Gene Regulatory Networks and Signaling Pathways in Palatogenesis and Cleft Palate: A Comprehensive Review

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Cells- 2502965

Comments to the Author This review attempts to describe gene regulatory networks and signaling pathways in palatogenesis and cleft palate. The authors have tried to go into detail and have provided nice illustrations. However, this review has a number of flaws, which will be described below:

Above all, we put the manuscript after the response letter because you won't have the option to upload a new version of the manuscript until you've responded to the reviewer's comments.

1. The review does not include recent work, although the authors used the words, “recent studies”, “recent”, and “recent research” in the manuscript. The authors have studied miRNAs and ZEB. The reviewer wondered why they did not include this information in the manuscript.

▶ We totally agree with the reviewer’s notion. We put in the recent references or change the word “recent” to “previous” in sentences. Also, as the reviewer commented, we have studied the interaction between miRNA and Zeb, Smad2, and Snai1. So, we wrote about that content as follows.

“The genetic process of palatal development has been described previously, and several studies have additionally shown the role of epigenetic factors such as microRNAs that regulate genes in the palatal fusion process [94,95]. For example, miR-200b, highly expressed in epithelial cells [96], was found in the MES during palatal fusion and its expression decreased as fusion progressed. Smad2, essential for Snai1 induction in Tgf-β signaling during palate development, was expressed in MEE and MES [94,95]. Snai1, crucial for palatal fusion via Tgf-β signaling, was present in mesenchyme and some MEE cells [95]. In addition, Ectopic miR-200b expression led to Zeb family suppression, E-cadherin upregulation, and alterations in cell migration and palatal fusion [94]. These findings indicate miR-200b's critical role in cell migration and palatal fusion during palate development by regulating Zeb1 and Zeb2 as a noncoding RNA, while also suggesting a potential interaction with TGF-β-mediated Smad2 and Snai1 signaling pathways in the context of normal palate development.”

 

2. Pages 2–3, The entire chapter of “2.2 Classification of cleft lip and palate”: Cleft palate phenotypes differ between humans and mice. It is not clear to the reviewer which species the authors are describing. Please clarify this in the manuscript and be especially careful with these comments: - “Involving only the hard palate or the soft palate” - “Either the hard palate or the soft palate”

▶ We totally agree with the reviewer’s notion. As the reviewer commented, “2.2 Classification of cleft lip and palate” is a description of cleft lip and palate in humans, so we changed the title to 2.2 Classification of cleft lip and palate in humans and added “human” to the sentence as follows.

“2.2 Classification of cleft lip and palate in human

In human, cleft lip and palate are common congenital disabilities that can affect the structure of the face. There are several classifications for describing clefts of the palate and lip. Cleft lip is a congenital condition that occurs when tissues of the upper lip do not fuse together properly during human embryonic development. This can result in a gap or opening in the lip, which can range in size and location. In addition, cleft lip can occur on one side of the lip (unilateral cleft lip) or on both sides of the lip (bilateral cleft lip) (Figure 1).

Unilateral cleft lip is further classified based on the extent of the cleft and location of the cleft within the lip. Unilateral cleft lip of human has three subtypes:

• Incomplete cleft lip: This type of cleft lip is characterized by a gap or opening in the lip that is smaller than a complete cleft lip. The location and size of the cleft can vary.
• Complete cleft lip: This type of cleft lip involves the entire width of the upper lip, extending from the base of the nose (boundary between the lip and the surrounding skin).
• Median cleft lip: This type of cleft lip is a rare type of unilateral cleft lip that occurs in the center of the upper lip, dividing the lip into two separate halves.

Bilateral cleft lip is a type of cleft lip that involves both sides of the upper lip. This type of cleft lip is less common than unilateral cleft lip. It can be more challenging to treat due to extent of deformity.

Cleft palate is an aberration that arises when the roof of the mouth fails to fuse correctly during human embryonic development.”

 

We clarified this in the manuscript and be especially careful with these comments: - “Involving only the hard palate or the soft palate” - “Either the hard palate or the soft palate”

▶ Based on the reviewer's advice, we changed the sentence in “2.2 Classification of cleft lip and palate in human” section as below.

“Cleft palate can occur as a complete cleft (involving both the hard palate and the soft palate) or an incomplete cleft (involving either the hard palate or the soft palate).”

 

3. Page 4, The entire chapter of “2.3 Morphological and molecular ……” : The reviewer assumes that the authors describe the development of the mouse. Please indicate this in the text.

▶We fully understood reviewer’s suggestion. We have included the sentence " Lip closure and palatal fusion during human gestation occur at the sixth and 12th weeks [12,18], respectively, necessitating the use of animal models to study normal and abnormal craniofacial development [11-13,17,19,20]. The mouse serves as the primary model organism for investigating orofacial cleft pathogenesis, due to its genetic homology with humans, similar embryonic facial and palate development processes, and the availability of mouse strains with spontaneous or engineered mutations causing cleft lip and/or cleft palate phenotypes [12,20,21].

As we begin this chapter, our focus will be on elucidating the mechanisms of palatogenesis through an in-depth exploration of palatal development in mice. " as the opening sentence of the paragraph.

 

4. Page 4, “Elevating palatal shelves eventually come into contact and fuse in the midline. The process typically occurs between E13.5 and E15.5” . : Please cite the reference for this sentence. At E13.5, two palatal shelves are two vertically oriented next to the growing tongue and could not fuse.

▶ We totally agree with the reviewer’s notion. As the reviewer commented, we put a reference for (Bush and Jiang, Development, 2012) as follows. “Elevating palatal shelves eventually come into contact and fuse in the midline. This process typically occurs between E13.5 and E15.5 [7].”

 

5. Page 4, “Mice with concurrent Hhat and Ptch1 mutations displayed SHH gradient disruptions during frontonasal process development, resulting in medial and lateral nasal process hypoplasia and ultimately causing CLP [16].”: What is CLP? Please review the phenotype described in the reference [16].

▶ We totally agree with the reviewer’s notion. We check again the phenotype of reference 16 and fixed the sentence as follows. “Mice with concurrent Hhat and Ptch1 mutations displayed SHH gradient disruptions during frontonasal process development, resulting in medial and lateral nasal process hypoplasia and ultimately causing cleft lip and remaining midline epithelial seam [16].”

 

6. Page 10, “a lack of Ikk-α or Tbx1 in mouse embryos results in aberrant oral adhesions between the tongue and palatal shelves[70].”: Please add reference(s) to the sentence for Ikk-α. Reference [70] focuses on Tbx1.

▶ We totally agree with the reviewer’s notion. As reviewer’s comment, we put in the reference for (Richardson et al, JCI, 2014) as follows. “A lack of Ikk-α or Tbx1 in mouse embryos results in aberrant oral adhesions between the tongue and palatal shelves [70,71]”

 

7. Page 11, “The lack of Tgf-β3 in embryonic mice allows palatal shelves to establish improper contact at the midline, resulting in persistence of the MES and formation of a full cleft palate. ”: Please include reference(s) in the sentence. As far as I know, Tgf-β3 knockout mice do NOT have a “full” cleft palate.

▶ We totally agree with the reviewer’s notion. We put in the references for (Proetzel, et al, Nat. Genet.), (Kaartinen et al, Nat. Genet., 1995), (Kaartinen et al, Dev. Dyn., 1997) and fixed sentence as follow. “The lack of Tgf-β3 in embryonic mice allows palatal shelves to establish improper contact at the midline, resulting in persistence of the MES [79-81]”

 

<Minor points>

1. Authors should carefully check and distinguish between genes (italics) and proteins. Please correct.

▶ We carefully checked the text and corrected the representation of genes and proteins in manuscript.

 

2. In Figure 1, the shape of “PP” is inappropriate (not an oval shape).

▶ We totally agree with the reviewer’s notion. We changed the primary palate shape from an oval shape to a semicircle and fixed the figure legend as well.

 

3. In Figure 2, the reviewer could not find the “Blue dotted line” described in the legend.

▶ We understand the reviewer’s notion. In section A of figure 2, there is a dotted light blue line surrounding the palate, which is yellow, so I changed it to a thicker dotted light blue line and changed the words "blue dotted line" to "light blue dotted line" in the figure legend.

 

4. The identification method for knockout mice is inconsistent. - Osr2–/– - Fgf10 KO

▶ We totally agree with the reviewer’s notion. We checked overall manuscript and corrected from KO to -/-.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript needs minor changes

1) The last lines of 2.2 section needs references as well as the whole section 2.3 related to orofacial cleft classification.

2) This is a good opportunity to intend to explain, from a cellular and molecular view, the causes of asymmetry in orofacial clefts considering the symmetry of the normal process of the middle line development of the maxillofacial territory. I suggest to include some topic regarding this issue or at least to comment the absent of evidence about it. 

Author Response

Cells- 2502965

This manuscript needs minor changes

Above all, we put the manuscript after the response letter because you won't have the option to upload a new version of the manuscript until you've responded to the reviewer's comments.

 

1) The last lines of 2.2 section needs references as well as the whole section 2.3 related to orofacial cleft classification.

▶ We totally agree with the reviewer’s notion. We put references in the last lines of 2.2section. And in section 2.3 on cleft lip and palate classification, we added the following sentences and references to connect the content as follows.

 

“However, some individuals might have an asymmetric cleft affecting one side of the lip/palate or both sides in opposite configurations, which could impact classification and management [14,15]. Evidence involving mouse models specifically for asymmetry in orofacial clefts is limited; however, the formation of orofacial structures can be regulated not only by genetic factors but also by epigenetics and environmental factors. Despite substantial advancements in understanding the genetic etiology of orofacial clefts and accelerated identification of candidate causal mutations through technological and bioinformatic progress, clinical care and prevention strategies remain largely unaffected. This is primarily due to the limited comprehension of the cellular, molecular, and developmental processes underlying cleft pathogenesis [16-18]. Therefore, elucidating the causes through various mouse models is crucial for the development of treatments.”

 

2) This is a good opportunity to intend to explain, from a cellular and molecular view, the causes of asymmetry in orofacial clefts considering the symmetry of the normal process of the middle line development of the maxillofacial territory. I suggest to include some topic regarding this issue or at least to comment the absent of evidence about it. 

▶ We understood the reviewer’s notion. We added a sentence stating that there is a lack of evidence for a cause of asymmetry in orofacial clefts.

“However, some individuals might have an asymmetric cleft affecting one side of the lip/palate or both sides in opposite configurations, which could impact classification and management [14,15]. Evidence involving mouse models specifically for asymmetry in orofacial clefts is limited; however, the formation of orofacial structures can be regulated not only by genetic factors but also by epigenetics and environmental factors. Despite substantial advancements in understanding the genetic etiology of orofacial clefts and accelerated identification of candidate causal mutations through technological and bioinformatic progress, clinical care and prevention strategies remain largely unaffected. This is primarily due to the limited comprehension of the cellular, molecular, and developmental processes underlying cleft pathogenesis [16-18]. Therefore, elucidating the causes through various mouse models is crucial for the development of treatments.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

N/A