new, posterior identity by integrating appropriately into the superior ganglion of the vagal nerve. However, r1/r2 neuroepithelium, which never expresses Hox genes, is not competent to respond in the same way [START_REF] Couly | Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development[END_REF][START_REF] Grapin-Botton | Plasticity of transposed rhombomeres: Hox gene induction is correlated with phenotypic modifications[END_REF]. Conversely, where Hoxb1expressing r4 is transplanted to r1-r3 regions, the neuroepithelium maintains its cell-autonomous Hoxb1 expression, but in half of the manipulated embryos, Hoxb1 is no longer transcribed in the grafted NC cells [START_REF] Kuratani | Rhombomere transplantation repatterns the segmental organization of cranial nerves and reveals cell-autonomous expression of a homeodomain protein[END_REF], correlating with variable effects on the morphologies of cranial ganglia V to VII and their nerves. Nonetheless, when r4-r6 neuroepithelium is transplanted to the level of the posterior mesencephalon, Hoxa2 is autonomously maintained in grafted NC cells within the mandibular arch (posterior PA1). Interestingly, this structure becomes hypoplasic by E4, and by E7-E9, all distal jaw and hyoid (in chick, entoglossal or basihyal tongue) skeleton is missing [START_REF] Couly | Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development[END_REF].

We have generated a conditional transgenic mouse line to examine the consequence of targeted ectopic expression of the Hoxb1 gene. To revisit and investigate the role of Hoxb1 in patterning of head and neck-derived tissues, we have crossed this new allele with the Wnt1-Cre line. Wnt1-Cre drives Cre recombinase expression in discrete areas of the presumptive dorsal brain and spinal cord and subsequently, in the majority of NC cells, including those of the Hox-negative regions of the head (Chai et al., 2000;[START_REF] Echelard | Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS[END_REF]. The resultant mice demonstrate a range of malformations affecting not only the craniofacial skeleton but also the forebrain, eyes, head and neck glands and the outflow tract of the heart. The milder end of the spectrum is reminiscent of human congenital malformations collectively known as multiple congenital anomaly syndromes, suggesting that their common phenotypic associations could stem from induction of temporally or spatially ectopic HOX gene expression in the NC lineage during embryonic development.

Results

Expression of CAG-Hoxb1 in neural crest cell lineages causes a range from hypomorphic to severe craniofacial phenotypes

We developed a mouse line, Tg(CAG-Hoxb1,-EGFP) 1Sza (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 as well as eGFP over-expression (Supplementary Figure 1). To drive expression within most of the cranial as well as all trunk NC lineages, we made use of the recently renamed H2afv Tg(Wnt1-cre)11Rth (Wnt1-Cre) line [START_REF] Danielian | Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase[END_REF]Goodwin et al., 2017). Lineage tracing at E9.5 showed that the Hoxb1 transgene was expressed within the entire Wnt1-Cre expression domain (Supplementary Figure 1; Chai et al., 2000;[START_REF] Echelard | Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS[END_REF].

At birth, 8 out of 46 pups died in the first day and had no belly milk spot. These were invariably of the Wnt1-Cre;CAG-Hoxb1 (mutant) genotype and had a cleft palate . Three had open eyelids. In comparison to their littermates of any other genotype, they had frontonasal hypoplasia and a rounded head, micrognathia, deep-set eyes behind small palpebral fissures and a reduced telencephalon. Although the number of mutants was somewhat lower than the 25% expected, their representation at birth was not significantly reduced (17%; p<0.12). Nonetheless, given the congenital malformations of the survivors, we decided to examine fetal stages, to determine if death also occurred prior to birth.

Prenatal phenotypes anticipate congenital malformations at two levels of severity

Between E11.5 and E19.5, Mendelian ratios were respected, and a full quarter of fetuses had a mutant genotype as expected. The signs observed in mutant perinatal pups were present in fetuses older than E15.5, including open eyelids that revealed coloboma of varying degrees of severity, a foreshortened snout, mild frontal bossing with a point at the caudal edge of the telencephalon, mandibular and external ear hypoplasia, and cleft palate (Figure 1e-h).

However, a subset of mutant fetuses observed from E15.5 on demonstrated a far more dramatic phenotype that had not been observed at birth (Figure 2a-h). This included dorsal or ventral exencephaly, frontonasal and maxillary agenesis, and striking mandibular aplasia or agnathia.

Remnants of arrayed whisker fields were often visible in the residual ectoderm of what would have become the lateral muzzle, and a small pointed tongue remained present. Severe coloboma or secondary anophthalmia, recognizable from deep remnant retinal pigmented epithelium, characterized all of these mutants (Figure 2b, 2f, 4g-h). No further gross anatomical defects were observed in the skeleton, limbs, adrenal glands or the gut.

Morphological differences distinguished hypomorphic from severe phenotypes at E11.5 as well; the former had both telencephalon and developing nostrils but were visibly smaller than non-mutant littermates (Figure 1i-l), and the latter lacked identifiable growth of the telencephalic vesicles or the frontonasal bud (Figure 2i-l). At E11.5, the maxillary and mandibular buds of the first pharyngeal arch were significantly less developed as measured by width in both mild (p<0.02) and severe phenotypes (p<0.001) respectively, with a greater effect on the mandible (p<0.005 in mild, p<0.00001 in severe).

Distances measured on photographs between the nostrils, or of the width of the telencephalon, were not significantly different between mildly affected and control littermates. However, vertical growth of the telencephalic vesicles was significantly reduced in even mildly affected mutants (p<0.01). Thus, newborn malformations reflected the consequences of a degree of phenotypic variation already present at E11.5, affecting derivatives and tissues adjacent to the frontonasal bud and first pharyngeal arch in all, as well as more posterior pharyngeal arches in some.

Cardiac defects reflect the differential penetrance of phenotype

Because NC cells of the hindbrain are critical for development of the caudal pharyngeal arches and migrate into the outflow tract of the heart, we examined mutants for cardiovascular malformations.

Correlating with the severity of prenatal craniofacial phenotypes, defects were frequently found between E15.5 and birth in terms of misaligned but septated aorta and pulmonary trunk (n=6/13) or as abnormally branched or connected pharyngeal arch arteries (n=5/13; Table 1). At E11.5, five pharyngeal arches have normally developed in controls, with the posterior arches patently connected to the dorsal aorta. However, two hypomorphic mutant embryos developed pericardial edema and patent connections at the level of hypoplasic anterior arches, while others appeared normal (Figure 3a,3c). Severely affected mutants only developed vascular connections to the level of the second aortic arch (Figure 3b, 3d) and had fully penetrant pericardial edema.

Misalignment of the great arteries was frequent (Figure 3E-F). By birth, severely affected embryos regularly presented pharyngeal arch artery defects such as interrupted aortic arch or abnormal connections between carotid and brachiocephalic arteries (Figure 3G-H). Bicuspid aortic valve being a common, but not always symptomatic, cardiac malformation, we examined valves in histological sections between E15.5 and birth (Table 1). Nearly two-thirds (9/14) of mutants had bicuspid aortic valves (Figure 3i-j), correlated with a dramatic reduction in their NCC component as shown by lineage tracing (Figure 3k-l). Bicuspid valves were observed in both mildly and severely affected mutant animals.

Differential phenotypes develop between E9.0 and E11.5

Mutant NCC follow normal migration patterns in head at E9.5

Early migratory patterns of NC cells into the pharyngeal arches at E9.5 appeared similar between control Wnt1-Cre; R26R-LacZ and mutant Wnt1-Cre; CAG-Hoxb1; R26R-LacZ embryos (Figure 4a-b) as well as between Wnt1-Cre; Rosa tdT and mutant Wnt1-Cre; CAG-Hoxb1; Rosa tdT embryos (Supplementary Figure 1). The dorsal diencephalon and mesenchyme around the dorsal optic vesicles, sometimes appeared less occupied by mutant cells (Figure 4b, asterisk), but this was not distinguishable from individual variation. The frontonasal bud was similarly occupied by mutant or WT NCC at E9.5. However, by E15.5, Wnt1-Cre;CAG-Hoxb1;R26R-LacZ mice consistently showed that NCC had not invested the proximal outflow tract as much as in their control littermates, particularly along the ventral pulmonary trunk (Figure 4c-d). This observation is consistent with the variable cardiovascular defects in mutants and reduced integration of mutant NC cells into the aortic valve, as described above (Table 1 andFigure 3).

Egr2/Krox20 and Fgf8 expression are not visibly affected at E9.0-E9.5

Egr2/Krox20 TF mRNA is normally expressed at E8.5-E9.0 in both rhombomeres r3 and r5, and disappears subsequently from the r3 neuroepithelium, although it remains transcribed in proximal NCC migrating away from both areas at E9.5. As such, Krox20 is a readout of the organization of rhombomeres r3, r4 and r5 during this critical window of NCC delamination and population of the pharyngeal arches.

The distribution of Krox20 transcripts seemed unaffected in the rhombomeres and the stream of NCC from r5/r6 towards posterior pharyngeal arches, in mutant (n=11) versus control (n=12) embryos from the same litters (Figure 4g-h).

Fgf8, encoding an important secreted growth factor needed for multiple processes of craniofacial development, is normally expressed at E9.5 in the developing brain at the midbrain-hindbrain constriction known as the isthmus, as well as at the rostral telencephalic midline (Figure 4i; [START_REF] Trumpp | Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch[END_REF]. These expression domains, as well as an ectodermal domain in the first pharyngeal arch (red arrow), are unperturbed in stage-matched Wnt1-Cre; CAG-Hoxb1 mutant embryos (Figure 4j; n=5). However, by E13.5 (n=3), normal Fgf8 expression at the edges of the nasal pits (Figure 4k) was absent from mutants, as was the frontonasal bud between them. Mutant embryos maintained vestigial ectodermal Fgf8 expression at the edges of the maxillary and mandibular buds, otherwise highly hypoplasic (arrowheads, Figure 4k).

Severe ocular malformations induced by Hoxb1-expressing periocular mesenchyme

NCC are critical for eye morphogenesis. As mesenchyme, they surround the optic vesicle as it grows towards the lateral ectoderm, contributing such periocular and anterior chamber structures as the matrix of the cornea, the melanocytes of the iris and ciliary body, the pericytes of the choroid, hyaloid and retinal blood vessels, and the sclera. In mice, the choroid is also heavily invested by NCCderived pericytes, in juxtaposition to the retinal pigmented epithelium (RPE; Chassaing et al., 2016).

Mildly affected Wnt1-Cre;CAG-Hoxb1 fetuses consistently have ocular malformations such as coloboma (Figure 1f), while severely affected animals undergo secondary microphthalmia, showing only rudiments of remaining RPE (Figure 2b). Examination of a Wnt1-Cre;CAG-Hoxb1;R26R-LacZ fetus at E18.5 in section shows that NC-accompanied hyaloid vasculature to the lens remains persistent well after its usual period of remodeling into the vascular plexus of the retina. Only small cartilaginous nodules appear to persist of the NC-derived sclera, while corneal NC cells have not stratified and thickened into the appropriate transparent structure (for comparison, adult Wnt1-Cre;R26R-LacZ cornea in Figure 4g). Ectopic differentiation of RPE all around the optic vesicle also occurs within the neural retina itself, which subsequently is not layered (arrow, Figure 4h).

Hypomorphic and severe mutant phenotypes reflect distinct molecular profiles

Both the timing of symptomatic onset and maintenance of rhombomere identity after induction of ectopic Hoxb1 expression by Wnt1-Cre are consistent with an initial effect on the NCC lineage rather than on the dorsal neuroepithelium as such. To orient future work on potential target effector molecules, we examined the relative global expression in the head and heart at E11.5 (cf. fragments featured in Figures 12, K-L) of transcripts representing candidate molecular pathways that we hypothesized might be affected in these areas by or within NCC mesenchyme. Normalizing to expression levels of the homogeneously and ubiquitously transcribed Tbp and Dhfr, we examined transcription of genes encoding bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4); Cyclin D2 (Ccnd2) and the cyclin-dependent kinase inhibitor p21 (Cdkn1a); the LIM and senescent cell antigenlike domains 1 adhesion protein, also known as Pinch1 (Lims1), the homeobox transcription factors Hoxa2, Msx1, Pax3, Pitx2 and Sox9; Shh and its principal dependence receptor and transcriptional target Ptch1; and the growth factors Fgf8 and Vegfa.

Embryos from two large litters were genotyped and classified into three phenotypic groups: control, hypomorphic (mild) mutant or severe mutant. The relative transcription levels of Bmp2, Bmp4, Vegfa, Ptch1, Lims1, Cdkn1a, Pax3 and Sox9 were significantly repressed in the severely affected mutants at E11.5, but not in their mildly affected mutant littermates, as compared with controls.

Three of these were in fact significantly more transcribed in the mildly affected mutants: Pax3, Sox9 and Ptch1, while Msx1 was unaffected in severe mutants and only somewhat, if significantly, reduced in hypomorphic mutants.

Discussion

Ectopic Hoxb1 expression in NC induces major molecular as well as morphological changes in the developing head, neck and heart

The lack of a significant change in Ccnd2 associated with a large decrease in Cdkn1a transcription is consistent with increased p53-independent apoptosis rather than reduced proliferation in severely affected mutants [START_REF] Gartel | The role of the cyclin-dependent kinase inhibitor p21 in apoptosis[END_REF]. This cell death may occur in the anterior NCC population forced to express Hoxb1, but also in the ocular, craniofacial, endocrine and brain tissues for which its inductive activity is needed. Indeed, older work has shown that experimental removal of NC cells in chick embryos induces brain and pituitary apoptosis, in part from the loss of the perineural vascular plexus, accompanied by NC-derived pericytes from dorsal to ventral forebrain, that will develop into the meninges of these areas [START_REF] Etchevers | Anterior cephalic neural crest is required for forebrain viability[END_REF].

As supported by changes in the transcription of multiple growth factors, many of the effects of ectopic Hoxb1 expression in cephalic NC are thus likely to be secondary. Complementary work has shown that Vegfa is also an essential growth factor secreted by Hox-negative NC mesenchyme, and we have shown here that this gene is less expressed in severely affected Wnt1-Cre;CAG-Hoxb1 mutant heads; its homozygous loss from the same population leads to cleft palate, reduced ossification of the premaxillary and frontal bones, and maxillary hypoplasia preceded by reduced vascular growth within PA1 [START_REF] Wiszniak | Neural crest cell-derived VEGF promotes embryonic jaw extension[END_REF]. Vegfa is also chemotactic for NC cells and expressed by the ectoderm of PA2, inciting Hoxb1-expressing NC from r4 to populate this arch [START_REF] Mclennan | Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo[END_REF]. Ectopic transcription of Hoxb1 may confer a measure of r4-origin identity on the Hoxa2-positive cephalic NC of r2/r3 and repress endogenous Vegfa, forcing cells to respond to exogenous signal from PA2 rather than to assume a less directed phenotype that would allow them to better disperse and colonize more rostral tissues (McLennan et al., 2015).

Another chemotactic factor for cephalic NC is Sonic hedgehog, in particular for mesencephalic NC surrounding the optic vesicles [START_REF] Tolosa | Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure[END_REF]. Shh is also a survival factor for Hox-negative NCC [START_REF] Ahlgren | Inhibition of sonic hedgehog signaling in vivo results in craniofacial neural crest cell death[END_REF]. As expected, Shh expression is not altered in Wnt1-Cre; CAG-Hoxb1 mutant embryo heads, but that of its dependence receptor and transcriptional target Ptch1 is. Human PTCH1 missense mutations are the second most common genetic cause of clinical anophthalmia (Chassaing et al., 2016) and their pathogenic mechanisms are incompletely understood. Shh produced by the foregut endoderm specifically amplifies the sublineage competent to give rise to mesenchymal derivatives such as vascular smooth muscle and skeletal elements in PA1 and 2, as opposed to neurons [START_REF] Brito | An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival[END_REF][START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF]. The Shh source in the ventral diencephalon and optic stalk may act similarly for maxillary and palatine mesenchyme [START_REF] Tolosa | Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure[END_REF]. Thus, effects on Ptch1 observed in our Wnt1-Cre; CAG-Hoxb1 mutants implicate Shh among the paracrine exchange of signals between NC-derived periocular mesenchyme and the ocular primordium, as an effector responsible for coloboma and secondary anophthalmia.

Fgf8 is a known survival factor for PA1 and PA2 mesenchyme [START_REF] Trumpp | Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch[END_REF]. As expected, its transcription in the oral ectoderm was not significantly affected by the ectopic expression of Hoxb1 in the underlying NC cells of PA1, as assessed both by qRT-PCR and in situ hybridization. However, although it did not reach significance, diminution in overall Fgf8 transcription in the most severely affected embryos at E11.5 likely reflects the secondary loss of the entire frontonasal bud by E13.5 and the nasal pits that also normally express Fgf8 in their ectoderm, as seen by in situ hybridization.

Ectopic Hoxb1 expression leads therefore to both direct and highly indirect transcriptional consequences for facial development, which in the future could be distinguished with an appropriate antibody developed for chromatin immunoprecipitation.

Hoxb1 overexpression in the NC lineage drastically decreases Bmp2 and Bmp4 transcription in severely affected mutants, correlated with a drop in chondrogenic Sox9 TF transcription. A Shhsoaked bead implanted into the frontonasal process of the chicken embryo not only increases NCderived mesenchyme but also the relative expression of Bmp2 and Bmp4 therein [START_REF] Hu | A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face[END_REF]. Like Ptch1, Sox9 shows a modest but significant increase in transcription in the hypomorphic embryos. These observations seem to implicate defective Shh signal transduction upstream of Bmp expression as effectors of the dramatic facial hypoplasia observed in severe mutant phenotypes.

Gain of Hox function restricts rostral mesenchymal NC competence

Distinct models have induced ectopic expression of paralog group 1 and 2 Hox genes in the heads of vertebrates, inducing skeletal but also additional malformations.

The injection of mRNA encoding Hoxa1 into fertilized eggs of zebrafish prevents the formation of the cartilages of the first pharyngeal arch and thereby, the lower jaw [START_REF] Alexandre | Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype[END_REF]. Such ectopic expression is induced in a mosaic manner in approximately a quarter of all cells, not only NC cells, but also not in all NC cells. Normally, as compared to the conserved expression domain of Krox20, the anterior limit of Hoxa1 expression in the hindbrain is at the r3/r4 boundary, as in other vertebrates. After injection, the hindbrain becomes mispatterned, with enlargement and displacement of r3. Mortality increases to 30% of Hoxa1-injected embryos. Survivors of this mosaic Hoxa1 gain-of-function lose the stream of Dlx2-expressing NCC to the 1 st pharyngeal arch.

Subsequently, the jaws do not develop. In addition, the ethmoid cartilage, derived from the frontonasal bud, but also cartilages from the posterior pharyngeal arches are severely reduced or fused, and microphthalmia is also evident. These morphological effects can be phenocopied by exogenous retinoic acid [START_REF] Alexandre | Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype[END_REF], a known activator of anterior Hox gene transcription and teratogenic both in deficiency and in excess.

Spontaneous Hox gene overexpression is associated with congenital cranial malformations. A study of late-stage death in over ten thousand commercial broiler chickens in ovo showed that calvarial aplasia, leading to exencephaly, accounted for nearly half of the axial skeletal defects and was a frequent birth defect at over 1% of the population. Exencephaly just before hatching correlated with a large increase in absolute and relative Hoxa1 transcription [START_REF] Jaszczak | Expression of Hoxa1 and Hoxd3 genes in chicken[END_REF]. The frontal bones in chicken are NC derivatives, as are their underlying meninges [START_REF] Couly | The triple origin of skull in higher vertebrates: a study in quail-chick chimeras[END_REF]. Supported by our work, regulatory regions of Hox paralog group 1 and 2 transcripts make appealing and novel genomic candidates to study in the context of rostral neural tube defects.

Experimentally forcing Hoxa2 expression in the Hox-negative forebrain and midbrain neural folds of the chick, before NC migration, yielded NC cells that normally colonized the facial primordia at a stage equivalent to mouse E9.5. Later, however, they did not produce upper or lower beak, and the fetuses became exencephalic, with loss of the calvaria and forebrain [START_REF] Creuzet | Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton[END_REF]. NC cells ectopically expressing Hoxa2 still contributed to the pericytes of facial vasculature, and the eyes were normal. Similar experiments carried out with ectopic Hoxa3 or Hoxb4 in Hox-negative NC mesenchyme again did not interfere with the initial dispersion of this mesenchyme throughout the frontonasal and PA prominences. Ectopic Hoxa3 induced severe midfacial hypoplasia, absence of the ocular sclera and loss of the lower jaw but retained a reduced telencephalon. In embryos carrying Hoxb4-transfected neural folds, the upper facial skeleton did not form at all, but the proximal portion of the lower jaw did. Co-introduction of exogenous Hoxa3 and Hoxa4 combined the most extreme effects of each on the craniofacial skeleton, but the ability of these modified NC to differentiate into Schwann cells of cranial nerves was maintained [START_REF] Creuzet | Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton[END_REF]. The eyes remained exempt from major malformations.

Later work induced Hoxa2 overexpression in murine NC with a similar approach to that described in this work: Wnt-Cre mice were crossed with a floxed CAG-Hoxa2 allele inserted at the R26R locus [START_REF] Kitazawa | Distinct effects of Hoxa2 overexpression in cranial neural crest populations reveal that the mammalian hyomandibular-ceratohyal boundary maps within the styloid process[END_REF]. The mutant mice also showed a short snout, exencephaly, cleft palate and apparent coloboma. There were distinct effects of Hoxa2 gain-of-function on the former Hoxexpressing NCC derivatives of PA1 compared to the naïve Hox-negative NCC rostral to that. The former group of cells underwent partial mirror image-duplications of hyoid structures in the proximal mandible, while the distal mandible (unlike the rest of the face) was not significantly affected. Similar to our findings, in mutant PA1-2, Fgf8 transcription was unaffected but Sox9 was significantly downregulated. PA2 developed malformed but identifiably appropriate hyoid skeletal elements and lost the stapedian artery, which were attributed to the relative cell-autonomous increase in Hoxa2 compared to other Hox genes such as Hoxb1 [START_REF] Kitazawa | Distinct effects of Hoxa2 overexpression in cranial neural crest populations reveal that the mammalian hyomandibular-ceratohyal boundary maps within the styloid process[END_REF].

The morphological transitions between new (rostral) and old (caudal) head and body are therefore encoded at a molecular level. The anterior end of the hindbrain beyond the future cerebellum is the source of some Hox-negative NC, derived from neuroepithelial levels having once expressed Hoxa1 and Hoxa2, which enter PA1 to form proximal bones of the jaw joint. In contrast, NC cells from the presumptive diencephalon, mesencephalon, isthmus and first rhombomere, never express Hox genes before their dispersal throughout the rostral head, and the tail end of this population contributes most mesenchyme to PA1. Caudal Hoxb1-expressing NC cells of PA2 form the hyoid bones, while NC mesenchyme of the posterior arches give rise to laryngeal, thyroid and cricoid cartilages as well as shape the branchial vascular network that originates in those arteries splitting off from the ventral aorta and irrigating the face and brain [START_REF] Etchevers | The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain[END_REF]. The structures from NC having once expressed Hox genes can undergo homeotic transformation (acquisition of a more posterior positional identity in concert with morphological boundaries; [START_REF] Burke | Hox genes and the evolution of vertebrate axial morphology[END_REF] and in this, are distinct from the rest of the Hoxnegative NC that came from regions never having expressed these transcription factors. Here, gain of Hox function leads to a homeotic shift in cellular potential relative to the mesectodermal ability to exert a paracrine influence on rostral head structures and to later differentiate into corresponding auxiliary support tissues. The present work introduces evidence that the inductive role of Hoxnegative cephalic NCC depends also on the continued absence of Hox transcription factors. This role normally precedes vascular remodeling and chondrogenesis, playing a vital role in growth and survival not only of facial bones and teeth but also, secondarily, of eyes, nose, external ears, pituitary, forebrain and facial muscles.

Loss of Hox function affects competence of caudal mesenchymal neural crest

We speculate that the widespread early death in Hoxa1-injected gain-of-function zebrafish embryos may be due to an uncharacterized cardiovascular defect in mosaics with greater allele load, since levels of murine Hoxa1 determines both mesodermal contributions to the outflow tract (Bertrand et al., 2011) and those of the hindbrain NC to the 4 th pharyngeal (aortic) arch in particular [START_REF] Roux | Hoxa1 and Hoxb1 are required for pharyngeal arch artery development[END_REF]. Hoxa1-null mice, and human patients with truncating mutations of HOXA1, also demonstrate cardiovascular malformations similar to, though more penetrant than, those we observed in our Hoxb1 gain-of-function model [START_REF] Makki | Cardiovascular defects in a mouse model of Hoxa1 syndrome[END_REF]. They also can be deaf, due to cochlear aplasia, and may have bilateral Duane syndrome, an oculomotor disorder, or central nervous anomalies affecting hindbrain nuclei (facial weakness, central hypoventilation) and intellectual abilitieswith a wide phenotypic spread [START_REF] Tischfield | Homozygous HOXA1 mutations disrupt human brainstem, inner ear, cardiovascular and cognitive development[END_REF].

Hoxb1-null mice have not been reported to have to defects in neural crest-derived skull bones or blood vessels, only in the facial motor nucleus and corresponding portion of facial nerves. Here, too, Krox20 expression and r4 neural crest cell migration were unaffected in mutants [START_REF] Goddard | Mice with targeted disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve[END_REF]. However, some homozygous mutant individuals had died at birth without a belly milkspot, consistent with a potential cleft palate. Skull defects may only have been subtle at the level of the jaw hinge, as in Hoxa2-mutant mice [START_REF] Rijli | A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene[END_REF], but were not examined. Human mutations in HOXB1 identified to date are missense mutations that, because of their similarities with the null mutant mouse phenotype, have been presumed to be loss-of-function. Here, too, the phenotypic spectrum includes facial nerve palsy and sensorineural hearing loss, but also includes esotropia (which can result from Duane syndrome), low-set ears or external ear malformations, midface retrusion (resulting from underdeveloped maxillary prominences), flat nasal bridge and upturned nose tip, and micrognathia [START_REF] Vogel | Homozygous HOXB1 loss-of-function mutation in a large family with hereditary congenital facial paresis[END_REF]Webb et al., 2012). Like HOXA1 mutations, HOXB1 mutations identified to date preferentially affect derivatives of the posterior pharyngeal arches or hindbrain, but have an additional, intriguing effect on the depth of PA1 and midline frontonasal derivatives, without affecting facial width.

Hoxb1 genetically interacts with other paralog group 1 and 2 genes to regulate morphogenesis in heart, great vessels and eyes

Recent studies on single and compound Hoxa1;Hoxb1 mutant embryos showed that reduction of Hoxa1 gene dosage in a Hoxb1-null genetic background results in abnormal PA and heart development and subsequently in great artery defects [START_REF] Makki | Cardiovascular defects in a mouse model of Hoxa1 syndrome[END_REF][START_REF] Roux | Hoxa1 and Hoxb1 are required for pharyngeal arch artery development[END_REF][START_REF] Roux | Hoxb1 regulates proliferation and differentiation of second heart field progenitors in pharyngeal mesoderm and genetically interacts with Hoxa1 during cardiac outflow tract development[END_REF]. Lineage tracing analysis shows that both Hoxa1 and Hoxb1 genes are expressed in hindbrain NCCs that contribute to PAs 3-6 and outflow tract development (Bertrand et al., 2011;[START_REF] Roux | Hoxa1 and Hoxb1 are required for pharyngeal arch artery development[END_REF]. Endothelial cells in PA4 differentiate normally in compound mutants, but the corresponding vascular smooth muscle is absent. Here, we have shown that NC-targeted Hoxb1 overexpression also results in defects such as interrupted aortic arch and misalignment of the great arteries. Lineage tracing analysis in this genetic context clearly shows reduced NC colonization of the outflow tract region (see Figure 3b,d), suggesting that cardiovascular defects observed in Wnt1Cre;CAG-Hoxb1 transgenic embryos is caused by insufficient numbers of NC cells. In the chick, ablation of the cardiac NC population leads to a similar if more severe phenotype along the same spectrum of anomalies [START_REF] Kirby | Neural crest cells contribute to normal aorticopulmonary septation[END_REF][START_REF] Waldo | Cardiac neural crest cells provide new insight into septation of the cardiac outflow tract: aortic sac to ventricular septal closure[END_REF]. However, we never observed an effect on thymus development, as is often associated with cardiac anomalies in ablation experiments.

We also detect NC deficiency at the level of the valve leaflets in Wnt1-Cre;CAG-Hoxb1 mice, correlated with a bicuspid aortic valve in nearly two-thirds of transgenic fetuses. Arterial valve development is susceptible to imbalances in the ratio of mesodermal to NC cells in the leaflets [START_REF] Odelin | Loss of Krox20 results in aortic valve regurgitation and impaired transcriptional activation of fibrillar collagen genes[END_REF][START_REF] Phillips | Neural crest cells are required for correct positioning of the developing outflow cushions and pattern the arterial valve leaflets[END_REF]. The Egr2/Krox20-expressing NC subpopulation emigrating from r5-r6 (see Figure 4g,h) is particularly important for arterial valve development [START_REF] Odelin | Loss of Krox20 results in aortic valve regurgitation and impaired transcriptional activation of fibrillar collagen genes[END_REF](Odelin et al., , 2018)). Thus, ectopic expression of Hoxb1 in NCCs from r5-r6 may compromise their fate by interfering with the transcriptional readout of other transcription factors such as Krox20 [START_REF] Desmazières | Krox20 controls the transcription of its various targets in the developing hindbrain according to multiple modes[END_REF].

The periocular mesenchyme of NC origin is particularly critical for eye development. It gives rise not only to most components of the anterior chamber (cf. Figure 4e): iris, corneal stroma and endothelium, but also to the cartilaginous sclera, the melanocyte-embedded choroid behind the retinal pigmented epithelium, and the smooth muscle and pericytes of the hyaloid and ophthalmic arteries and retinal vascular plexus (reviewed in [START_REF] Creuzet | Neural crest derivatives in ocular and periocular structures[END_REF]. Lineage tracing in mice confirms these contributions [START_REF] Gage | Fate maps of neural crest and mesoderm in the mammalian eye[END_REF]. NC-targeted ablation of Pitx2 induces anterior segment defects as well as secondary anophthalmia, similar to the effect of ectopic expression of Hoxb1 in the same cells [START_REF] Evans | Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development[END_REF]. However, Pitx2 expression levels were not changed in our Hoxb1 gain-of-function mutants as compared to control embryos, suggesting that these two transcription factors act independently within NC periocular mesenchyme to control its paracrine effects on eye development. Indeed, one such effect of NC-specific Pitx2 deficiency is to expand the Pax2-expressing domain of the future optic nerve within the neuroepithelium at the expense of retinal cell fate, and subsequently at the expense of the RPE [START_REF] Evans | Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development[END_REF]. Although NC-targeted Hoxb1 overexpression also results in secondary anophthalmia, we observed a RPE around the entire ocular globe remnant, incorporation of the lens but obliteration of the pupil by neural retina itself differentiating into RPE, accompanied by a similar incapacity of the periocular mesenchyme to differentiate into the sclera or cornea.

We also consistently observed low-set external ears and loss of the nasal capsule, philtrum and the olfactory bulbs and cortex. These phenotypes emphasize that repression of Hoxb1, in addition to that of other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for optimal development and subsequent protection of ancestral cephalic sensory functions such as audition, olfaction and vision in vertebrates. This repression and widespread paracrine dependencies necessary for cephalic development may explain the particular sensitivity of the Hox-negative NC cell population to either retinoic acid toxicity (Matt, [START_REF] Ghyselink | Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm[END_REF] or deficiency (e.g. the syndromic microphthalmia due to STRA6 deletions; [START_REF] Chassaing | Phenotypic spectrum of STRA6 mutations: from Matthew-Wood syndrome to non-lethal anophthalmia[END_REF] in utero and their phenotypic overlap. Ultimately, the human embryo must actively repress Hox gene activation in rostral cephalic NC cells or risk developing a spectrum of multiple congenital anomalies. pregnant (C57Bl/6) foster mothers. We obtained offspring from each of the five positive founder lines and subsequently selected the line with the highest expression.

Lineage tracing and immunofluorescence

After dissection, hearts were fixed in 4% buffered paraformaldehyde during 10 minutes at room temperature, embedded in OCT and cryosectioned at 12 µm. Standard immunofluorescence procedures were used [START_REF] Ryckebüsch | Decreased levels of embryonic retinoic acid synthesis accelerate recovery from arterial growth delay in a mouse model of DiGeorge syndrome[END_REF] with anti-CD31 (PECAM) (1:100, BD Pharmingen 9 A , a Ale a Fluor™-555 conjugated goat anti-rat antibody (1:500, Life Technologies), and DAPI ′, -diamidino-2-phenylindole) was used at 300 nM as a counterstain to distinguish cell nuclei. Recombined cells were observed using complementary techniques. eGFP is produced by Creexpressing cells on a CAG-Hoxb1 or Rosa mT/mG background and was visualized, like for immunofluorescence, in cryostat section at 12 µm on a Zeiss Apotome fluorescent microscope. In order to use light microscopy in whole mount, we used the floxed R26R-LacZ reporter allele as an additional lineage indicator of Cre recombination. We compared embryos at the same numbers of somite pairs +/-1 pair. -galactosidase is produced by Cre-expressing cells and precipitates an insoluble dark turquoise-blue chromogen in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-Dgalactopyranoside), as described [START_REF] Gierut | Whole-mount X-Gal staining of mouse tissues[END_REF]. Whole-mount photographs were taken on a Zeiss Axiozoom dissecting microscope. The head and heart of a Wnt1-Cre;CAG-Hoxb1;R26R-LacZ embryo at E18.5 were embedded, sectioned at 10 µm on a cryostat, developed in X-Gal, and counterstained with hematoxylin. To study aortic valves between E15.5 and birth, hearts were sectioned at 10 µm and alternate sections stained with hematoxylin-eosin according to standard protocols. Microphotographs were taken with a Zeiss AxioScan.Z1 slide scanner.

In situ hybridization

Whole-mount in situ hybridization was carried out according to standard protocols for mouse embryos at E9.5 and E13.5 [START_REF] Trumpp | Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch[END_REF]. The Krox20 probe template plasmid was linearized with BamHI and transcribed with T3 RNA polymerase; the Fgf8 probe template plasmid was linearized with BglII and transcribed with SP6 RNA polymerase.

qRT-PCR and statistics

Groups of 3 WT (including carriers of individual Wnt1-Cre or floxed CAG-Hoxb1 alleles) or 4 mildly or severely affected mutant embryos were constituted after individual qRT-PCR analysis of the expression levels of 16 genes (primers in Table S1 of the online supplementary materials). Total RNA was extracted using the NucleoSpin RNA extraction kit (Macherey-Nagel). First-strand cDNA was reverse transcribed from 500 ng total RNA with the AffinityScript kit (Agilent) using a mix of random hexamer and oligo-dT primers, and standard qPCR with the LightCycler 480 SYBR Green I Master mix (Roche) carried out on the corresponding LightCycler, according to manufacturers' recommendations.

Expression was normalized to the geometric mean of Tbp and Dhfr expression in each group. Oneway analysis of variance demonstrated that these groups were differentially variable, such that means of the mildly or severely affected groups were compared individually to the control group, using the two-tailed Student's t-test under conditions of unequal sample size and variance, and a significance threshold of p<0.03. Measurements of distances on frontal/ventral photographs of these embryos before lysis (examples shown in Figures 1 and2, k-l) were taken using ImageJ 1.51w and groups of three of each genotype were compared using a two-tailed Student's t-test under conditions of equal sample size and variance, with a significance threshold of p<0.03. was supported by an Association Française contre les Myopathies Strategic Project award to SZ and postdoctoral fellowships from the Fondation Lefoulon-Delalande to GO, SS and FL. A. A transgenic construct was designed with the CAG promoter (CMV enhancer/chicken beta-actin promoter/splice acceptor from the rabbit beta-globin gene), a loxP-flanked STOP sequence (Chloramphenicol Acetyl Transferase [CAT] followed by a SV40 polyadenylation signal), the entire mouse Hoxb1 cDNA (900bp), an internal ribosomal entry site (IRES), and the cDNA of the enhanced green fluorescent protein (eGFP) gene. Prior to Cre-mediated excision of the "floxed" STOP sequence, no ectopic expression of Hoxb1 is observed in cells. When bred to Cre recombinase-expressing mice, the STOP sequence is removed in Cre-expressing cells of the resulting offspring, allowing transcription of both Hoxb1 and eGFP. B. Schematic vertebrate embryo at approximately mouse E9.5 representing neural crest colonization of the face, heart, foregut and somites in red (first panel); the typical Hoxb1 expression domain in green in rhombomere 4, neural crest cells colonizing the second pharyngeal arch and multiple tissues caudal to the third pharyngeal arch (second panel); and the superposition of transgenic and endogenous Hoxb1 expression in Wnt1-Cre; CAG-Hoxb1-GFP mutant embryos, inducing ectopic expression in the roofplate, isthmus and neural crest cells investing the head, cardiac outflow tract and face. C. Reporter (red, Tomato fluorescence) expression at E9.5 after recombination by Wnt1-Cre. D. Reporter (red, Tomato fluorescence) expression at E9.5 in Wnt1-Cre; CAG-Hoxb1-GFP mutant embryo. D'. Expression of GFP reporter for the Hoxb1 transgene in same embryo.

Stage

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Table 1 ,

 1 Supplementary Figure1and TableS1are separate files Supplementary Figure1.

Table 1 .

 1 Cardiovascular (CV) malformations observed in a subset of Wnt1-Cre;CAG-Hoxb1 or control littermates. PAA, pharyngeal arch artery (comprises interrupted aortic arch); arterial trunk defects include double-outlet right ventricle and over-riding aorta; PE, pericardial edema; BAV, bicuspid aortic valve; NC, non-coronary

		CV	PAA	Arterial	PE with	No CV	Valve	BAV	Small	TAV
		defects,	defects	trunk	PAA	defect	defects,		NC
		controls		defects	defects		controls		leaflet
	E11.5	0/8	5/15		7/15	3/15	n/a	n/a	n/a	n/a
		(0%)	(33%)		(47%)	(20%)
	E15.5	0/5	2/8	5/8		1/8	0/2	2/3	1/3	0/3
		(0%)	(25%)	(62%)		(13%)	(0%)	(67%)	(33%)	(0%)
	E16.5-	0/4	3/5	1/5		1/5	0/2	4/11	2/11	5/11
	birth	(0%)	(60%)	(20%)		(20%)	(0%)	(36%)	(18%)	(45%)

Table S1 .

 S1 Primers for genotyping and qRT-PCR in mice, used in this work

	Transcripts 5'	3'
	Bmp2	TAAACCGTCTTGGAGCCTGC	TCCGAATGGCACTACGGAAT
	Bmp4	TGCTTCTTAGACGGACTGCG	CTGGGGAAGCAGCAACACTA
	Ccnd2	GACCTTCATCGCTCTGTGC	ATCCTGCTGAAGCCCACA
	Cdkn1a	GCAGACCAGCCTGACAGATT	GAGGGCTAAGGCCGAAGA
	Dhfr	TTTATCCCCGCTGCCATCAT	ACCAGATTCTGTTTACCTTCCACT
	Fgf8	GCTGAGCTGCCTGCTGTT	AGCTCGGAGCAGGGAAGT
	Hoxa2	GAAGGCGGCCAAGAAAAC	CATCAGCTATTTCCAGGGATTC
	Lims1	AAATGCCATGCCATCATTG	CCGAGCATCAGCAGTTAGC
	Msx1	TCAAGCTGCCAGAAGATGCT	GGGACTCAGCCGTCTGGC
	Pax3	ACTACCCAGACATTTACACCAGG	AATGAGATGGTTGAAAGCCATCAG
	Pitx2	CCTTACGGAAGCCCGAGT	AAAGCCATTCTTGCACAGC
	Ptch1	CTTCGCTCTGGAGCAGATTT	TCTTTTGAATGTAACAACCCAGTT
	Shh	TCCACTGTTCTGTGAAAGCAG	GGGACGTAAGTCCTTCACCA
	Sox9	TCGGTGAAGAACGGACAAGC	TGAGATTGCCCAGAGTGCTCG
	Tbp	CCCCACAACTCTTCCATTCT	GCAGGAGTGATAGGGGTCAT
	Vegfa	TTAAACGAACGTACTTGCAGATG	AGAGGTCTGGTTCCCGAAA
	Transgenes 5'	3'
	Cre	GGCGCGGCAACACCATTTTT	CCGGGCTGCCACGACCAAG
	CAG-Hoxb1 GCCCAGTTCCATCACCTCTT	GACCAGGATGGGCACCAC
	LacZ	GCATCGAGCTGGGTAATAAGCGTTGGCAAT GACACCAGACCAACTGGTAGCGAC
	GFP	GAAGCAGCACGACTTCTTCAAG	ATTCTAGACTACAGCTCGTCCATGCCGAGAGC

Acknowledgments

We thank Elise Plaindoux, Nathalie Eudes and Adeline Spiga-Ghata for their precious technical assistance and Brigitte Laforest and Emilie Faure for helpful discussions. The following colleagues kindly provided probe template plasmids: Piotr Topilko (Krox20) and Robert Kelly (Fgf8). This work

Methods

Embryo collection

Timed pregnant mice were euthanized with CO2 at chosen days following embryonic day (E)0.5, determined based on the presence of a vaginal plug after mating. Progeny were analysed at E9.5, E11.5, and individually euthanized by decapitation at E15.5, E16.5, E18.5 and P0 (day of birth). Mice and embryos were genotyped using primers in Table S1.

Research with vertebrate animals

Wnt1-Cre [H2afv Tg(Wnt1-cre)11Rth ], Rosa mT/mG [Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo ], Rosa tdT [Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze ] and Rosa LacZ [Gt(ROSA)26Sor tm1Sor ] outbred to a Crl:CD-1 background, were purchased from The Jackson Laboratory and Charles River Laboratories respectively, and maintained under authorizations 32-08102012 and 37-08102012 provided by the regional animal care and use committee (C2eA-14) overseeing our institution.

Generation of the floxed CAG-Hoxb1-IRES2-eGFP transgenic mouse line

Complete cDNA from mouse Hoxb1 was provided by Dr. Anthony Gavalas (Biomedical Research Foundation of the Academy of Athens, Greece). The coding sequence was cloned into the BamHI site of a pCIG-IRES-eGFP plasmid [START_REF] Megason | A mitogen gradient of dorsal midline Wnts organizes growth in the CNS[END_REF]. Briefly, pCIG was made by linker insertion of three nuclear localization sequences into pIRES2-EGFP (Clontech) and transferring this insert to pCAGGS [START_REF] Niwa | Efficient selection for high-expression transfectants with a novel eukaryotic vector[END_REF]. The Hoxb1-IRES2-eGFP fragment was digested with EcoRV-NotI, blunted and cloned into the EcoRV site of a plasmid containing CAG promoter-loxP-CAT-loxP-cloning site-poly(A) (pBSKISecI CAG-CAT-poly(A), a kind gift from Dr. Yumiko Saga, National Institute of Genetics, Japan). The final construct was linearized with SecI and i roi je ted i to fertilized C Bl/ DBA/ F eggs at a o e tratio of appro . g/μl, using standard techniques. Injected eggs were re-implanted the day after the injection into pseudo-


Abstract

The employment of physiological measurements to perform on-line assessment of operators' mental states is crucial in the field of human-robot interaction (HRI) researches, and is still an open topic to the best of our knowledge. In order to progress towards systems that would dynamically adapt to operators' mental states, a first step is to determine an adequate protocol that elicits variations in engagement.

To this purpose, this work focuses on analyzing operator's physiological data streams recorded during a human-robot mission executed in a original virtual environment. In detail, the conceived mission consists in a mutual cooperation of a firefighter robot and its human operator to extinguish fires. A high level of complexity is obtained by the number and the random nature of events to be handled during the mission. As an example, guiding the robot, managing its water tank level and taking care of its electric charge are all tasks to be accomplished simultaneously, which can randomly be assigned to autonomous or manually operative mode. In addition to that, an extra task consisting in keeping an adequate level of an external water tank to allow robot refill is assigned only to the human operator for continuously soliciting his attention. Anyone can experience this mission by visiting the website robot-isae.isae.fr set up to collect a large amount of behavioral data by crowdsourcing.

The mission is accomplished through a remote human-machine interface made of controllers and a screen displaying different areas corresponding to each task. Figure 1 shows the graphical user interface, with the 5 areas of interest (AOIs). The control station is equipped with sensing devices for human data collecting. The sensing devices which allows for human data collection are an eye-tracker (SMI), located on the bottom bar of the display and a portable bluetooth electrocardiograph (eMotion Faros 360). A specific procedure for the experiment has been defined to guarantee a statistical significance of the recorded dataset.

In detail, each different human operator has to complete at least three times a ten minutes mission aiming to the best score in terms of fires extinguished. An absolute resting is imposed between missions to get a baseline for the cardiac activity. The data are collected on 17 participants of mixed sex (9 females) with average age 28.5 (S.D. = 4.52). The number and the duration of fixations per area of interest are extracted from the eye-tracker. The length of inter-beat intervals, the Heart Rate Variability (HRV) and instant Heart Rate Variability (IHRV) are computed from the ECG.

Preliminary results show a lower HRV and IHRV during the mission than during the rest session: Student and Wilcoxon statistical tests ensure a difference at least equal to 6 (p < 0.05). This evidence, according to the literature, highlights that the created mission succeeded in engaging the participants.

The impact of each mode of operation (manual/autonomous) on the human markers is also observed and analyzed. Contrarily to expectations the operator results more engaged (lower HRV) during autonomous than manual mode (p¡0.05). This is in accordance with the tasks' difficulty. In fact, when the autonomous mode takes over, the human priority is the only task that he has to accomplish by himself (external tank filling), which is also designed to be the hardest one. This is confirmed by IHRV that in average is greater during manual mode. Moreover, finding that HRV and IHRV behaving in the same way, a real-time Human-Robot team supervision application can be foreseen. The effect of the current mode of operation is observable also on the number and durations of fixations on the two main AOIs: video streamed from the robot and external water tank level. The first attracts the operator attention mainly when in manual mode, the second when in autonomous mode (p < 0.05).

Spearman correlations of data sample per second confirm previous results. Indeed, markers on these AOIs are correlated with the mode of the robot (ρ = 0.22, p < 0.05) as well as IHRV (ρ = 0.03, p < 0.05). Several kind of correlation have been identified analyzing the recorded dataset, of which the most significant to describe the human operator engagement are found to be: the number and durations of fixations on AOIs corresponding to the two main tasks are negatively correlated (ρ = -0.6, p < 0.05) which describes the fact that the human tends to switch attention mainly between these two tasks; the correlation of IHRV marker to the markers on the AOIs (tank: ρ = 0.1, p < 0.05; video: ρ = -0.06, p < 0.05) demonstrates that the main tasks are perceived as so from the human operator, while its correlation to the remaining mission time (ρ = 0.05, p < 0.05) expresses a higher engagement as the mission progresses. Moreover, IHRV is also negatively correlated to performance indexes as the number of extinguished fires (ρ = -0.07, p < 0.05) and the external tank level (ρ = -0.14, p < 0.05) meaning that the human operator has higher engagement level when successfully accomplishing the mission.

The outcomes of the proposed research confirm that human-robot interaction mission implies mental states variation that corresponds to levels of engagement. The demonstrated link between the sampled correlations and the global statistical analyses, returning relevant information on the human operator behavior, validates the possibility of using these markers for on-line applications. The effect of the alternation of manual and autonomous mode during the mission has been quantified on the markers and paves the way for automatic tasks allocation by a decisional system based on physiological data classification.

Finally, the results obtained through these experiments demonstrate the validity of the overall approach proposed and the designed virtual environment. Further statistical analyses and the employment of additional physiological measurements such as electroencephalogram (EEG) are foreseen for the next future.

Acknowledgements

This work was supported by Dassault Aviation patronage chair CASAC.